Metabolic Biochemistry, Volume 4 (Biochemistry and Molecular Biology of Fishes) (Vol 4)

Preface The idea of editing a series of volumes on The Biochemistry and Molecular Biology of Fishes was born out of the...

Author: T.P. Mommsen | Peter W. Hochachka

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Preface The idea of editing a series of volumes on The Biochemistry and Molecular Biology of Fishes was born out of the present-day lack of a forum for state-of-the-art review articles in this rapidly expanding field of research. On the one hand, researchers and students in this area always find themselves combing the literature on general (rat-dominated) biochemistry before discovering short and usually incomplete and disappointing coverage of the situation in the piscine setting. On the other hand, the rapidly expanding volume and quality of the primary literature in fish biochemistry and molecular biology supply convincing evidence for a maturing field. This discipline is no longer the younger sibling of rat or human biochemistry but has recently led to a number of major conceptual breakthroughs; for this reason, and because its activity domain is sometimes nonoverlapping with 'mainstream' biochemistry, the field is certainly ripe and ready for a review series of its own. Comparative biochemistry and molecular biology and comparative physiology as disciplines by definition use organisms as a special kind of experimental parameter for probing general mechanisms and principles of function. In theory this approach is relatively blind to phylogenetic boundaries, but in practise the realities of funding and availability of experimental material greatly narrow the field of play. As a result, two phylogenetic groups - - the insects and the fishes - - have over the last several decades provided the bulk of the experimental data base in these disciplines. Interestingly, although comparative biochemistry in many ways grew out of comparative physiology, the growth and development of these two activities in the insect field have to major extent proceeded along independent paths. By contrast, the comparative physiology and biochemistry of fishes have not been so independent of one another and the tendency has been for the former to envelope the latter. We believe that the current conceptual developments in the fields as well as the simple logistics of dealing with massive data bases make this the right time for the reality of independence to match the perception of independence, which we feel is another important rationale for this review series. Our goal is to provide researchers and students with a pertinent information source from theoretical and experimental angles. To be useful to students, theoreticians, and experimentalists alike, contributing authors are urged to emphasize concepts as well as to relate experimental results to the biology of the animals, to point out controversial issues, and todelineate as much as is possible directions for future research. Peter W. Hochachka Thomas P. Mommsen Vancouver and Victoria, B.C.

Contributors

Hiroki Abe, Department of Food Science and Nutrition, Kyoritsu Women's University, 1- 710 Motohachioji, Hachioji, Tokyo 193, Japan (Chapter 14) James S. Ballantyne, Department of Zoology, University of Guelph, Guelph, Ontario, Canada NI G 21/11 (Chapter 10) Andrew H. Bass, Section of Neurobiology and Behavior, Cornell University, Ithaca, New York 14853, USA (Chapter 12) Ralf Bastrop, Universiti~tRostock, Fachbereich Biologie, Zoologisches Institut, Universitiitsplatz 2, D-02500 Rostock 1, Germany (Chapter 7) Richard W. BriU, Southwest Fisheries Science Center, Honolulu Laboratory, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, Honolulu, Hawaii 96822-2396, USA (Chapter 1) Stephen EJ. Brooks, Nutrition Research Division, Health Canada, Tunney's Pasture, Ottawa, Ontario, Canada K1A OL2 (Chapter 13) C.G. Carter, Department of Aquaculture, University of Tasmania, PO Box 1214, Lauceston, Tasmania 7250, Australia (Chapter 8) Nathan L. Collie, Department of Biological Sciences, Texas Tech University, Lubbock, Texas 79409-3131, USA (Chapter 9) Ronaldo P. Ferraris, Department of Physiology, University of Medicine and Dentistry of New Jersey,, New Jersey Medical School, Newark, New Jersey 07103-2714, USA (Chapter 9) Glen D. Foster, Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada KIN 6N5 (Chapter 4) Edward M. Goolish, National Oceanic and Atmospheric Administration, Southwest Fisheries Science Center, La Jolla, California 92038, USA and Scripps Institution of Oceanography, Center for Marine Biotechnology and Biomedicine, University of California, San Diego, La Jolla, California 92093, USA (Chapter 15) Joaquim Guti6rrez, Departament de Bioquimica i Fisiologia, Universitat de Barcelona, Unitat de Fisiologia Animal F, Av. Diagonal, 645, E-08071 Barcelona, Spain (Chapter 17) Peter W. Hochachka, Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada V6T 2A9 (Chapter 1)

viii

Contributors

D.E Houlihan, Department of Zoology, University of Aberdeen, Aberdeen, TiUydrone Avenue, Aberdeen AB9 2TN, Scotland, UK (Chapter 8) Karl Jiirss, Universiti~t Rostock, Fachbereich Biologie, Zoologisches Institut, Universitiitsplatz 2, 1)-02500 Rostock 1, Germany (Chapter 7) Odile Mathieu-Costello, Department of Medicine, Universityof California, San Diego, La Jolla, California 92093-0623, USA (Chapter 1)

I.D. McCarthy, Department of Zoology, University of Aberdeen, Aberdeen, Tillydrone Avenue, Aberdeen AB9 2TN, Scotland, UK (Chapter 8) Thomas P. Mommsen, Department of Biochemistry and Microbiology, University of

Victoria, P.O. Box 3055, Victoria, British Columbia, Canada VSW3P6 (Chapter 12) 9Thomas W. Moon, Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada KIN 6N5 (Chapter 4) Christopher D. Moyes, Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6 (Chapter 16) Isabel Navarro, Departament de Bioquimica i Fisiologia, Universitat de Barcelona,

Unitat de Fisiologia Animal F,, Av. Diagonal, 645, E-08071 Barcelona, Spain (Chapter 17) Bernd Pelster, lnstitut ffir Physiologie, Ruhr-Universitdt Bochum, D.44780 Bochum,

Germany (Chapter 5) Jean-Francois Rees, Laboratory of Animal Physiology, Catholic University of Louvain,

Croix du Sud 5, B-1348 Louvain-la-Neuve, Belgium (Chapter 18) Kenneth B. Storey, Departments of Biology and Chemistry, Carleton University,

Ottawa, Ontario, Canada KIS 5B6 (Chapter 13) Eric M. Thompson, Laboratoire de Biologie Cellulaire, Unit~ de Biologie du D~veloppement, Institut National de la Recherche Agronomique, F-78352 Jouy-en-Josas, France (Chapter 18) Guido van den Thillart, Institute of Evolutionary and Ecological Sciences, Animal

Physiology, Gorlaeus Laboratories, University of Leiden, PO Box 9502, 2300 RA Leiden, The Netherlands (Chapter 3) Douglas R. Tother, NERC Unit of Aquatic Biochemistry, School of Natural Sciences,

University of Stirlinb StirlingFK9 4LA, Scotland, UK (Chapter 6) Marcel van Raaij, Institute of Evolutionary and Ecological Sciences, Animal Physiology, Gorlaeus Laboratories, University of Leiden, PO Box 9502, 2300 RA Leiden, The Netherlands (Chapter 3) Patrick J. Walsh, Marine Biology and Fisheries Division, Rosenstiel School of Marine and Atmospheric Sciences, Universityof Miami, 4600 Rickenbacker Causeway, Miami, Florida 33149-1098, USA (Chapter 12)

Contributors

ix

Jean-Michel Weber, Biology Department, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada KIN 6N5 (Chapter 2) Timothy G. West, Department of Zoology, Cambridge University, Downing Street, Cambridge, CB2 EJ3, UK (Chapter 16) Harold H. Zakon, Department of Zoology, Patterson Laboratory, The University of Texas, Austin, Texas 78712, USA (Chapter 11) Georges Zwingelstein, Laboratoire Maritime de Physiologie, Institut Michel Pacha, Universit~ de Lyon, 1337 Corniche Michel Pacha, Tamaris, F-83500 La Seyne sur Mer, France (Chapter 2)

Abbreviations

Amino acid(s) Acetylcholine receptor Adrenocorticotropic hormone Alanine aminotransferase Aldolase Ammonia quotient Atlantic salmon cell line Aspartate aminotransferase Brushborder membrane vesicles Branched-chain amino acid aminotransferase BCKAD Branched-chain a-ketoacid dehydrogenase Bluegill fry cell line BF-2 Immunoglobulin binding protein BiP Basolateral membrane vesicles BLMV Y,5'-cyclic adenosine-monophosphate cAMP Cytochrome C oxidase CCO CHSE-214 Chinook salmon epithelium cell line Creatine phosphokinase CPK Carnitine palmitoyl transferase CPT Citrate synthase CS Diacylglycerol DAG 5a-Dihydrotestosterone DHT Dimethylformamide DMF Dimethylsulfoxide DMSO 17fl-Estradiol E2 EAA Essential amino acid(s) Ethylenediaminetetraacetic acid EDTA Electric organ EO Electric organ discharge EOD Erythropoietin EPO Free amino acid(s) FAA Fatty acid binding protein FABP Fructose 1,6-bisphosphatase FBPase Free fatty acid(s) FFA Fast glycolytic (muscle fiber) FG Fathead minnow cell line FHM Fast oxidative glycolytic (muscle fiber) FOG Glucose 6-phosphatase G6Pase Glucose 6-phosphate dehydrogenase G6PDH Gamma-aminobutyrate GABA GAPDH Glyceraldehyde 3-phosphate dehydrogenase Glutamate dehydrogenase GDH Glucagon-like peptide GLP Glycogen phosphorylase GPase oeGPDH ~-Glycerophosphate dehydrogenase Glycogen synthase GSase High density lipoproteins HDL 12-Hydroxyeicosapentaenoate HEPE 12-Hydroxyeicosatetraenoate HETE AA AChR ACI'H AIaAT ALD AQ AS AspAT BBMV BCAAT

HK HPLC HSP IDL LCAT LDH LDL LT LX ME MT NEAA NMJ NMR ODC PAF PC PCA PCr PDG 6PGDH PEPCK PFK-I PG PG I PGK PK PKA PKC PMN PtdA PtdCho PtdEtn Ptdlns PtdSer PUFA RQ RT-2 RTG SDA SO T3 TAG TF TPI TRH TX VHDL VLDL XDH XO

Hexokinase High performance liquid chromatography Heat-shock protein Intermediate density lipoproteins Lecithin:cholesterol acyl transferase Lactate dehydrogenase Low density lipoproteins Leukotrienes Lipoxins Malic enzyme 17oe-Methyltestosterone Non-essential amino acids Neuromuscular junction Nuclear magnetic resonance Ornithine decarboxylase Platelet activating factor Pyruvate carboxylase Perchloric acid Phosphocreatine Phosphate-dependent glutaminase 6-Phosphogluconate dehydrogenase Phosphenolpyruvate carboxykinase Phosphofructokinase- 1 Prostaglandins Phosphoglucose isomerase Phosphoglycerate kinase Pyruvate kinase Protein kinase A Protein kinase C Pacemaker nucleus Phosphatidic acid Phosphatidylcholine Phosphatidylethanolamine Phosphatidylinositol Phosphatidylserine Polyunsaturated fatty acids Respiratory quotient Rainbow trout germ cell line Rainbow trout gonad cell line Specific dynamic action Slow oxidative (muscle fiber) 3,5,3? -Triiodo-L-thyronine Triacylglycerol Turbot fin cell line Triosephosphate isomerase thyrotropin releasing hormone Thromboxanes Very high density lipoproteins Very low-density lipoprotein Xanthine dehydrogenase Xanthine oxidase

Hochachka and Mommsen (eds.), Biochemistry and molecular biology of fishes, vol. 4 9 1995 Elsevier Science B.V. All rights reserved. CHAPTER 1

Design for a high speed path for oxygen: tuna red muscle ultrastructure and vascularization ODILE MATHIEU-COSTELLO, RICHARD W. ]]RILL * AND PETER W. HOCHACHKA **

Department of Medicine, University of California, San Diego, La JoUa, CA 92093-0623, U.S.A., * Southwest Fisheries Science Center, Honolulu Laboratory, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, Honolulu, HI 96822-2396, U.S.A. and **Department of Zoology, University of British Columbia, Vancouver, B.C., Canada V6T 2,49

I. Introduction II. Materials and methods 1. Animals 2. Tissue preparation 3. Morphometry III. Results and discussion Acknowledgements IV. References

I. Introduction Because it is one of the most aerobic muscles in fish, the red muscle of tuna is of particular interest to study strategies and constraints in structural designs for high 02 flux from capillary to muscle fiber mitochondria. Tuna can maintain extremely high aerobic metabolic rates and reach high swimming speeds 4. The tuna red muscle is well known to operate at higher than ambient water temperature by conserving heat via the central counter-current heat exchange (for review, see ref. 36), and white muscle lactate turnover rates after exercise are known to be closer to those found in mammals than in other fish 1'39. In this chapter, we summarize our morphometric findings on the three-dimensional arrangement of the capillary network and its relationships with fiber ultrastructure in red muscle of skipjack tuna, Katsuwonus pelamis, in comparison to highly aerobic skeletal muscles of birds and mammals. Muscles designed for high sustainable activity (hummingbird and bat flight muscles as well as the red muscle of tuna) are all composed of only one population of very highly aerobic fibers, instead of the mosaic of fiber types with different metabolic pattern found in the vast majority of skeletal muscles. This homogeneity allows one to specifically examine capillary-fiber geometrical relationships across species, in particular vascular supply in relation to muscle

2

O. Mathieu-CosteUo, R.W. Brilland RW. Hochachka

fiber aerobic capacity in cases of very high demand for 02 flux. As summarized further in this chapter, previous studies showed striking similarities in structural design for high 02 flux in hummingbird and bat flight muscles despite several differences in capillary-fiber geometry2s,29. In fish as in birds, red blood cells are nucleated and less deformable than mammalian red cells, but they can be larger than bird red cells, and fishes operate at different body temperature than both birds and mammals. Thus, it is of particular interest: (1) to examine capillary-fiber structural arrangement in the red muscle of one of the most athletic fishes known; and (2) to compare it with that in highly aerobic skeletal muscles of birds and mammals.

II. Materials and methods While the details of methods used here have been described elsewhere 22, it is important to briefly highlight aspects that are relevant to properly explain the results.

1. Animals Five Skipjack tuna (Katsuwonus pelamis); body mass 1.5-2 kg; fork length 43-44 cm) were purchased from local commercial fishermen and held in outdoor 10 m diameter holding tanks supplied with continuously flowing seawater (25 4- 1~ at the Kewalo Research Facility (National Marine Fisheries Service, Honolulu, Hawaii).

2. Tissuepreparation After the tunas had been netted and anesthetized, muscle peffusion fixation with glutaraldehyde fixative (four animals) or infusion with Batson's casting material (one animal) were performed following procedures and subsequent tissue processing described elsewhere in detail22. Transverse and longitudinal sections (1 /zm thick) of perfusion-fixed tissue were used for light microscopy morphometry of capillarity and fiber size. Ultrathin transverse sections (50-70 nm) were examined with a Zeiss 10 transmission electron microscope and sampled for morphometry of fiber ultrastructure. Samples injected with casting material were examined with a Stereoscan 360 scanning electron microscope (Cambridge Instrument).

3. Morphometry Sarcomere length was measured on longitudinal sections, after careful control of the angle of each section 19. Fiber cross-sectional area, capillary diameter and capillary number around a fiber were measured on transverse sections with an image analyzer. Capillary numbers per fiber sectional area in transverse and longitudinal sections were collected by point-counting, and the data were used to estimate

Design for a high speed path for oxygen: tuna red muscle ultrastructure and vascularization

3

the degree of orientation of capillaries and capillary length per fiber volume 18. Capillary-to-fiber ratio (i.e. capillary number per fiber number) was computed as the product of capillary density (i.e. number per fiber cross-sectional area) and mean fiber cross sectional area. Capillary surface per fiber volume was obtained by intersection-counting on vertical (i.e. longitudinal) sections using a cycloid grid 2. Capillary-to-fiber perimeter ratio in transverse section, which is an index of the size of the capillary-fiber interface 25 was measured by intersection-counting in transverse sections 21, and capillary surface per fiber surface estimated as the product of capillary-to-fiber perimeter ratio and an orientation coefficient c'(K',O) as described elsewhere 25. The volume of mitochondria per volume of muscle fiber was estimated by standard point-counting 22, and mitochondrial volume per/zm fiber length calculated as the product of mitochondrial volume density and fiber cross-sectional area. Where appropriate, data on fiber size and capillary density were normalized to sarcomere length, in order to compare morphological data between muscles, independent of the particular length at which each sample was fixed and therefore examined. A normalizing sarcomere length of 2.1/zm was chosen because it is in the mid-range of the sarcomere lengths where maximal tension is developed in skeletal muscles, and it is within the range of operating sarcomere lengths in hindlimb muscles of mammal during terrestrial locomotion (range, 1.7-2.7 ~m) 6, wing muscles of bird during wing beat cycle (1.7-2.3/zm) 5 and red muscle in fish during swimming at slow speed (1.9-2.2/zm) 35.

III. Results and discussion Figure la-c illustrates the high capillary density, small fiber size and high mitochondrial volume density previously reported in red muscle of tuna 3,1~ In longitudinal sections (Fig. lb), we found a large number of capillaries cut in transverse or oblique section, as well as branches running perpendicular to the muscle fiber axis. This suggested the presence of capillary manifolds in tuna red muscle, as previously found in the highly aerobic pectoralis muscle of pigeon 2~ Figure 2a,b illustrate the remarkable similarity between the appearance of capillary manifolds in tuna red muscle (Fig. 2a) and pigeon pectoralis muscle (Fig. 2b). In that study, Potter and coworkers 34 showed that these capillary branches oriented perpendicular to the muscle fiber axis are venular capillaries which form dense manifolds around groups of muscle fibers. The examination of microcorrosion casts of tuna red muscle also showed that capillaries form a dense envelope of blood around muscle fibers (Fig. 2c). The functional implications of the particular arrangement of venular capillaries in those muscles are not fully understood. Capillary manifolds could facilitate an increased vascular supply to and from the muscle fibers at the venular end of the network where substrates and 02 content are lowest and metabolite concentration highest. They could also be related to other functional aspects such as heat dissipation and/or the blood pumping action of the muscle during flight in

4

O. Mathieu-Costello, R.W. BriU and RW. Hochachka

Fig. 1. Fine structure of tuna red muscle, a and b: light micrographs of portions of muscle bundles in transverse and longitudinal sections, respectively, c: electron micrograph of transverse section of muscle fibers and adjacent capillaries (c). Capillaries are empty after the fixation by vascular perfusion. Note large capillary density and small fiber size (a-c), large number of capillary branches running perpendicular to the muscle fiber axis (b) and high density of mitochondria, M (c). From ref. 22.

birds. Interestingly, however, capillary manifolds were found in flight muscle of hummingbird ~, but not in bat 24,29. The fact that they were found in tuna red muscle also suggest possible rheological implications since in fish, as in bird, red blood cells are nucleated and less deformable than mammalian red cells. Another possibility in tuna is transfer of heat from the muscle at the venular end of the network, as it possibly favors heat removal in bird flight muscle 2~ Table 1 summarizes morphometric data on capillarity and fiber ultrastrueture in red muscle of tuna compared with tuna white muscle, and aerobic muscles of birds and mammals with large differences in aerobic capacities. In tuna red muscle, fiber cross-sectional area was small (~500/~m 2) but not as small as in ultimate cases of high aerobic capacity in bird and mammal. In hummingbird and bat flight muscles, average fiber cross-sectional area was ~200 and 300/~m 2, respectively, in tissues similarly prepared. Note that the number of capillaries per number of fibers was similar in tuna red muscle and hummingbird flight muscle (~1.6). However,

Design for a high speed path for oxygen: tuna red muscle ultrastructure and vascularization

5

Fig. 2. Examples of capillary manifolds, a: light micrograph in a longitudinal section of tuna red muscle. b and c: scanning electron micrographs of vascular corrosion casts examined perpendicular to the surface of the manifold in pigeon flight muscle (b) and in cross-section in tuna red muscle (c). Note the remarkable similarity between the appearance in tuna (a) and pigeon (b) muscles, and the dense envelope formed by capillaries around muscle fibers (c). Based on fiber dimensions, two muscle fibers (A and B) could be contained in the empty space in c. From refs. 22 (a,c) and 34 (b).

because of the difference in fiber size, there was a huge difference in capillary numerical density between the muscles. The number of capillaries per mm 2 fiber cross-sectional area at 2.1/zm sarcomere length was 3400 in tuna red muscle and 8000 in flight muscle of hummingbird. Capillary length density is an important estimate of capiUarization which accounts for capillary geometry, and determines capillary volume and surface area available for exchange per unit volume of fiber and mitochondria. Figure 3 shows estimates of the degree of capillary orientation, expressed as the percentage added

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Sarcomere length,/~m Fig. 3. Plot of the degree of orientation of capillaries (expressed as the percentage added to capillary length by tortuosity and branching, compared with straight, unbranched capillaries oriented parallel to the muscle fiber axis) against sarcomere length in red muscle of tuna (solid circle) compared with group mean values (:I:SE) in highly aerobic muscles of birds and mammals. From refs. 22 (tuna), 28 (hummingbird) and 29 (bat). Relationships in rat hindlimb (solid line) and pigeon pectoralis muscles (broken line) are from refs. 25 and 20, respectively. to capillary length by tortuosity and branching, against sarcomere length in the muscles listed in Table 1. It is now well established that both fiber size 7,11,26 and the degree of orientation of capillaries 19'27,33 are functions of sarcomere length. As a consequence, capillary density in transverse sections can underestimate capillary length per fiber volume by a different percentage (e.g. 10-70% in rat muscles; see solid line in Fig. 3) depending on the sarcomere length at which samples are fixed and therefore examined. Figure 3 also shows that the degree of orientation of capillaries can vary between muscles and/or animals, for example bat flight muscle compared with rat soleus, or bird compared with mammal. Therefore, it may not be appropriate to compare muscle capillarity based on capillary densities in transverse sections alone even if all samples are fixed at the same sarcomere length 2~ It is interesting to note that in tuna red muscle, the contribution of tortuosity and branching to capillary length was similar to that in rat muscles (Fig. 3) in spite of the different geometry. In contrast to tuna and bird muscles 22,28 skeletal muscles of mammals showed no evidence of capillary manifolds 29. The different contribution of capillary tortuosity and branching to capillary length in tuna red muscle compared with bird muscles (Fig. 3) may be related to differences in fiber size, yielding differences in length and/or number of branches in manifolds between the muscles. In addition, capillary length density in tuna red muscle and pigeon pectoralis muscle were remarkably similar (4100 mm/mm 3) in spite of the different degree of capillary orientation, capillary-to-fiber ratio and fiber size. It was less than half the capillary length density seen in ultimate cases such as the flight muscles of hummingbird and small bats (Table 1).

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Our measurements of fiber size in red and white muscle of skipjack tuna are within the range of values reported by others, although direct comparison is often difficult because of differences in tissue preparation or because sarcomere length is not reported. To our knowledge, capillary density (e.g. capillary-to-fiber ratio and number per fiber cross-sectional area) or geometry in tuna red muscle had never been reported prior to our studies. Comparison with data in red muscle of other fishes 8,17,3~ revealed that neither was fiber size the smallest, nor capillary number around a fiber or capillary density the highest in red muscle of tuna. Similarly, mitochondrial volume density in tuna red muscle (28.5-35%; this study and ref. 16) was high, but not the highest, for fish muscle. The highest mitochondrial volume density for fish (45.5%) has been reported in red muscle of anchovy 17. The comparison of capillary length per fiber volume at a given mitochondrial volume density showed that values in tuna red muscle were as great as in mammalian heart and about half those in highly aerobic muscles of bird and mammals (Fig. 4). For example, capillary length per fiber volume at 30% mitochondrial volume density was 4300 mm -2 in tuna red muscle 22 compared with 7600 mm -2 in bat and rat muscles 29. It is interesting to note that in flight muscle of bird (hummingbird and pigeon), capillary length per unit volume of mitochondria was similar to that in bat and rat hindlimb (Fig. 4). There were about 25 km capillaries per ml of mitochondria in those muscles compared to 14 km in tuna red muscle. The different capillary geometry does not account for the different relationship between capillary length per fiber volume and mitochondrial volume density in tuna compared with

Design for a high speed path for oxygen: tuna red muscle ultrastructure and vascularization

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highly aerobic muscles of birds and mammals (capillary manifolds were found both in tuna red muscle and bird flight muscle). On average, about one third of fiber mitochondrial volume was subsarcolemmal in tuna red muscle. This fraction was less than in flight muscles and more than in rat soleus, where subsarcolemmal mitochondria represented about one half and less than one fifth of the fractional volume of mitochondria, respectively (Table 1). In other words, comparison of highly aerobic muscles in fish, bird and mammal shows that the proportion of subsarcolemmal mitochondria is not greater in muscle with greater fiber size. Rather the opposite is observed, bat and hummingbird flight muscles (with the smallest fiber size) showing the greatest relative proportion of subsarcolemmal mitochondria. Interestingly the red muscle of anchovy, with the greatest reported volume density of mitochondria for fish skeletal muscle (45.5%), also showed a much greater fiber cross-sectional area (1115 /tm2; ref. 17) than tuna and other highly aerobic muscles (Table 1). This also indicated that intrafiber diffusion distances to mitochondria are not necessarily reduced in highly aerobic muscles of fish. A relatively large proportion of subsarcolemmal mitochondria (25% of total mitochondrial fractional volume) was found in tuna white muscle (Table 1). It was similar to that in bat hindlimb and almost as large as in pigeon pectoralis and tuna red muscle, i.e. muscles with much smaller fiber size and much greater proportion of interfibrillar mitochondria than in white muscle of tuna. Thus, a great ratio of subsarcolemmal relative to interfibrillar mitochondria is not necessarily a characteristic of highly aerobic muscles. Another important parameter to consider when assessing the three-dimensional arrangement of capillaries relative to the muscle fibers and its impact on the geometry of blood-tissue exchange, is capillary-fiber surface. Traditionally, muscle potential for 02 flux had been viewed in terms of intercapillary and diffusion distances. In contrast, recent experimental and theoretical evidence (see ref. 13 for review) suggested an important role of the capillary-fiber interface in determining 02 flux rates in working red muscles. Cryomicrospectroscopy measurements of myoglobin saturation in quick-frozen red muscles have shown that the major pO2 drop from capillary into a cross-section through the muscle fiber occurs within a few microns subjacent to the capillary and further decline towards the center of the fiber is very shallow because of myoglobin facilitated diffusion 9. In this context, capillary-to-fiber surface, i.e. the size of the capillary-fiber interface, is an aspect of capillary-fiber structure which needs to be also considered when assessing muscle capacity for 02 flux from capillary to fiber mitochondria. As pointed out by Sullivan and Pittman 3s, matching 02 supply and demand in muscles can be achieved by nature via different strategies. It can change fiber size (which affects capillary surface per fiber volume) or capillary-fiber contact area (i.e. capillary-fiber surface) or both. Figures 5 and 6 show the relationships between capillary surface per fiber volume and mitochondrial volume density (Fig. 5) and capillary-fiber surface and mitochondrial volume per unit length of fiber (Fig. 6) in red muscle of tuna compared with highly aerobic muscles of bird and mammal. Capillary surface density at a given volume density of mitochondria was smaller in tuna red muscle

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e===

0 0

e 9149

0.2 s o s 0

0

r

o 0.0 ,,. 0

.--_ Q. C)

o

6 <>

0

~

0 50

1 O0

150

Mitochondrial volume //~m

200

250

fiber length

Fig. 6. Plot of capillary surface per fiber surface against mitochondrial volume per unit length of muscle fiber (i.e. mitochondrial volume density multiplied by fiber cross-sectional area) in tuna red muscle (solid circle) compared with rat soleus (open diamond), flight muscle of hummingbird (solid triangle) and bat (solid diamond) and group mean value (:I:SE) in bat hindlimb. From refs. 22 (tuna), 28 (hummingbird) and 29 (bat and rat).

Design ]'or a high speed path ]:or oxygen: tuna red muscle ultrastructure and vascularization

11

(Fig. 5). This was due to the smaller capillary length density in tuna (Fig. 4) while capillary diameter was similar ("4/xm) among muscle groups. It is also interesting to note the similar capillary surface per unit volume of mitochondria in highly aerobic flight muscles (bat and hummingbird) and in bat hindlimb and rat soleus muscles ('-'3400 cm 2 per ml of mitochondria). In comparison, the value in tuna red muscle was only "~1800 cm 2 (Fig. 5). In contrast, capillary surface per fiber surface at a given mitochondrial volume per unit length of fiber was similar in tuna red muscle and rat M. soleus and it was about half that in the flight muscles of bat and hummingbird (Fig. 6). Interestingly, the ratio between capillary-to-fiber surface and mitochondrial volume per unit length of fiber in the most highly aerobic muscle in fish, i.e. the red muscle of anchovy (calculated from ref. 17; see ref. 22) was also close to that in tuna red muscle and it was more than half those in flight muscles of bat and hummingbird. This suggests consistent differences in the size of the capillary-to-fiber interface relative to the mitochondrial volume to be supplied per unit length of fiber in extremely highly aerobic muscle of fish compared with bird and mammal. The greater capillary-fiber surface ratio in flight muscles at a given mitochondrial volume per unit length of fiber suggests an increased capacity for 02 flux. It is consistent with the greater respiratory rates of mitochondria in flying hummingbirds 37 (7-10 ml 02 per ml mitochondria per min) compared with locomotory muscles of mammals running at VO2max (ref. 15) (5 ml O2/ml mitochondria/min). It supports the idea of an important role of the capillary-fiber interface in determining 02 flux rates in working red muscles 9. In tuna red muscle, capillary-fiber surface at a given volume of mitochondria per unit length of fiber was similar to that in rat soleus (Fig. 6), in spite of the lower capillary surface per unit volume of mitochondria in tuna (Fig. 5). Measurements of maximal respiratory rates of tuna red muscle mitochondria in vitro 31, yielded estimates of maximal in vivo mitochondrial respiratory rates at least 3-5 times lower in tuna red muscle than in mammals 22. The reason for this difference is not fully understood. The differences in operating temperatures between the muscles could play a role, since accounting for plausible Q l0 values yield maximal respiratory rates in tuna close to those in mammal 22. However, other explanations are also possible including the up-regulation of protein and amino acid metabolism in fish muscle compared with other vertebrates 12 which may require greater mitochondrial volume densities for the enzymes of amino acid and protein turnover. Both substrate and heat transfer may also require an increased capillary-fiber surface in tuna independently of 02 transfer per se 22. In summary, examination of capillary-to-fiber geometry in tuna red muscle displays both similarities and differences with features found in the most highly aerobic muscles of birds and mammal. Three features seem prominent in the design for high flux paths for oxygen: (1) small fiber size; (2) high capillary density; and (3) high mitochondrial density, but in tuna these are not as pronounced as in hummingbird and bat flight muscles. Additionally, a particular arrangement of capillary manifolds seem required in birds and tuna but not in mammals. Perhaps because of constraints of function at different temperatures, capillary length per unit volume of mitochondria is substantially shorter in red muscle of tuna than in

12

O. Mathieu-Costello, R. W. Brill and R W. Hochachka

skeletal muscles of both bird and mammal over a wide range of aerobic capacities. Similarly, capillary-to-fiber surface appears to be systematically smaller in highly aerobic muscles of fish than in flight muscle of birds and mammals for the volume of mitochondria to be supplied per unit length of fiber. Whether those differences are related to differences in mitochondrial properties or capillary function or both, remains to be determined.

Acknowledgements. Supported by Grant 5PO1 HL-17731 from the National Institutes of Health, U.S.A.

II4. References 1. Arthur, P.G., T.G. West, R.W. Brill, P.M. Schulte and P.W. Hochachka. Recovery metabolism of skipjack tuna (Katsuwonus pelamis) white muscle: rapid and parallel changes in lactate and phosphocreatine after exercise. Can. I. Zool. 70: 1230-1239, 1992. 2. Baddeley, A.J., H.J.G. Gundersen and L.M. Cruz-Orive. Estimation of surface area from vertical sections./. Microsc. 142: 259-276, 1986. 3. Bone, Q. Myotomal muscle fiber types in Scomber and Katsuwonus. In: The Physiological Ecolo~ of Tunas, edited by G.D. Sharp and A.E. Dizon, New York, Academic Press, pp. 183-205, 1978. 4. Bushnell, P.G. and R.W. Brill. Responses of swimming skipjack (Katsuwonus pelamis) and yellowfin (Thunnus albacares) tunas to acute hypoxia, and a model of their cardiorespiratory function. PhysioL Zool. 64: 787-811, 1991. 5. Cutts, A. Sarcomere length changes in the wing muscles during the wing beat cycle of two bird species, l. Zool. London (.4) 209: 183-185, 1986. 6. Dimery, N.J. Muscle and sarcomere lengths in the hind limb of the rabbit (Otyctolagus cuniculus) during a galloping stride, l. Zool. London (tl) 205: 373-383, 1985. 7. Dulhunty, A.E and C. Franzini-Armstrong. The relative contributions of the folds and caveolae to the surface membrane of frog skeletal muscle fibres at different sarcomere lengths. I. Physiol. (London) 250: 513-539, 1975. 8. Dunn, J.E, W. Davison, G.M.O. Maloiy, P.W. Hochachka and M. Guppy. An ultrastructural and histochemical study of the axial musculature in the African lungfish. Cell Tissue Res. 220: 599-609, 1981. 9. Gayeski, T.E.J. and C.R. Honig. O2 gradients from sarcolemma to cell interior in red muscle at maximal VO2. Am. I. Physiol. 251: H789-H799, 1986. 10. George, J.C. and E.D. Stevens. Fine structure and metabolic adaptation of red and white muscles in tuna. Env. Biol. Fish. 3: 185-191, 1978. 11. Gray, S.D. and E.M. Renkin. Microvascular supply in relation to fiber metabolic type in mixed skeletal muscles of rabbits. Microvasc. Res. 16: 406-425, 1978. 12. Hochachka, P.W. and G.N. Somero. Biochemical Adaptation, New Jersey, Princeton University Press, 1984. 13. Honig, C.R., Gayeski, T.E.J. and Groebe, IC Myoglobin and oxygen gradients. In: The Lung, edited by R.G. Crystal, J.B. West, P.J. Barnes, N.S. Cherniack and E.R. Weibel. New York: Raven Press, p. 1489-1496, 1991. 14. Hoppeler, H. and S.R. Kayar. Capillarity and oxidative capacity of muscles. News Physiol. Sci. 3: 113-116, 1988. 15. Hoppeler, H. and S.L. Lindstedt. Malleability of skeletal muscle in overcoming limitations: structural elements. J. Exp. BioL 115: 355-364, 1985. 16. Hulbert, W.C., M. Guppy, B. Murphy and P.W. Hochachka. Metabolic sources of heat and power in tuna muscles. I. Muscle fine structure, l. Exp. Biol. 82: 289-301, 1979. 17. Johnston, I.A. Quantitative analyses of ultrastructure and vascularization of the slow muscle fibres of the anchovy. Tissue Cell 14: 319-328, 1982. 18. Mathieu, O., L.M. Cruz-Orive, H. Hoppeler and E.R. Weibel. Estimating length density and quantifying anisotropy in skeletal muscle capillaries. I. Microsc. 131: 131-146, 1983.

Design for a high speed path for oxygen: tuna red muscle ultrastructure and vascularization

13

19. Mathieu-Costello, O. Capillary tortuosity and degree of contraction or extension of skeletal muscles. Microvasc. Res. 33: 98-117, 1987. 20. Mathieu-Costello, O. Morphometric analysis of capillary geometry in pigeon pectoralis muscle. Am. J. Anat. 191: 74-84, 1991. 21. Mathieu-Costello, O. Morphometry of the size of the capillary-to-fiber interface in muscles. Adv. Exp. Med. Biol, 345: 661-668, 1994. 22. Mathieu-Costello, O., P.J. Agey, R.B. Logemann, R.W. Brill and P.W. Hochachka. Capillary-fiber geometrical relationships in tuna red muscle. Can. J. Zool. 70: 1218-1229, 1992. 23. Mathieu-CosteUo, O., P.J. Agey, R.B. Logemann, M. FIorez-Duquet and M.H. Bernstein. Effect of flying activity on capillary-fiber geometry in pigeon flight muscle. Tissue Cell, 26: 57-73, 1994. 24. Mathieu-Costello, O., P.J. Agey and J.M. Szewczak. Capillary-fiber geometry in pectoralis muscles of one of the smallest bats. Respir. Physiol., 95: 155-169, 1994. 25. Mathieu-Costello, O., C.G. Ellis, R.E Potter, I.C. MacDonald and A.C. Groom. Muscle capillaryto-fiber perimeter ratio: morphometry. Am. J. Physiol. 261: H 1617-H 1625, 1991. 26. Mathieu-Costello, O., D.C. Poole and R.B. Logemann. Muscle fiber size and chronic exposure to hypoxJa.Adv. Exp. Med. Bio1248: 305-311, 1989. 27. Mathieu-Costello, O., R.E Potter, C.G. Ellis and A.C. Groom. Capillary configuration and fiber shortening in muscles of the rat hindlimb: correlation between corrosion casts and stereological measurements. Microvasc. Res. 36: 40-55, 1988. 28. Mathieu-Costello, O., R.K. Suarez and P.W. Hochachka. Capillary-to-fiber geometry and mitochondrial density in hummingbird flight muscle. Respir. Physiol. 89:113-132, 1992. 29. Mathieu-Costello, O., J.M. Szewczak, R.B. Logemann and P.J. Agey. Geometry of blood-tissue exchange in bat flight muscle compared with bat hindlimb and rat soleus muscle. Am. J. Physiol. 262: R955-R965, 1992. 30. Mosse, P.R.L. The distribution of capillaries in the somatic musculature of two vertebrate types with particular reference to teleost fish. Cell Tissue Res. 187: 281-303, 1978. 31. Moyes, C.D., O. Mathieu-Costello, R.W. Brill and P.W. Hochachka. Mitochondrial metabolism of cardiac and skeletal muscles from a fast (Katsuwonus pelamis) and a slow (Cyprinus carpio) fish. Can. J. Zool. 70: 1246-1253, 1992. 32. Poole, D.C. and O. Mathieu-Costello. Analysis of capillary geometry in rat sub-epicardium and sub-endocardium. Am. J. Physiol. 259: H204-H210, 1990. 33. Potter, R.E and A.C. Groom. Capillary diameter and geometry in cardiac and skeletal muscle studied by means of corrosion casts. Microvasc. Res. 25: 68-84, 1983. 34. Potter, R.E, O. Mathieu-Costello, H.H. Dietrich and A.C. Groom. Unusual capillary network geometry in a skeletal muscle, as seen in microcorrosion casts of M. pectoralis of pigeon. Microvasc. Res. 41: 126-132, 1991. 35. Rome, L.C. and A.A. Sosnicki. The influence of temperature on mechanics of red muscle in carp. J. Physiol. (London) 427: 151-169, 1990. 36. Stevens, E.D. and Neill, W.H. Body temperature relations of tunas, especially skipjack. In: Fish Physiology. Vol. VII, Locomotion, edited by W.S. Hoar and D.J. Randall, New York, Academic Press, p. 315-359, 1978. 37. Suarez, R.K., J.R.B. Lighton, G.S. Brown and O. Mathieu-Costello. Mitochondrial respiration in hummingbird flight muscles. Proc. Natl. Acad. Sci. USA 88: 4870-4873, 1991. 38. Sullivan, S.M. and R.N. Pittman. Relationship between mitochondrial volume density and capillarity in hamster muscles. Am. J. Physiol. 252: H149-H155, 1987. 39. Weber, J.-M., R.W. Brill and P.W. Hochachka. Mammalian metabolite flux rates in a teleost: lactate and glucose turnover in tuna. Am. J. Physiol. 250: R452-R458, 1986.

Hochachka and Mommsen (eds.), Biochemistryand molecularbiology of fishes, vol. 4 9 1995 Elsevier Science B.V. All rights reserved. CHAPTER 2

Circulatory substrate fluxes and their regulation JEAN-MICHEL WEBER AND GEORGES ZWINGELSTEIN *

Biology Department, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada K l N 6N5 and * Laboratoire Maritime de Physiologic, Institut Michel Pacha, Universit~ de Lyon, 1337 Comiche Michel Pacha, Tamaris, F-83500 La Seyne sur Mer, France

I. II. III. IV. V. VI. VII. VIII.

Introduction Why and how to measure metabolite fluxes in vivo? Basic regulatory mechanisms Lactate fluxes Glucose fluxes Amino acid fluxes Lipid fluxes References

I. Introduction Multicellular life can only be sustained if selected metabolic fuels, end-products, and anabolic precursors are transported between cells at the appropriate rates and times. In vertebrates, most inter-tissue metabolite exchange depends on the cardiovascular system and, consequently, the regulation of circulatory substrate fluxes plays a crucial role in achieving homeostasis. Fishes are no exception. They must constantly adjust rates of blood metabolite turnover to coordinate biochemical processes involved in maintenance, growth, reproduction, locomotion and various responses to environmental stresses. As a group of vertebrates, however, they must use distinct metabolic strategies mainly imposed by their aquatic environment and high protein intake 48,83. Both, proteins and lipids dominate fish energy metabolism because low amounts of carbohydrates are ingested and their absorption is rather limited 42. Surprisingly, most of the detailed information concerning metabolite fluxes of fish deals with carbohydrates even though they often represent a very small fraction of these organism's total energy budget. This bias can be explained by: (1) an imitation of mammalian studies where carbohydrates can play a major role; and (2) the relative simplicity of carbohydrate biochemistry compared with lipids and proteins. In this chapter, we examine the main metabolic substrates found in the systemic circulatory system of fish (Fig. 1) and review what is presently known about the modulation of their fluxes. Plasma concentrations of these major substrates are

16

J.-M. Weber and G. Zwingelstein TRANSPORT

EXCHANGE WITH ENVIRONMENT

RFtEE 90LUW3~" INTESTINE

[~GLYCEROL ACIOiS ~T~A~

GILLS SKIN

~DNEY

STORAGEAND TRANSFORMATION

ADIPOSE SKi~ETAL I~SCI, F.S

~iOTEIV.BOUNQ

FATTYACIDS 1 TRIACYLGLYCEROLS PHOSFtlOUPIOS STEROLESTERS " I I

HEART BRAIN

! GONADS

..... i

Fig. 1. Major soluble and protein-bound circulatory fuels in fish: sources and destinations.

summarized in Table 1. The secondary circulation47.119 is not discussed separately because no metabolite measurements from this compartment are yet available. Also, the fluxes of several systemic substrates have never been measured directly. In such cases, we suggest important avenues for future work and provide indirect estimates whenever possible.

II. Why and how to measure metabolite fluxes in vivo ? All body constituents are constantly produced and utilized 44, and circulatory metabolites are therefore kept in a dynamic state, undergoing constant turnover. For decades, however, changes in plasma concentration have been used to draw quantitative conclusions about rates of substrate release into the circulation and uptake therefrom. Such conclusions are often not valid because concentration changes only indicate an imbalance between release and uptake, and major variations in flux can potentially occur while concentration stays constant 12s. Fortunately, flux and concentration of individual metabolites usually vary in parallel and some qual. itative information about flux can be gained from the direction of concentration changes. Modem access to various metabolic tracers has opened the door for the direct measurement of fluxes in vivo on a routine basis and mammalian biology has greatly benefited from this approach. In contrast, relatively few whole organism turnover studies have been attempted in fish with the two major techniques presently available: bolus injection and continuous infusion. The terminology, experimental procedures and calculations necessary to carry out reliable flux measurements have

Circulatory substrate fluxes and their regulation

17 TABLE 1

Resting concentrations of major plasma substrates in fish Metabolite

g 1-1

/zmol m1-1

Directly available (no hydrolysis required) Glucose 123,131 Lactate 9,26,71,123 Amino acids 54,77,92,95 Fatty acids 59,64,131

0.20.010.3 0.1 -

2.5 0.2 2.3 1.5

1 -14 0.1- 2 2 -14 0.1- 5.4

Only available after hydrolysis Triacylglycerols64'94,131 Phospholipids 19,94 Total proteins 1~

1 4 28

-11 -10 -35

1 -12 7 -12 m

Molar concentrations were calculated using average molecular weights of 160 (amino acids), 280 (fatty acids), 880 (triacylglycerols), and 780 (phospholipids).

been described in detail by Hetenyi 44, Katz 51-53, Okajima 81 and their coworkers and by Wolfe 128, amongst others. The bolus injection technique has almost been used exclusively in fish studies because it only requires a single catheter for both, tracer injection and blood sampiing. In contrast, continuous infusion takes two catheters to allow simultaneous infusion and sampling, and the added difficulties associated with surgical placing and maintenance of two lines have encouraged fish biologists to opt for the simpler experimental design of bolus injection 123. This is unfortunate because much more information could be obtained from continuous infusion where consecutive measurements of flux are possible in a single experiment under steady or non-steady state conditions (i.e. even when metabolite concentration varies during the experiment). A more complete understanding of flux regulation in fish will require common use of continuous infusion and the development of easier double catheterization techniques should make this possible.

III. Basic regulatory mechanisms How does the organism alter metabolite turnover rate in response to different stresses? In a study on the regulation of plasma metabolite fluxes in exercising Thoroughbred horses, blood flow and plasma metabolite concentration were proposed as the coarse and fine control, respectively 125,126. There is no reason to believe that flux regulation follows different principles in teleosts. Blood flow is the coarse control because its changes will affect all plasma fuels to the same extent. Metabolite concentration represents the fine control because modifying it for individual substrates will allow the modulation of flux for each fuel independently. This way, the respective contribution of each substrate to total metabolism can be affected by its relative concentration in the circulation, and a positive correlation between circulating concentration and turnover rate has been demonstrated for a

18

J.-M. Weber and G. Zwingelstein

LACTATE

[•'• X =) _J It.

SWIMMING

c~,.s. I TROUT

I ~ST

IrLouNoER~

~,.o,, I

CONCENTRATION Fig. 2. Relationships between plasma lactate concentration and flux in several species of teleosts. Note the positive correlation between the slope of this relationship and cardiac output.

variety of metabolites in all species studied to date. In addition, the slope of the relationship between concentration and flux increases as cardiac output rises 126. The regulating roles of cardiac output and circulating metabolite concentration can be demonstrated for teleosts in the case of lactate. Figure 2 shows how the slope varies between species and experimental conditions. Slopes range between 0.7 and 3.6 in resting teleosts where changes in lactate concentration were elicited by hypoxia or previous heavy exercise21,29,71. In contrast, during exercise, rainbow trout (Oncorhynchus mykiss) show a slope of 5.2 (ref. 123): almost twice the value found during hypoxia29. This large difference can be explained by the fact that cardiac output is much higher in swimming animals than in resting hypoxic fish. Finally, it is not surprising to find the highest slope of 15.1 in skipjack tuna (Katsuwonus pelamis), a species with a cardiac output more than 7 times higher than rainbow trout 32. There is no doubt that the above analysis of flux regulation is still extremely primitive. Potentially important biochemical signals and direct neural effects have not even been mentioned here because their influence has not been investigated in fish. Presumably, some of these factors will affect fluxes indirectly by changing blood flow or circulating concentration of the metabolite of interest. Several hormones are bound to play important regulating roles and their investigation should be a priority in future research.

IV. Lactate fluxes Lactate has occupied a prominent position in studies of hypoxia and muscle metabolism for a very long time. In the last 20 years, tracer experiments have allowed to establish that its fluxes were much higher than for other plasma substrates, even in resting organisms, and that it could become an important metabolic fuel

Circulatory substrate fluxes and their regulation

19 TABLE 2

Lactate turnover rate in post-absorptive teleosts Species

Mass (kg)

Rt (/zmol kg -1 min -1)

Predicted Rt for mammal of same size

Predicted mammal Rt/ Measured fish Rt

Anguilla rostrata 26 Platichthys stellatus 71 Oncorhynchus kisutch 71 Ictalurus punctatus 21 Oncorhynchus mykiss 29 Oncorhynchus mykiss 123 Katsuwonus pelamis TM

O.180 0.335 0.275 0.800 0.350 0.322 1.420

0.50 0.76 1.33 2.25 2.80 4.41 112

145 112 122 78 110 114 61

290 147 92 35 39 26 0.54

Predicted values for mammals of equivalent body mass were calculated as follows: Rt -- 70.78 Mb 0'42, where Rt = lactate turnover rate in ttmol kg -I min -1, and Mb ffi body mass in kg (modified from reference 123).

for oxidative tissues in mammals 76,122. This new picture of lactate metabolism has attracted the attention of fish biologists, and resting lactate turnover rates have been measured in several teleost species (Table 2). Except for tuna, the lactate fluxes of fish range from 0.5 /~mol kg -1 rain -1 in eels (Anguilla sp.) to 4.4/tmol kg -1 min -1 in rainbow trout (Oncorhynchus mykiss). Enough information is available from mammals to derive an allometric equation expressing the relationship between resting lactate turnover rate and body mass in this vertebrate group 123. We have used this equation to compare lactate fluxes in teleosts and mammals of equivalent size (Table 2). This comparison shows that turnover rate is 26 to 290 times lower in fish than in mammals, but skipjack tuna (Katsuwonuspelamis), the only scombrid measured to date, stands out as a clear exception with fluxes exceeding those of mammals

TM.

Ratios between lactate turnover and oxygen consumption rates of teleosts and mammals of the same size are similar, suggesting that the metabolic role of lactate is equivalent in all resting vertebrates when the effect of body mass is taken into account 123. This conclusion may not hold during exercise because patterns of lactate exchange between skeletal muscle and the circulation are so strikingly different in fish and mammals. After strenuous swimming for example, lactate is released extremely slowly from fish white muscle 113 and this typical pattern of retention found in all teleosts is exaggerated in bottom-dwelling, sedentary s p e c i e s 114'121. Therefore, during exercise, lactate oxidation may account for different proportions of total VO2 in mammals, pelagic and benthic fish. Stresses of different kinds are known to stimulate lactate fluxes in mammals, but little information is available for fish. Nonetheless, fasting, hypoxia and exercise have all been shown to increase lactate turnover rate in some teleosts. In American eels, long-term fasting causes a 2.5-fold rise in turnover rate 26, but the effect of food restriction has never been quantified in other fish species. Similarly, the effect of hypoxia has only been measured in rainbow trout where turnover rate increased by

20

J.-M. Weberand G. Zwingelstein

seven-fold at an environmental p O 2 of 4 kPa (ref. 29). Exercise studies have been limited by the steady state assumption of the bolus injection technique. Two steady state situations have been investigated to date: prolonged aerobic swimming and recovery from strenuous, anaerobic exercise. During sustained exercise, circulatory lactate transport between tissues could be a convenient way to shuttle carbohydrate energy between body compartments 76, and white to red muscle lactate exchange was hypothesized as a potential mechanism to support energy metabolism in active red muscle of fish. However, lactate flux only increases by two-fold during sustainable swimming in trout, showing that such a mechanism does not play a significant role in this species 123. In recovery from exhaustive exercise, fluxes are also elevated to accelerate the disposal of large lactate loads accumulated during anaerobic work. After strenuous swimming, a 3- and 9-fold increase in turnover rate was measured in flounder and salmon, respectively71. In neither species were recovery fluxes sufficiently high to explain the time course of decrease in muscle lactate concentration, suggesting that a fraction of the total lactate load never leaves white muscle and is metabolized in situ.

V Glucosefluxes Rates of glucose turnover have been measured in several species at rest (Table 3). They were determined under steady state conditions and therefore represent both, rates of glucose production (Ra) and disappearance from the circulation (Rd) at the whole organism level. The liver accounts for most of the glucose produced, but fish kidneys can probably also make a significant contribution unlike their mammalian counterpart 55.7s,1~ The relative importance of liver and kidney has not been quantified in vivo, and is likely to depend upon species, diet, and level of activity. Measured rates of glucose t u r n o v e r (Rt) are upper estimates of glucose oxidation TABLE 3 Glucose turnover rate (Rt) in resting, post-absorptive teleosts Species

Rt

Dicentrarchus labrax3s Oncorhynchus mykiss 27'3s Oncorhynchus mykiss 2 Paralabrax sp. 14,15 Oncorhynchus kisutch 65 Hemitriptems ameticanus 118 Hoplias malabaricus67 Pleuronectes platessa 12 Katsuwonus pe/.am/s124 Anguilla rostrata26

0.6 1.0 1.1 2.1 2.2 3.6 3.9 5.7 15.3 56

Turnover rates are given in/~mol glucose kg -1 min -1.

Circulatory substrate fluxes and their regulation

21

(Rox) because not all glucose leaving the circulation is usually oxidized. Rox glucose has not been quantified directly in vivo, but comparative recovery of expired 14CO2 after bolus injection of different 14C-substrates shows that glucose is oxidized at much lower rates than fatty acids and amino acids except for glycine 37,115. Unfortunately, the experimental approach used in these studies does not provide absolute rates of oxidation. Continuous infusion of 14C-substrates after priming the CO2/bicarbonate pool, and monitoring 14CO2 production will be needed to measure such rates. Then, comparing R~,, t and Rox values will allow to determine what percentage of total glucose turnover is oxidized. Except for eel and tuna, glucose turnover rates of fish range between 0.6 and 5.7 /zmol kg -1 min -1 (Table 3). These values are 20-100 times lower than for resting mammals of equivalent size ~24. The lower body temperature and lower metabolic rate of fish may account for this difference. The high glucose fluxes of tuna can be explained by their 'mammalian' metabolic rates and greater reliance on carbohydrates for energy metabolism, but it remains unclear why eels should have the ability to support even higher turnover rates than tuna or mammals. Species showing high turnover rates appear to have a better ability to maintain steady blood glucose concentrations 1~ However, the main factors involved in the regulation of glucose fluxes have not been investigated thoroughly. The evidence available to date suggests that the regulatory mechanisms of fish operate very slowly (hours) compared with mammals (minutes). Hepatic glucose production only shuts down 1-2 h after glucose loading in Paralabrax 14. Also, indirect evidence from changes in circulating glucose concentration suggests that insulin45,1~~and glucagon ~11 take at least 30 min to start modifying fluxes and that their effect lasts for several hours. Elevated plasma cortisol has no effect on the glucose turnover of rainbow trout (cortisol injection) 2 and sea raven (high cortisol induced by chronic stress) 118. Similarly, subjecting trout to 3 h of low water pO2 (4 kPa) had no effect on their glucose flux29, mainly because elevated plasma catecholamines tend to abolish the inhibitory effect of hypoxia 129. In future work, quantifying the respective effects of circulating glucose, insulin, glucagon and other hormones will require the use of continuous infusion and, eventually, the 'glucose clamp' technique 44 should be adapted for fish experiments. Because several tissues rely exclusively on glucose for energy metabolism, some attention has been devoted to potentially limiting glucose fluxes during fasting. The most dramatic effect has been shown in American eels where a 10-fold decrease in glucose turnover was measured after 15 months of food deprivation 26. Shorter studies in other species provide conflicting results. A 30% reduction in glucose flux was observed in Paralabrax 14 and Hoplias67,but Hemitripterus 118 and Dicentrarchus 38 showed an 80 and 320% increase, respectively. These species differences are quite puzzling and a closer look at the combined effects of several factors including size, age, diet, locomotory habits, and temperature may provide an explanation. The effect of exercise on glucose turnover rate has not been investigated in fish. However, West et al. 127 have recently used deoxyglucose to quantify glucose uptake of individual tissues from the circulation. This exciting approach will allow to determine the relative contribution of different organs to whole-animal glucose turnover

22

J.-M. Weber and G. Zwingelstein

and it opens the door for a detailed investigation of fish glucose metabolism in vivo. In a first series of experiments on trout, these authors have shown that exercise causes a 28-fold increase in red muscle glucose utilization but has no effect on cardiac muscle. Interestingly, glucose utilization only accounts for less than 10% of the oxidative metabolism of these two tissues during swimming 127.

VI. A m i n o acid fluxes Proteins represent a very important source of energy in teleosts 16, and rates of nitrogen excretion have been used to quantify protein catabolism 117. Different studies have concluded that amino acid oxidation accounts for 14-85% of total 1(/IO2 depending on species, feeding status, and level of activity 18'56'57'116. Despite this well-known dependence on protein for energy metabolism, very few researchers have tried to measure rates of circulatory amino acid turnover and oxidation. Furthermore, reports to date are qualitative only, providing relative rates between substrates or experimental conditions. Borer and colleagues estimated that alanine, glutamate, and aspartate fluxes of Paralabrax were equivalent to mammalian values 15, confirming the much higher relative importance of amino acid catabolism to total MO2 in fish than in mammals, because of the large metabolic rate difference between these two groups of animals. The turnover rate of the three amino acids measured was not affected by 72 days of fasting 15. Measurements of 14CO2production after injection of t4C-substrates show that circulating glutamate, alanine, leucine, and phenylalanine are oxidized much more rapidly than glucose in resting fish37. This is also true in swimming trout where leucine becomes the preferred amino acid substrate for oxidation in active muscles nS. As expected, non-essential amino acids are generally favored over essential amino acids 120. A significant fraction of total flux is channeled through gluconeogenesis. Teleosts have evolved a relatively high capacity for converting amino acids to glucose and this has been interpreted as a strategy to synthesize enough mucopolysaccharides for mucus production in organisms with little dietary carbohydrates 15. Two very interesting situations where amino acid fluxes should be particularly high have not been investigated so far: elasmobranchs and migrating salmon. Elasmobranchs have no significant ability to oxidize lipids outside the livers.1~ Therefore, during sustained locomotion, they should derive most of their energy from amino acid oxidation as indicated by their high capacity to metabolize glutarnine in muscle mitochondria 22. Similarly, salmon is known to depend almost exclusively on protein catabolism in the last stages of long migrations, after carbohydrate and lipid reserves have been depleted 3.16. In sockeye salmon (Oncorhynchus nerka), several amino acids appear to be converted to alanine before inter-organ transport 77, suggesting that alanine fluxes are much higher in migrating than in non-migrating teleosts. In addition, amino acid fluxes should increase throughout migration as white muscle proteins are progressively catabolized via the indirect action of androgens 4,s, and proteolytic agents such as cathepsins 130.

23

Circulatory substratefluxes and their regulation

Finally, no amino acid flux measurement should be attempted in fish without considering the large concentration gradient between red cell and plasma. Amino acids are three 11 to over 200 times 34 more concentrated inside fish erythrocytes than in plasma, and, therefore, the specific activity measured in plasma or whole blood will be different, leading to the calculation of distinct flux rates. Each experimental situation should be considered individually before selecting plasma or whole blood because concentration gradients and red cell membrane transport kinetics are so variable between species and amino acids.

I~I. Lipid fluxes To our knowledge, plasma lipid fluxes have never been measured in fish at the whole organism level even though fat represents a critical source of ATP in these animals. The following analysis will focus on indirect and qualitative information to point out promising directions and potential difficulties for future research. The major circulatory lipids of f i s h - free fatty acids (FFA) and triacylglycerols (TAG) - and their sites of appearance and disappearance are summarized in Fig. 3. Also, many important aspects of circulatory lipid transport in fish have been reviewed by Sheridan 98. Lipid substrates are shuttled between tissues either as FFA (rapid delivery) or as TAG and phospholipids (slow delivery). Most of our discussion will deal with INTESTINE DIETARY UPIDS STORAGE

CATABOLIC TISSUES

i

TISSUES

TG+PL

_

..........

~'

F

~

FFA ~

TG + PL

UVER

Fig. 3. Source, destination and composition of plasma lipids. FABP ffi fatty acid binding protein; FFA = free fatty acids; HSL = hormone sensitive lipase; LPL = lipoprotein lipase; PL = phospholipids; TG = triacylglycerols; VLDL = very low density lipoproteins.

24

J..M. Weber and G. Zwingelstein

the relatively simple situation of FFA rather than with other lipids (TAG, glyceryl ethers, phospholipids, free and esterified cholesterol) whose complex circulatory transport involves ehylomierons, VLDL, HDL, and LDL9s. Even in mammals, the present understanding of these compounds' kinetics is still very limited. In fish, 20--30% of circulating FFA are unsaturated with chain lengths of 20 and 22 carbons 1~176 The major function of these polyunsaturated FFA is to act as precursors of membrane phospholipids 13,1n and eicosanoid compounds 6,66,88,while shorter chain fatty acids (C18 and less) are used primarily for energy metabolism. Therefore, C20 and C22 acids should have much lower turnover rates than the FFA involved in oxidative pathways. Also, one would expect that swimming will have a much more pronounced effect on the flux of 'short' acids than on C20 and longer FFA. The choice of an appropriate marker fatty acid for measuring FFA fluxes for different purposes and under different conditions should take the above considerations into account. Plasma FFA concentration is approximately one order of magnitude higher in teleosts than in elasmobranchs and holocephalans (see Table 4). In addition, and contrary to eyclostomes and teleosts28,4~ elasmobranchs lack albumin-like plasma proteins 33, and they are incapable of oxidizing fatty acids in other tissues than in liver7,22,1~ The FFA fluxes of sharks, skates and rays should therefore be significantly reduced in view of their remarkably limited capacity to transport and metabolize lipids. The high cardiac output of elasmobranehs (53 v e r s u s 17 ml kg-lmin -1 in resting dogfish and trout, respectively5~ can only partially compensate for their low plasma FFA (approximately 0.15 v e r s u s 1.2 mM). With the same relative extraction from plasma, dogfish would only be able to support less than half the FFA delivery rate of trout. TABLE 4 Total plasma free fatty acid concentration in teleosts, elasmobranchs, and a holocephalan Species

FFA (~mol m1-1)

Teleosts Oncorhynchus mykiss 43 Salvelinus alpinus 39 Dicentrarchus labrax 131 Mullus surmuletus 131 Scomber scombrus 131 Gadus aeglefinus 59 Gadus morhua 59

1.52 2.11 1.10 1.42 1.22 1.54 1.28

Elasmobranchs Scyliorhinus canicula 131 Squalus acanthias 131 Raja rad/ata 59 Etmoptems spinax59

0.15 0.15 0.09 0.29

Holoeephalan Chimaera monstrosa 59

0.17

25

Circulatory substrate fluxes and their regulation

T h e effect of exercise on p l a s m a FFA fluxes should be very different b e t w e e n species b e c a u s e teleosts show very diverse swimming abilities and lipid s t o r a g e s t r a t e g i e s 1~ S o m e species store m o s t of their T A G in l o c o m o t o r y muscles (e.g. herring, Clupea harengus), o t h e r s c o n c e n t r a t e T A G in liver (e.g. cod, G a d u s m o r h u a ) or in a d i p o s e tissue 98. Table 5 lists a few species to illustrate muscle/liver s t o r a g e TABLE 5 Total lipid content of liver and muscle (% lipid per g tissue wet weight) Species

Liver

Muscle

Oncorhynchus mykiss 1 Oncorhynchus nerka 17 Clupea harengus 17 Scomber scombrus 17 AnguiUa anguiUa70 Dicentrarchus labrax a Gadus morhua 36 Gadus aeglefinus 82

5 7 2 8 12 18 63 63

4 15 11 13 18 3 0.3 0.4

a

G6rard Brichon, unpublished results. TABLE 6 Effects of hormones on plasma FFA concentration indicating similar changes in FFA fluxes

Hormone

Effect on plasma FFA

Insulin 63,74

Decrease

Glucagon46 Glucagon23,60,91,109

Increase No effect

Catecholamines3~ Catecholamines31 Catecholamines86

Increase Decrease No effect

ACTH 75 ACTH 3~176

Increase No effect

Somatostatin99,101

Increase

Urotensin I199,1~

Increase

Arginine vasotocin49,68 Arginine vasotocin 68

Increase Decrease

Thyroid hormone8~176 Thyroid hormone96

Increase No effect

Cortisol 2~ Cortiso196

Increase No effect

Sex steroid analog 1~

Increase

Growth hormone69,75

Increase

Prolactin61,75

Increase

J..M. Weber and G. Zwingelstein

26

options. During swimming, teleosts favoring hepatic and adipose storage will have to supply most FFA to their working muscles v/a the circulation. Such species should therefore increase plasma FFA fluxes to a much larger extent than fish with considerable TAG reserves in their muscles. A variety of hormones are potentially involved in the regulation of plasma FFA fluxes. In mammals, flux and concentration are positively correlated 41,s4 and fish should be no different. The direction of hormonal effects on FFA concentration and flux are probably also identical in this group of vertebrates. Table 6 summarizes the potential effects of several hormones on the turnover rate of circulating FFA. It is interesting to note that some hormones will not regulate FFA fluxes in all species because their effects are tissue specific 9s. For example, catecholamines stimulate lipolysis in fish hepatocytes 97, but have no effect on their adipocytes 79. Consequently, catecholamines should increase FFA turnover in species storing TAG in the liver, but they should play no regulating role in species using mostly adipose tissue for lipid storage. Finally, the study of circulating triacylglycerol, phospholipids, and cholesterol promises to be extremely complex, but experimental difficulties should be overcome TABLE 7A

Total lipoprotein concentration and respective percent contribution of VLDL, LDL and HDL in plasma. All values were measured by ultracentrifugation methods Species Oncorhynchus mykiss juvenile 1~ adult 24,25.35

Oncorhynchus nerka93 Sardinops caerulae 62 Myxine glutinosa 72 Latimeria chalumnae 72 Scyliorhinus canicula 72 Centrophorus squamosus 73 Conger vulgaris72

Lipoproteins

VLDL

LDL

(gl -~)

(~)

(~)

13-17

18

64

18

23-26 7 8 29 14 2 7 7

7 26 13 57 77 14 61 67

36 38 15 24 14 75 34 33

57 37 72 19 9 11 6 -

HDL

TABLE 7B

Average lipid and protein composition of fish lipoproteins (% lipoprotein wet weight) Proteins

Phospho-

Free

lipids a

cholesterol

..... Cholesteryl

Triacylglycerols

esters ,,

Chylomicron VLDL LDL HDL

2 13 26 49

8

1

2

15 23 26

7 9 6

18 14 12

84 43 25

10

a Phospholipids (mostly phosphatidylcholine and sphingomyelin). In Latimeria, Scyliorhinus, and Conger, 13 tO 70% of the triacylglycerol fraction is made of alkyldiacylglycerols72,73. Values calculated from references 24, 25, 35, 62, 72, 73, 93, 100, and 105.

Circulatory substrate fluxes and their regulation

27

because, in certain species, these compounds can represent 10 to 40 times the energy stored in circulating FFA (see Table 1). They are first transported as chylomicrons before being stored in liver or adipose tissue where they can be converted to lipoproteins and released back in the circulation 98. Measuring the fluxes of these compounds will be a real challenge because the relative contribution of VLDL, LDL, and HDL to total plasma lipoproteins varies greatly between species (Table 7A), and each class of lipoproteins has a different composition (Table 7B). For these two major reasons, great care will have to be taken in choosing adequate lipid tracers and modes of administration to decipher specific aspects of plasma lipoprotein kinetics.

VIII. References 1. Abdul Malak, N., G. Zwingelstein, J. Jouanneteau and J. Koenig. Influence de certains facteurs nutritionnels sur la pigmentation de la truite arc en ciel par la cantaxanthine. Ann. Nutr. Alim. 29: 459-475, 1975. 2. Andersen, D.E., S.D. Reid, TW. Moon and S.E Perry. Metabolic effects associated with chronically elevated cortisol in rainbow trout (Oncorhynchus mykiss). Can. J. Fish. Aquat. Sci. 48: 1811-1817, 1991. 3. Ando, S., M. Hatano and K. Zama. Deterioration of chum salmon (Oncorhynchus keta) muscle during spawning migration -I. Changes in proximate composition of chum salmon muscle during spawning migration. Comp. Biochem. Physiol. 80B: 303-307, 1985. 4. Ando, S., M. Hatano and K. Zama. Deterioration of chum salmon muscle during spawning migration - VI. Changes in serum protease inhibitory activity during spawning migration of chum salmon (Oncorhynchus keta). Comp. Biochem. Physiol. 82B: 111-115, 1985. 5. Ando, S., E Yamazaki, M. Hatano and K. Zama. Deterioration of chum salmon (Oncorhynchus keta) muscle during spawning migration - III. Changes in protein composition and protease activity of juvenile chum salmon muscle upon treatment with sex steroids. Comp. Biochem. Physiol. 83B: 325-330, 1986. 6. Anonymous. Potential importance of arachidonate in gill function of marine fish. Nutrit. Rev. 45: 58-60, 1987. 7. Ballantyne, J.S., M.E. Chamberlin and TD. Singer. Oxidative metabolism in thermogenic tissues of the swordfish and mako shark. J. Exp. Zool. 261: 110-114, 1992. 8. Ballantyne, J.S., H.C. Glemet, M.E. Chamberlin and TD. Singer. Plasma nonesterified fatty acids of elasmobranch fishes. Mar. Biol., 166: 47-52, 1993. 9. Bange-Barnoux, R. Evolution de I'acide lactique et du glucose plasmatiques chez la Tanche lors de rasphyxie par confinement. C.R. S~ances Soc. Biol. 159: 400-403, 1965. 10. Bange-Barnoux, R., C. Bange, H. Vanel and J. Pottu. Influence du jeQne sur la protid6mie et les compos6s alpha-amin6s du sang de la tanche (Tinca tinca L.). Ann. Inst. Michel Pacha 4: 77-101, 1971. 11. Barnoud, R. and G. Peres. Recherches sur la protid6mie de la tanche soumise ~ r asphyxie par confinement. III Aminoacid6mie. J. Physiol. (Paris) 55: 193-194, 1963. 12. Batty, R.S. and C.S. Wardle. Restoration of glycogen from lactic acid in the anaerobic swimming muscle of plaice Pleuronectes platessa L.J. Fish Biol. 15: 509-519, 1979. 13. Bell, M.V., R.J. Henderson and J.R. Sargent. The role of polyunsaturated fatty acids in fish. Comp. Biochem. Physiol. 83B: 711-719, 1986. 14. Beret, K., M. Chenoweth and A. Dunn. Glucose turnover in kelp bass (Paralabrax sp.): in vivo studies with (6-3H, 6-14C)glucose.Am. J. Physiol. 232: R66-R72, 1977. 15. Beret, K., M. Chenoweth and A. Dunn. Amino acid gluconeogenesis and glucose turnover in kelp bass (Paralabrax sp.). Am. J. Physiol. 240: R246-R252, 1981. 16. Bilinski, E. Biochemical aspects of fish swimming. In: Biochemical and Biophysical Perspectives in Marine Biology, edited by D.C. Malins and J.R. Sargent, New York, Academic Press, pp. 239-288, 1974. 17. Braekken, O.R. Reports on Technological Research Concerning Norwegian Fish Industry. Norwegian

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95.

96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112.

113. 114. 115. 116. 117.

31

- comparative observations in serranides and sparides. Comp. Biochem. Physiol. 99B: 251-255, 1991. Schlisio, W. and B. Nicolai. Kinetic investigations on the behaviour of free amino acids in the plasma and of two aminotransferases in the liver of rainbow trout (Salmo gairdnerii Richardson) after feeding on a synthetic composition containing pure amino acids. Comp. Biochem. Physiol. 59B: 373-379, 1978. Sheridan, M.A. Effects of thyroxine, cortisol, growth hormone and prolactin on lipid metabolism of coho salmon, Oncorhynchus kisutch, during smoltification. Gen. Comp. Endocrinol. 64: 220-238, 1986. Sheridan, M.A. Effects of epinephrine and norepinephrine on lipid mobilization from coho salmon liver incubated in vitro. Endocrinology 120: 2234-2239, 1987. Sheridan, M.A. Lipid dynamics in fish: aspects of absorption, transportation, deposition and mobilization. Comp. Biochem. Physiol. 90B: 679-690, 1988. Sheridan, M.A. and H.A. Bern. Both somatostatin and the caudal neuropeptide, urotensin II, stimulate lipid mobilization from coho salmon liver incubated in vitro. Regul. Pept. 14: 333-344, 1986. Sheridan, M.A., J.K.L. Friedlander and W.V. Allen. Chylomicrons in the serum of postprandial steelhead trout (Salmo gairdneri). Comp. Biochem. Physiol. 81B: 281-284, 1985. Sheridan, M.A., E. Plisetskaya, H.A. Bern and A. Gorbman. Effects of somatostatin-25 and urotensin II on lipid and carbohydrate metabolism of coho salmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol. 66: 405-414, 1987. Singer, TD. and J.S. Ballantyne. Absence of extrahepatic lipid oxidation in a freshwater elasmobranch, the dwarf stingray Potamotrygon magdalenae: evidence from enzyme activities. J. Exp. Zool. 251: 355-360, 1989. Singer, TD. and J.S. Ballantyne. Metabolic organization of a primitive fish, the bowfin (Amia calva). Can. J. Fish. Aquat. Sci. 48: 611-618, 1991. Singer, TD., V.G. Mahadevappa and J.S. Ballantyne. Aspects of the energy metabolism of lake sturgeon, Acipenserfulvescens, with special emphasis on lipid and ketone body metabolism. Can. J. Fish. Aquat. Sci. 47: 873-881, 1990. Skinner, E.R. and A. Rogie. The isolation and partial characterization of the serum lipoproteins and apolipoproteins of the rainbow trout. Biochem. J. 173: 507-520, 1978. Suarez, R.K. and T.P. Mommsen. Gluconeogenesis in teleost fish. Can. J. Zool. 65: 1869-1882, 1987. Takashima, E, T Habiya, N. Phan-Van and K. Aid. Endocrinological studies on lipid metabolism in rainbow trout. -II. Effects of sex steroids, thyroid powder, adrenocorticotropin on plasma lipid content. Bull. Jap. Soc. Sci. Fish. 38: 43-49, 1972. Tashima, L. and G.E Cahill. Fat metabolism in fish. In: Handbook of Physiology, Section 5: Adipose tissue, edited by A.E. Renold and G.E Cahill, Washington D.C., American Physiological Society, pp. 55-58, 1965. Tashima, L. and G.E Cahill. Effects of insulin in the toadfish Opsanus tau. Gen. Comp. Endocrinol. 11: 262-271, 1968. Thorpe, A. and B.W. Ince. Effects of pancreatic hormones, catecholamines, and glucose loading on blood metabolites in the Northern pike (Esox lucius L.). Gen. Comp. Endocrinol. 23: 29-44, 1974. Thorson, T The partitioning of body water in Osteichthyes: phylogenetic and ecological implications in aquatic vertebrates. Biol. Bull. 120: 238-254, 1961. Tocher, D.R. and J.R. Dick. Incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids in phospholipid classes in cultured Atlantic salmon (Salmo salar) cells. Comp. Biochem. Physiol. 96B: 73-79, 1990. Turner, J.D., C.M. Wood and D. Clark. Lactate and proton dynamics in the rainbow trout (Salmo gairdneri). J. Exp. Biol. 104: 247-268, 1983. Turner, J.D., C.M. Wood and H. H6be. Physiological consequences of severe exercise in the inactive benthic flathead sole (Hyppoglossoides elassodon): a comparison with the active pelagic rainbow trout (Salmo gairdneri). J. Exp. Biol. 104: 269-288, 1983. van den Thillart, G. Energy metabolism of swimming trout (Salmo gairdneri). J. Comp. Physiol. 156: 511-520, 1986. Van den Thillart, G. and E Kesbeke. Anaerobic production of carbon dioxide and ammonia by goldfish, Carassius auratus (L.). Comp. Biochem. Physiol. 59A: 393-400, 1978. Van Waarde, A. Aerobic and anaerobic ammonia production by fish. Comp. Biochem. Physiol. 74B:

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675--684, 1983. 118. Vijayan, M.M. and T.W. Moon. The stress response and the plasma disappearance of corticosteroid and glucose in a marine teleost, the sea raven. Can. I. ZooL 72: 379-386, 1994. 119. Vogel, W.O.P. Systemic vascular anastomoses, primary and secondary vessels in fish, and the phylogeny of lymphatics. In: Cardiovascular Shunts: Phylogenetic, Ontogenetic and Clinical Aspects, edited by K. Johansen and W. Burggren, Copenhagen, Munksgaard, pp. 143-159, 1985. 120. Walton, M.J. and C.B. Cowey. Aspects of intermediary metabolism in salmonid fish. Comp. Biochem, PhysioL 73B: 59-79, 1982. 121. Wardle, C.S. Non-release of lactic acid from anaerobic swimming muscle of plaice Pleuronectes platessa L.: a stress reaction. 3`. Exp. Biol. 77: 141-155, 1978. 122. Weber, J.-M. Design of exogenous fuel supply systems: adaptive strategies for endurance locomotion. Can. I. Zool. 66: 1116-1121, 1988. 123. Weber, J.-M. Effect of endurance swimming on the lactate kinetics of rainbow trout. 1. Exp. Biol. 158: 463-476, 1991. 124. Weber, J.-M., R.W. Brill and P.W. Hochachka. Mammalian metabolite flux rates in a teleost: lactate and glucose turnover in tuna. Am. 3`. Physiol. 250: R452-R458, 1986. 125. Weber, J.-M., G.E Dobson, W.S. Parkhouse, D. Wheeldon, J.C. Harman, D.H. Snow and P.W. Hochachka. Cardiac output and oxygen consumption in exercising Thoroughbred horses. Am, 3'. PhysioL 253: R890-R895, 1987. 126. Weber, J.-M., W.S. Parkhouse, G.P. Dobson, J.C. Harman, D.H. Snow and RW. Hochachka. Lactate kinetics in exercising Thoroughbred horses: regulation of turnover rate in plasma. Am. 3'. Physiol. 253: R896-R903, 1987. 127. West, T.G., P.G. Arthur, R.K. Suarez, C.J. Doll and P.W. Hochachka. In vivo utilization of glucose by heart and locomotory muscles of exercising rainbow trout (Oncorhynchus mykiss). I. Exp. Biol. 177: 63-79, 1993. 128. Wolfe, R.R. Tracers in Metabolic Research. Radioisotope and Stable Isotope~Mass Spectrometry Methods, New York, Alan R. Liss, 1984. 129. Wright, P.A., S.E Perry and T.W. Moon. Regulation of hepatic gluconeogenesis and glycogenolysis by catecholamines in rainbow trout during environmental hypoxia. 3`. Exp. Biol. 147: 169-188, 1989. 130. Yamashita, M. and S. Konagaya. High activities of cathepsins B, D, H and L in the white muscle of chum salmon in spawning migration. Comp. Biochem, Physiol. 95B: 149-152, 1990. 131. Zammit, V.A. and E.A. Newsholme. Activities of enzymes of fat and ketone-body metabolism and effects of starvation on blood concentrations of glucose and fat fuels in teleosts and elasmobranch fish. Biochem. I. 184: 313-322, 1979.

Hochachka and Mommsen (eds.), Biochemistryand molecular biologyof fishes, vol. 4 9 1995 Elsevier Science B.V. All rights reserved. CHAPTER 3

Endogenous fuels; non-invasive v e r s u s invasive approaches GUIDO VAN DEN THILLART AND MARCEL VAN RAAIJ

Institute of Evolutionary and Ecological Sciences, Animal Physiology, Gorlaeus Laboratories, University of Leiden, P.O. Box 9502, 2300 RA Leiden, The Netherlands

I. II.

Introduction Quantifying endogenous fuels: destructive methods 1. Handling stress 2. Tissue damage 3. Tissue extraction 4. Storage 5. Measurement III. Non-destructive approaches 1. Cannulation 2. Calorimetry 3. Nuclear magnetic resonance spectroscopy IV. Storage of endogenous fuels 1. High energy phosphates 2. Carbohydrates 3. Lipids 4. Proteins and amino acids V. Mobilization of endogenous fuels 1. Hypoxia and anoxia 2. Exercise 3. Starvation and migration VI. Summary VII. References

I. Introduction Life is a condition that requires non-equilibrium conditions, since all processes proceed only when free energy is converted into entropy. A state of non-equilibrium is kept at the expense of free energy: energy consumption for maintenance and activity ultimately result in heat production, since the organism itself hardly changes. The conversion of energy forms such as from chemical energy to kinetic energy are always coupled with increase of entropy, normally resulting in heat production. In the case of exercise on a hometrainer the conversion-efficiency from chemical to kinetic energy is about 25%, so 75% is lost as heat. Of course, kinetic energy dissipates in the end as heat, so all free energy is then lost as heat. For metabolic pathways the conservation of the free energy in the form of ATP has not always the same efficiency37. We can distinguish high and low efficiency pathways depending on the amount of free

34

G. ,,an den ThiUartand M. van Raaij

energy loss22. We may ask ourselves why nature did not develop only high efficiency pathways in order to minimize energy losses. The answer to this question is: reaction rate. The higher the energy loss, the faster the reaction can proceed. So, high efficiency processes are necessarily slow because they proceed near the equilibrium condition, and low efficiency processes are fast and are hardly influenced by changes in substrate and product levels because they operate far from equilibrium 37. In order to keep an organism in a state of non-equilibrium and to enable a large number of physiological processes, a constant energy input is needed. The sources for this energy input are the substrates for fermentation and oxidative processes. From a thermodynamic point of view, cells possess three types of reactions: endergonic, equilibrium and exergonic reactions. Most anabolic and homeostasis reactions are endergonic and require reactions coupled with ATP hydrolysis. While instead most exergonic reactions are coupled with ATP synthesis of which the most important are: the creatine kinase reaction; the pyruvate kinase and the 3-phosphoglycerate kinase reactions of the glycolysis; and the oxidative phosphorylation. The substrates for these three processes are respectively: creatine phosphate, glycogen (glucose) and NADH. Although the first two can be considered as fuels, the last substrate is in fact an intermediate occurring at fairly low concentrations normally below 10 ~M 31,n9 and can therefore hardly be viewed as a fuel. Instead, the substrates for the NADH generating processes should be considered as fuels for the oxidative phosphorylation. NADH is generated mainly by three processes: (1) the/~-oxidation of fatty acids; (2) the Krebs cycle; and (3) the glycolytic pathway. The substrates for these processes are lipids, proteins and sugars, and can therefore be considered as the fuels for the oxidative phosphorylation. When we define a fuel as a compound that acts as a substrate for an ATP producing pathway, and that can be stored to some extent, we should include anaerobic processes as well, and consider both ATP and PCr as fuels. Biochemically speaking we know only two types of ATP synthesis: (1) chemically driven reactions like the pyruvate kinase reaction (substrate phosphorylation); and (2) electrochemically driven reactions like the H+-driven ATP synthesis in the inner mitochondrial membrane. The latter is more important from a quantitative point of view, i.e. 18 times as much ATP is produced by the mitochondria than by glycolysis during complete degradation of glucose. However, the energy generating processes under anaerobic condition are for most animals crucial for survival, since oxygen shortage is a regularly occurring phenomenon either on the tissue level (due to ischemia or high consumption level) or particularly with fish on the organismal level (due to low environmental 02) 22,43,1~176 Therefore, although the total ATP production capacity of anaerobic processes is limited in comparison with that of oxidative processes, we feel compelled to discuss the fuels for both pathways separately.

II. Q u a n t i f y i n g e n d o g e n o u s fuels: destructive m e t h o d s

The quantification of endogenous fuels and metabolites especially for the purpose of describing physiological processes, is difficult because measurement always implies

35

Endogenous [uels; non-invasive versus invasive approaches

interference. It is therefore crucial for the interpretation of the data to know to what level the process under study is disturbed by the determination of a certain parameter. Thus far most measurements are based on destructive methods (chemical and enzymatic reactions), for which an extract is required. Few people realize the problems associated with tissue sampling, extraction, and metabolite measurement. Depending upon the metabolite in question, the applied method of sampling, and the method of extraction, the concentration may vary by more than an order of magnitude, which should make us at least very cautious with respect to the interpretation of the results. The number of different procedures indicate already how difficult it is to obtain reliable metabolite concentrations. Since most metabolite measurements are based on chemical reactions with and/or purification of the compound in question, a solution of the metabolite must be obtained from a previous tissue extraction. We can distinguish 3 phases in an extraction procedure, and within each phase a number of steps (Fig. 1). Phase I refers to the way the animal is handled: how it is taken out of its box and manipulated in order to obtain tissue samples. Animals may be killed by electrocution 76, by a blow on the head, by decapitation, by anesthesia, a combination of these or even by immersion in liquid nitrogen a9,136 (see also Table 1). The major problem in this phase is to prevent struggling of the animal, particularly when one is interested in resting values. Certainly electrocution is the poorest and anesthesia the best way to reach resting values. On the other hand, one should take into account that during the period necessary to reach anesthesia, animals (after exposure to anoxia or exercise) have time for recovery (5-10 min). Phase II is the sampling phase. In this phase samples are taken by a biopsy needle, or by dissection. This takes time, depending on the skill of the operator

I Precauti~ I I sampling I I freezing I I extracti~ 1

l~r+ ~AI

[~wder-~AI

/

I~I

1 , high speed mixer/centrifugation

I

Fig. 1. Steps in tissue extraction. At every step artifacts may develop, disturbing the final metabolic picture. Only very critical consideration of the procedures may result in an acceptable estimation.

36

G. van den Thillart and M. van Raaij

TABLE 1 Muscle lactate under 'resting' conditions Species

Lactate (/zmol g-l)

lmmobilisation

Sampling

Trout49 Trout/carp 95 Trout98 Trout1~ Trout25 Tuna 5 Cod16 Perch76 Human 1~ Eel 124 Goldfish112 Dog 17

15 14 13 1 6 7 5 3.5 3 2.4 1.5 1.2

Liquid N2 A + blow a Blow A Decapitation A + blow Blow Electrocution A + biopsy A + curare A A + isol. prep.

Excision at-20~ Tissue in liquid N2 Tissue in liquid N2 Freeze clamp Freeze clamp Freeze clamp Freeze clamp Tissue in liquid N2 Liquid N2, freeze dry Freeze clamp Freeze clamp Freeze clamp

a A = anaesthetic.

and the number and kind of tissues that have to be sampled. To reduce the loss of precious time in this phase, dissection is sometimes carried out on frozen tissue. Normally the sampling phase is terminated by freeze-clamping the tissue at liquid nitrogen temperature (-195.8~ 1~ Sometimes this step is left out, and the tissue is extracted immediately in cold perchloric acid (PCA). Freezing is however the best way to 'freeze' metabolism and bring it within a few milliseconds to a complete stop 138. Obviously this is important when one is dealing with processes that have high reaction rates. Phase III includes extraction and denaturation of the sample. In order to extract and denaturate the sample properly, it is necessary to pulverize the frozen sample together with the extraction medium to a fine powder 1~2,~5. This way the time for denaturation is minimized, and total surface area for extraction is maximized. This step can be left out only when slow metabolic processes are studied. Although acid will eventually hydrolyze compounds like ATP and PCr, the rate of hydrolysis is only a few percent per day, and therefore in most cases negligible. Extraction and denaturation occur during thawing, therefore it is obligatory to mix the powdered tissue with the PCA during thawing thoroughly. At each step artifacts may be introduced, sometimes leading to spurious results. We can distinguish five conditions where artifacts are likely to develop: handling stress, tissue excision, tissue extraction, tissue and extract storage, and metabolite measurement. 1. Handling stress

Except for blood sampling from cannulated fish, tissue sampling leads to handling stress, since few animals will voluntarily give up a part of their body for scientific

Endogenous fuels; non-invasive versus invasive approaches

37

inspection. So the animals have to be anesthetized and/or killed quickly in order to prevent extreme struggling which will otherwise certainly lead to significant changes of the metabolite profile. Large animals offer the possibility to use biopsy needles, although this type of sampling has its restrictions, too, because of local tissue damage (see below). Handling stress is a behavioral type of stress, a stress reaction initiated by the central nervous system either via direct stimulation or indirectly via hormones (adrenaline and cortisol). A large number of physiological reactions are activated under these conditions, all aimed at preparing the animal for an outburst of activity by redirecting bloodflow to the muscles, stimulating heartbeat and ventilation, increasing muscle tone, etc. All these activities have their effects on tissue metabolism, the more so if the animal is already engaged in struggling. Handling stress can be overcome only if the animal is not able to respond to the sampling; this can be reached either by surprise, by anesthesia, or by a probing technique that is not sensed by the animal. Animals are not easily surprised, certainly not on the level of tissue sampling. The fastest method is needle biopsy, this technique is often used with experiments on humans and larger mammals 1~ but also on fish this technique has been applied 5. It can be carried out fast enough to reduce handling stress, certainly if a local anesthetic is applied, although due to local tissue damage sampling-artifacts cannot be completely prevented (see below). The major problem with anesthesia is the delay; it takes time for the anesthetic to take effect. During this delay recovery processes may take place, especially when they are fast, the original metabolic picture may change completely during a delay of 5-10 minutes. Besides this delay, anesthesia has also side-effects on metabolism such as erythrocyte swelling 96. Repeated anesthesia also changes markedly the levels of blood borne metabolites 12. In the case of resting metabolism anesthesia is the best approach, since the disturbance will be minimal, and no recovery processes are to be expected. In the case of exercise, a fast blow, followed by decapitation, should be preferred in order to prevent recovery during the delay of the anesthetic. The delay problem may be overcome by infusion of an anesthetic via a cannula inserted in the aorta. Cannulation of the dorsal aorta is widely used for acid-base and blood-gas studies, its application for metabolic studies is restricted since only blood can be sampled. As far as we know cannulation has never been used in order to reach a very fast anesthesia after exercise. In some papers on fish metabolism, the experimental fish were inactivated/killed with electrical shock, obviously this method has its limitations, since it will likely stimulate the whole animal and particularly the muscles, thus leading to significant changes in the metabolite profiles. It has been described several times that premortem stress not only reduces the levels of glycogen, PCr and ATP, but also dramatically accelerates the postmortem degradation rate in comparison with anesthetized fish 29'103. The best way to overcome handling stress is to use non-invasive and nondestructive (physical) probing techniques which are not sensed by the animal. The technique currently available is in vivo NMR (nuclear magnetic resonance), which will be discussed separately.

38

G. van den Thillart and M. van Raaij

2. Tissue damage To acquire a piece of tissue, it must be excised, which obviously causes damage to the cells. In addition to local damage, nerves are cut and/or damaged, leading to stimulation of the adjacent tissue via spinal reflexes, thus resulting in general activation of the excised tissue. The metabolic rate of certain tissues can thus be increased enormously, therefore one should employ two different strategies: (1) suppress activation as much as possible; and (2) inactivate the sample as fast as possible. Activation particularly of muscle and neural tissue can be reduced significantly by anesthesia, and muscle relaxants. For example, muscle from anesthetized eel (Anguilla anguilla) responds immediately to incision, only by intracardial injection of curare the spinal reflexes can be suppressed 124. Recently, Arthur and collaborators 5 applied lidocaine at the spinal cord of skipjack tuna (Katsuwonus pelamis) - after previous MS 222 anesthesia - to suppress spinal reflexes during biopsy sampling. To inactivate the sample, freeze-clamping is the ultimate procedure. The aim of sampling is to have a momentary view of the metabolite levels. Using aluminium blocks cooled by liquid nitrogen (-195.8~ Wollenberger and colleagues 138 demonstrated that a piece of tissue can be metabolically put to a standstill within a few msec. This seems fast enough, especially since muscle contractions are slower. The dimensions of the tongs determine the sample size, normally 0.1-2.0 g. With enlarged tongs even whole animals can be freeze-clamped to a weight of about 6 g (refs. 2, 60, 87, 136). Freeze-clamping is not always used, some authors immerse the samples 9s or even whole animals in liquid nitrogen 36.49. This technique is inferior to the clamping method, because the time needed to completely deep-freeze a sample may take several minutes or longer depending on the size. Temperature equilibration depends on distance, temperature-difference and heat-transfer capacity. The equilibration time is exponentially related to both distance and heat transfer capacity, so the distance should be minimal (<2 mm), and the heat transfer should be maximal (copper or aluminium clamps). Particularly the conductance of heat through ice or water appears very slow, which makes the freezing rate extremely size dependent ~3s. An important problem that remains, is the time period needed for cutting out the tissue, a procedure that takes 10-200 s. A problem still hard to overcome since most tissues are difficult to excise. The application of needle-biopsy is mainly suited for muscle and possibly liver of large animals. Tissue preparation after freezing is not advisable, because freezing of a whole animal cannot be carried out fast enough.

3. Tissue extraction In order to extract metabolites from the tissue properly, one should make a very fine homogenate to reduce the diffusion barriers (maximal surface area), to break the protein-substrate bonds, and most importantly to block all enzyme activities. The technique often used is the one described by Williamson and Corkey137; this technique employs ethanol as an antifreeze and perehlorie acid (PCA) as a denaturation agent. The (frozen and powdered) tissue is then homogenized at

Endogenous fuels; non-invasive versus invasive approaches

39

-15~ A low temperature is necessary to minimize enzyme activities during the interval between the onset of thawing and complete denaturation. Too often this technique is not consistently applied, probably because its rationale is not well understood. Between -0.8 and -5~ glycolysis and hydrolysis of high energy bonds proceed at higher rates than at room temperature 9,42,77,8~ The reason for this is that between -0.8 and -5~ the intracellular water is only partially frozen. Ice crystals rupture the membranes, resulting in a release of Ca 2+ from the sarcoplasmatic reticulum and the extracellular space. The presence of Ca 2+ causes a striking activation of glycolysis and of myosin ATPase. The formation of ice (crystals) also increases the concentrations of enzymes, ions, substrates and modulators to very high values in the remaining water phase, which of course greatly stimulates the enzymatic reactions. The rate of ATP hydrolysis of fish muscle in the 'critical freezing zone' can be 200 times as high as the rate at room temperature 9,77. Denaturation at low temperature can be achieved by the use of an ethanol/ perchloric acid mixture, which should be pulverized in liquid nitrogen together with the tissue. When the concentration of ethanol is sufficiently high (30% v/v) the medium melts between -15 and -20~ without the formation of ice crystals. Denaturation thus starts at a low temperature below the critical freezing zone. Still, we should realize that at the moment a protein can be denaturated, it can at same time catalyze its own reaction. Since the denaturation process takes time, it will never be possible to completely exclude enzymatic reactions during denaturation. Denaturation is a stochastic process, so it will take time to denaturate all proteins. Therefore we should try to reduce the reaction rates as much as possible, this holds especially for ATPases and creatine kinase since the activities of these enzymes are rather high. Enzymes that use ATP as a substrate, need Mg 2+ as a cofactor. It is possible to chelate both Ca 2+ and Mg 2+ to F- and EDTA. The addition of these substances to the extraction medium results in almost 100% recovery of ATP and PCr which was checked by adding standards to the frozen tissue/PCA powder 115. This procedure is however not applicable to whole animal extractions. The reason appears to be the presence of Ca 2+ and Mg 2+ salts from the skeleton and the high concentrations of digestive enzymes. The problem can be solved by extracting separately the carcass and the intestines, adding the appropriate amount of EDTA and F - to the extraction medium (van den Thillart and Nieveen, unpublished). Another solution is to freeze dry the sample at -40~ followed by PCA extraction. This way the interfering enzymes are dehydrated and therefore not active before denaturation. Low lactate levels and high PCr levels are found with this technique in muscle 1~ as well as in whole animals 87. 4. Storage

Metabolites in frozen tissues are not stable at -20~ nucleotide levels and free fatty acid contents 28 change at this temperature, apparently due to enzyme activities. Therefore storage in liquid nitrogen or at liquid nitrogen temperature is advisable, since even at temperatures lower than -20~ conversions are reported. In most extracts, we have found trace amounts of enzyme activities, particularly

40

G. van den Thillart and M. van Raaij

activities of ereatine kinase were always present. This means that after some time equilibrium conditions will be reached in the extract. For example, in the presence of ATPases and ereatine kinase, ATP will be buffered by creatine phosphate: [PCr] decreases, while [ATP] remains constant. Metabolite levels in the extract are not stable often due to the presence of enzymes, therefore the best way to store extracts is in liquid nitrogen. Of course some metabolites are more stable than others, and extracts are not always the same with respect to traces of enzyme activity. In the tissue extracts we have analyzed for nueleotides by HPLC, we found <2% change in nueleotide concentration over a 12 hour period at room temperature, indicating a rather stable preparation. However this is not always the ease. The stability of the extracts, and the efficiency of the extraction procedure has to be checked frequently, particularly since many artifacts may occur.

5. Measurement There are many different ways of measuring metabolites. The techniques are mostly well described and tested, but under restricted conditions, and with pure solutions. Extracts, however, contain a large number of mostly unknown compounds which were never tested before. Also, the presence of trace amounts of enzymes may disturb the measurements. Therefore the use of internal standards at least in a few samples of a series is highly recommended in order to test the recovery. Recovery should be tested in two ways: (1) addition of internal standards to the tissue powder to test the overall recovery; and (2) addition of internal standards to the extract, to test the efficiency of the analysis. In the discussion about (destructive) measurements and the impact of the different artifacts, it is important to take into account the relative level of disturbance. For example, when glyeolysis was active during the sampling and extraction procedure, then it is obvious that the relative change of glycogen is much smaller than the change in the level of lactate. Glycogen may have decreased from 10 to 9/zmol 1-1 (-10%), while at the same time lactate increased from 0.5 to 2.5/zmol 1-1 (+400%). Similarly, a significant change in the concentration of free fatty acids can develop in frozen tissues, because the concentrations are very low, while in contrast the fluxes and also the concentrations of triglyeerides are normally high 12~ Thus the impact of the sampling and extraction procedure on the final data is not always the same.

111. Non-destructive approaches As we have seen above, destructive techniques have quite a few drawbacks; apart from the handling stress and the extraction artifacts, an additional problem is the fact that for each single measurement one animal is needed. Thus, usually a large number of experimental animals is required for statistical reasons. There are a few techniques that allow us to overcome these problems: eannulation, calorimetry, and in vivo NMR.

Endogenous fuels; non-invasive versus invasive approaches

41

1. Cannulation

For cannulation, an indwelling catheter is usually inserted in the dorsal aorta under anesthesia according to the method of Soivio et al. 97. After a two day recovery in a narrow box, experiments can be carried out on the fish, and blood samples can be drawn without disturbing the animal. The low levels of cortisol and catecholamines found under these conditions indicate that there is indeed a low stress level 121. Cannulation particularly of the dorsal aorta is often applied in acid/ base studies, other applications are rare. For metabolic studies cannulation can be used for turnover studies, i.e. lactate and glucose 13, but also to follow certain blood constituents as metabolic parameters. For example, changes in lactate, glucose, amino acids, fatty acids, and hormones have been used in this respect to follow the metabolic response of fish upon exercise, hypoxia, and acid exposure. More complicated cannulation applications, such as the determination of A - V differences over certain organs, are almost exclusively found in the mammalian literature. This is likely due to the difficulty of reaching small blood vessels in fish. Still, for evaluating the metabolic function of organs, such studies are obligatory. Results from studies on isolated organs, and certainly on isolated cells, have always some bias in comparison to whole animal studies; perfusion is never the same, neural and hormonal control is cut off, and cell surface is changed, etc. Cannulation can of course not be applied for the measurement of metabolites within the different tissues. However, the technique is important for the determination of the turnover of both endogenous and exogenous substrates. 2. Calorimetry

According to the laws of thermodynamics, all the available energy liberated by conversion of substrates is converted to heat, other products, and activity. Under steady state conditions, activity dissipates as heat, and we have to consider only the substrates and products. By means of calorimetry, it is possible to calculate the amount and the kind of substrates used by the animal for its heat production, i.e. the use of sugars, proteins, and lipids can be calculated. This is based on the equations that can be elaborated from the different fermentation and oxidation reactions taking place within the animal 36,126. Calorimetry refers to the measurement of heatflow, which can be carried out directly and indirectly; for direct calorimetry one has to use a calorimeter, while for indirect calorimetry one calculates the heatflux from oxygen consumption, and CO2 and NH3 excretion. Calorimetry is a non-invasive and non-destructive method that can be carried out without excessive stress for the experimental animals. Recently, it has been shown that non-invasive glycogen determination is possible from accurate respiration studies 72. Normally the animal may stay alive, although for some calculations additional end products should be measured like urea, lactate, and ethanol. Under conditions when the animal is not in an aerobic steady state, a combination of direct and indirect calorimetry reveals the fraction of each pathway for the total energy production 128.

42

G. van den Thillart and M. van Raaij

Actually, this is the only way to quantify with a direct method the total contribution of anaerobiosis to the energy production. Calorimetry has some restrictions, which makes this technique not easily accessible. In the first place, the animals must be in a steady state, because it takes a while before a thermal, gas, and metabolite equilibrium is reached. In the second place, we must assume that no other reactions take place other than those used for the calculations. And finally the method is slow, and expensive. Unlike mammalian respirometry, RQ (respiratory quotient) measurements of fish do not give sufficient information on substrate utilization. This is because protein oxidation by NH3 excreting animals results in RQ = 0.96, which is not distinguishable from carbohydrate oxidation with RQ = 1.00. Therefore also NH3 excretion should be measured. Measurement of CO2 and NH3 production by fish is difficult. CO2 measurements are disturbed by the HCO 3 buffer in the water. The NH3 production is about 10% of the 02 consumption. Furthermore, the accumulation of CO2 and NH3 is directly related to the O2 concentration change in the water, which is small due to the low solubility of oxygen. Thus, some skill is essential to measure CO2 and NH3 accurately enough in order to obtain reliable RQ and AQ values (RQ, respiratory quotient = ~'CO2/'v'O2; AQ, ammonia quotient =

VNH3/VO2). In contrast, oxygen consumption measurements are relatively easy to obtain, because of the high sensitivity of the currently available oxygen electrodes 38. The oxygen consumption data are mostly used as a measure of the energy flow of the animal. This is based on the assumption that the animal is completely aerobic, and that the animal uses the same mixed substrate for its oxidation reactions. Of course both conditions will never be completely fulfilled; animals do use different substrates, anaerobic pathways, and incomplete oxidation reactions from time to time. However, for an estimation of energy flow oxygen consumption can be used, as long as its limitations are not forgotten. Both direct and indirect calorimetry on fish is seldom applied. Based on the few available data, an overview has recently been published 127. 3. Nuclear magnetic resonance spectroscopy

With in vivo NMR we are able to look inside tissues without incurring damage or changing the metabolite pattern. The technique is based on radiowave absorption/ emission in a high magnetic field (see Volume 3 of this series, Chapter 50). As with all spectroscopic techniques, NMR is non-destructive: the chemical composition of the sample does not change during the measurement. Because the radiowaves and magnetic fields penetrate through living tissue, the technique is suited for in vivo applications. For metabolic studies three different types of nuclei are available: 1H, 13C and 31p. These nuclei occur in numerous metabolites, which then in principle can be measured with NMR spectroscopy. There is, however, a great difference between high resolution NMR (analysis of a solution in a test tube) and in vivo NMR (whole animal or tissue within the sensitive area) with respect to resolution and sensitivity. Mainly because of peak broadening, the detection threshold for

Endogenous fuels; non-invasive versus invasive approaches

43

the different compounds that can be detected by in vivo NMR is much lower than with high resolution NMR. The biggest problems occur with 1H- and 13CNMR. With 1H-NMR there is an enormous interference of water and lipids on the metabolite signals: a 104-fold difference in signal strength. In addition, the spectral band available for all the different signals is small, resulting in overlap of most signals. New pulse editing techniques are being developed which will make 1H-NMR available for many applications in the near future. The drawback of 13CNMR is the low sensitivity of the nucleus, and the fact that the natural abundance of 13C is only 1.1%. The sensitivity can be enlarged by 13C-coupled 1H-NMR, and the concentration of 13C can be increased by the use of 13C enriched compounds. Still, the techniques is difficult and expensive. There are a few applications for very small animals and in vitro systems (not for fish). The above mentioned problems do not occur with 31P-NMR, which makes this the most widely used technique for in vivo NMR at the moment. While in vivo 1Hand 13C-NMR have a high potential for future metabolic studies, the applicability of in vivo 31p-NMR for fish studies has been demonstrated already by several publications 15,11~ In vivo 31p-NMR spectroscopy allows non-invasive and non-destructive measurement of phosphorus-containing compounds such as sugar phosphates, inorganic phosphate, IMP, phosphodiesters, creatine phosphate, and ATP. Furthermore, from the spectral position of the inorganic phosphate peak, the intracellular pH can be calculated 11~ and from pH, ATP, Pi, and PCr, the free concentration of ADP can be calculated 11~ The most striking observations with in vivo NMR are the low Pi (1-0.5 mM) and the high PCr/total creatine ratio (>90%) in muscle tissue in comparison with standard measurements. The low Pi value, sometimes even below the detection limit of 0.5 mmol 1-1 (Fig. 1), indicates a high phosphorylation potential, which is to be expected under aerobic and resting conditions 31,11~ A high phosphorylation potential should go together with a high PCr/total creatine ratio. The low PCr/total creatine ratios observed in the literature, led to the suggestion that creatine should play a role in osmoregulation 7, however, it has been found that with very careful sampling and extraction of muscle tissue a much higher degree of creatine phosphorylation can be obtained 5,87,1~ Figure 2 shows the inorganic phosphate section of the in vivo 31P-NMR spectrum of eel (A. anguiUa) muscle, after 24 h in a flow-cell under resting and normoxic conditions. It demonstrates the effect of exposure to respectively 100, 10, 6 and 3% air-saturated water. Evidently, under normoxia, the Pi is almost invisible, which means a concentration lower than 0.5 mM. Under hypoxic conditions, the Pi peak increases, shifts and broadens, indicating respectively increasing concentration, decreasing pH, and compartments with different pH. In addition to the low Pi level, the high degree of phosphorylation of creatine (90%) is typical for in vivo 31P-NMR spectra. In comparison with 'destructive' methods, the NMR technique shows that the phosphorylation potential of the cytosol of resting aerobic tissues is very high. The effect of an anesthetic on the energy balance of a fish is clearly demonstrated by Chiba and coworkers is. In a simple in vivo NMR experiment, these authors showed that the phosphorylation potential of loach (Cobitis biwae) muscle increases markedly during anesthesia.

44

G. van den Thillart and M. van Raaij

, I,, 5.5

j,

I,,,, 5.0

L , , , , i , , PPN

4.5

4.0

Fig. 2. Part of a series of in vivo 31p-NMR spectra from the rump muscle of eel. The eel was exposed to 100, 10, 6, and 3% air-saturated water, respectively. Under normoxic conditions the Pi signal was absent; during hypoxia the Pi peak increases markedly. The shift to the right indicates a decrease of the

intracellular pH from 7.605 to 7.100.

When a piece of muscle tissue is excised, we find with in vivo NMR a significant lower PCr level together with a higher Pi concentration than in the muscle of an intact animal 115. This indicates that the sampling procedure must be responsible for this difference. Even the use of an anesthetic appears insufficient to prevent activation of muscle catabolism during and following excision. This follows also from the skipjack tuna (IC pelamis) experiments of Arthur and colleagues 5" Although the application of the biopsy sampling coupled with a spinal block is impressive, and creatinc phosphorylation reaches 75% (the highest so far), the levels of PCr are not high enough and the lactate level is not low enough. The shortfall from the expected values, based on NMR data, can be caused by tissue damage, freezing time, and extraction technique. Since we observed metabolic activation in excised tissue with NMR, tissue damage appears to be the most likely reason. Similar biopsy studies on humans 1~ show lactate levels around 3 raM, and crcatine phosphorylation of 60-70%. Freeze clamping a whole animal 2~176 and muscle pieces, result in lactate levels below 1 mM. Further, blood lactate levels in fish are low under

Endogenous fuels; non-invasive versus invasive approaches

45

resting conditions (<0.5 mM, refs. 13, 16, 121). In conclusion, it is likely that the effects of tissue damage cannot be completely overcome by the application of anesthesia. Since PCr/total creatine ratios are seldom presented, the lactate concentration appears to be a sensitive indicator for the level metabolic rest of the muscle tissue. In Table 1, a list is presented of lactate levels in muscle from several species, obtained under different conditions. The lowest levels are obtained when an anesthetic is used in combination with very fast sampling and freeze clamping.

IV. Storage of endogenousfuels 1. High energy phosphates When endogenous fuels are considered as those substances that contribute to cellular energy metabolism, the ultimate forms of energy are phosphorylated adenylates (especially ATP) and creatine-phosphate. ATP levels may vary considerably between various tissues and species. Values of about 1/zmol g-I are normally found in brain and liver25,98,131 while heart and red muscles contain ATP levels in the range of 2-5/zmol g-1 (refs. 25, 98, 111). However, the highest ATP levels (up to 10/zmol g-l) are mostly observed in white muscle 5,25,76. In addition, white muscles also contain considerable amounts of PCr (20-30/zmol g-1)5,25,76, whereas in heart and red muscle moderate levels are observed (10-20/zmol g-l). PCr is also present in significant amounts in the brain; almost no PCr is found in the liver25,~. In vertebrates, PCr is a directly available high-energy phosphate reservoir stored generally in the cytosol although in muscle it may be locally concentrated near myosin ATPase 8,86. As discussed earlier, PCr stabilizes [ATP] via the creatine kinase reaction 11~ Although this pathway can generate ATP at high velocity, which is favourable in fast twitch white muscle specialized for burst work, the amounts of ATP and PCr provide only a minor reserve in terms of useful energy equivalents.

Even high levels of ATP and PCr can provide only enough energy to support a few seconds of burst activity. Standard metabolic rate, however, can be supported for a few hours.

2. Carbohydrates Especially in carnivorous fish species, carbohydrates are generally not a major fuel for cellular energy metabolism. However, during hypoxia or burst activity carbohydrate may become the preferential substrate. Carbohydrates are essentially stored in the form of glycogen, a polymer of glycosyl units with linear t~(1-4) and branched u(1-6) linkages and is generally found in the cytosol in the form of small granules called fl-particles. Glycogen can also be stored in larger a-particles and specialized glycogen bodies which are observed only in tissues which store huge amounts of glycogen. As in mammals, the primary storage site in fish is the liver or hepatopancreas. Hepatic glycogen contents are extremely variable between fish species and even between individuals; values from about 2/zmol glycosyl units g-1 wet weight

G. van den Thillan and M. van Raaij

46 TABLE 2

Glycogen contents of fish liver and muscles Species Atlantic c o d 54 Atlantic cod 54 European eel 124

American eel69 Flounder79 S u n f i s h 111

trout79 Rainbow trout25 Common carp1~176 Cutthroat trout 1~1 Mullet2 Mackerels2 Goldfish111 (20~ Goldfish132 (20~ Goldfish132 (4~ Crucian carp45 (summer) Crucian carp45 (winter) Rainbow

Liver White muscle Red muscle (ttmoles glucosylunits per gram of tissue wet weight) 2

-

39 15 49 50

2 12 6

111

120 134 556 667 750 816 750 967 1366 139 1806

3

6 18 4 9 13 26 27 22 194

14 -

16 14 41 32 -

to extraordinary levels of 1800/~mol g-1 are observed (Table 2). Liver glycogen may serve to provide exogenous blood glucose as is demonstrated in the anaerobic goldfish 93 but not in hypoxic rainbow trout 25. White and red muscles contain significantly less glycogen than the liver, sometimes more than a magnitude in difference. Although the white muscle is specialized for burst type exercise which can be regarded as typically anaerobic, this tissue does not necessarily store more glycogen than the red fibers 25'69'111. However, in very active pelagic fish (e.g. tuna) white muscle glycogen levels may reach values above 100/~mol g-1 (ref. 5). In heart muscle, glycogen levels are mostly 2-4 times higher to those in the red muscle 25.52,Ts.s3,n4. It has been observed that, in contrast to the situation in mammals, the fish heart may use carbohydrates as a major energy fuel 93,94. Other tissues may all contain variable amounts of glycogen and the concentration in the brain in particular may have a high impact on the hypoxia tolerance of fish species. In a comparative study on rainbow trout (Oncorhynchus mykiss) and brown bullhead catfish ( I c t a l ~ nebulosus), the brain of the anoxia-tolerant catfish contained about five times as much glycogen as the anoxia-intolerant rainbow trout 23. This implicates the importance of glycogen as an endogenous fuel in brain, heart and muscles during emergency situations like environmental hypoxia, anoxia or exhaustive exercise. On the basis of glycogen contents and the total mass of the tissue, both liver and white muscle glycogen stores respectively are important as energy reserves. Glycogen contents of fish tissues are highly influenced by environmental factors including O2-availability, temperature, season and dietary composition. Hypoxia acclimated goldfish store about twice as much glycogen in their muscle tissues

Endogenousfuels; non-invasiveversusinvasiveapproaches

47

compared to normoxic individuals although liver glycogen is not affected, possibly because maximal glycogen deposition was already established 111. In flounder (Platichthys flesus), the deposition of heart glycogen was markedly increased after three weeks of moderate hypoxia63. Glycogen in liver and white muscle of cold-adapted goldfish (Carassius auratus, 4"C) was significantly higher than that in 20"C acclimated animals 132. This is probably of ecological relevance since Crucian carp (Carassius carassius), a highly anoxia tolerant species from the same genus, which can survive for months in anoxic ponds during winter, store huge amounts of glycogen in their muscles and liver at the beginning of the winter but deplete these stores during summer 45 (Table 1). Beside these abiotic factors, glycogen levels are affected also by the composition of dietary intake. When trout (O. mykiss) were forced fed orally with high doses of glucose, liver and muscle glycogen levels were rapidly increased 18 while increased levels of dietary protein also stimulated glycogen storage probably by increased gluconeogenesis 13~

3. Lipids Lipids are a major energy fuel for fish tissues and most tissues posses the necessary machinery for catabolism of fatty acids at high rates 51. As in other vertebrates, lipids used for energy metabolism are stored in fish mainly as triglycerides, although some species use wax-esters or alkoxydiacylglycerols exclusively or in combination with triglycerides 91. Lipids appear to be stored in several depot organs which contrasts to the situation in mammals in which lipids are stored almost exclusively in adipose tissues. The most important storage sites in fish are mesenteric fat, liver and muscles. The deposition, mobilization and composition of lipids in fish tissues have been reviewed by Sheridan 91 and Henderson and Tother 41 and the reader is directed to these publications for detailed information (el. chapter 6, this volume). Adipose tissue is found in the viscera of a number of species including rainbow trout (Oncorhynchus mykiss), coho salmon (Oncorhynchus kisutch), perch (Perca fluviatilis), carp (Cyprinus carpio) and goldfish (Carassius auratus) and high lipid contents which amount to more than 80% on a wet weight basis may be observed occasionally 41. Visceral lipid contents of 15-45% wet weight are frequently observed, although it must be mentioned that most of the literature deals with fish fed commercial diets 41. In contrast to these fatty viscera, relatively lipid-poor viscera are found in snakeheads 9~ Mesenteric fat deposits are markedly increased by excessive dietary intake 134. Considering the high lipid concentrations and the relative contribution to total body weight (up to 10%), visceral lipid depots are quantitatively a major fuel reserve which releases free fatty acids (FFA) to the blood thus supplying exogenous fuels for most other tissues. Dietary lipids and other lipogenic substrates are processed in the liver which is the primary 'lipogenic factory' of the fish body 91. Part of this lipid is transported to other body compartments, but a significant portion is stored directly in the liver. Fat contents of fish livers generally range from 3% to about 25% wet weight. Although lipid contents reaching as high as 50% may occasionally be observed in fresh water fish, really 'fatty' livers are found in benthic marine species like cod (60-70% wet

48

G. van den ThiUan and M. van Raaij

weight) 41. Liver lipids can be mobilized readily, thereby providing lipoproteins and FFA for utilization in peripheral tissues. Lipid levels in muscle tissues are generally lower than those in liver, although some species exhibit extreme triglycefide stores in their muscles 85. It is known for some time now that red muscle stores about 5 times more lipid than white muscle and triglycedde deposits in the form of lipid droplets are found in the cytosol as well as the interstitial space 33'34'91. Although generally overlooked, in several species a relative large amount of lipid is also present superposed on the anterior red muscle below the skin. While liver and mesenteric fat may be regarded as storage sites for the total body, the lipid which is stored in and around the red muscle probably has a direct endogenous function. Since this tissue is well peffused, contains a high density of mitochondria, displays substantial tdglyceride lipase activity and may posses a [FFA] higher than the blood, the lipid in this muscle is used for the energy metabolism in situ, especially during low speed sustained swimming (see below). Other tissues like kidney, heart and brain are all highly capable of burning fatty acids St, but their lipid contents are mostly moderate supporting local metabolism only. 4. Proteins and amino acids

Proteins contribute an important part to the general fish diet, especially in carnivorous species. After feeding a protein rich diet, the concentration of amino acids (AA) is increased in all tissues with the rate of protein synthesis, gluconeogenesis and AA catabolism increasing accordingly Is. The deposition of proteins is difficult to measure although some techniques using radioactive labeled AA have been employed 11,27,46. The study of protein catabolism is hampered by the absence of effective techniques but some insights can be obtained from the differences between the rates of synthesis and growth6,1s. Since the fish body contains a large amount of protein, changes in concentrations are mostly small and may not reach statistical significance or are simply the result of other changes in the tissue (e.g. water or lipid content). Instead, most studies are directed to measurement of AA metabolism or NH~ production. However, it must be noted that the latter can also be influenced by adenylate breakdown during hypoxia or high intensity exercise 122. Amino acids are an important energy-source for fish tissues and the literature on this subject is vast and numerous. This conclusion is based on amino acid decarboxylation 74,1~ deamination 122,123, enzyme patterns and activities 2,1s,~,7~ and nutrition studies 47,13~ However, in spite of all these data, the quantitative impact of AA on total oxidative energy metabolism is incompletely understood, especially since large species differences are observed. The protein content of the fish body amounts generally to 10 to 20% of fresh weight and is therefore the main body constituent since 60-80% of body weight is water. The liver protein content is generally lower than 10% wet weight although slightly higher amounts have been observed in eels21,69. The rate of protein synthesis in the liver of rainbow trout was found to be the highest of all tissues studied

Endogenous fuels; non-invasive versus invasive approaches

49

(liver > intestine > gills > muscle) 133, however, a considerable part of these proteins are designated to the plasma. The levels of free AA are highest in the liver and kidney and amount to approximately 20-60/zmol g-I (taurine excluded), although levels below 10/zmol g-1 have also been reported 68. The major amino acids in the liver are glutamate, glycine, proline and alanine, as well as taurine. The amount of AA is very sensitive to the nutritional status of the animal and varies with species, dietary history and the time of sampling after feeding 18. Nevertheless, within an individual, the [AA] is mostly higher in the liver than in other tissues which suggests a major role for the liver in AA metabolism. It was observed that 50-70% of the total NH3 production originates from the liver 81 and, moreover, hepatectomized eels showed a reduced capacity to deaminate an excess of AA which may occur after feeding 56. In accordance with the role of the liver with regard to carbohydrate and lipid stores, amino acid deamination will provide the necessary substrates for either gluconeogenesis or lipogenesis within the liver. Although liver protein turnover is relatively high, the proteins of fish liver are not often mobilized and since the liver mass does not exceed 3% of total body weight, liver proteins cannot be regarded as a major energy store (see mobilization section). Protein levels in muscles are mostly higher than those observed in other tissues. Values up to 20% wet weight are normally observed and generally white muscle contains more protein than the red muscle (Table 3). Muscle amino acids are mostly present in slightly lower concentrations compared to liver ranging from 6 to approximately 50/zmol g-I (Table 3). Glutamate, alanine and glycine are the major contributors to the total AA pool similar to liver. Also asparagine, glutamine and in particular histidine may be present in high amounts in muscle 117,119. Beside high levels of histidine, muscles may also contain high levels of other histidinederived imidazole compounds, trimethylamine, sarcosine and sulphur-containing polypeptides. However, these compounds serve primarily as biological buffers or osmoregulators and are probably not important as fuels for energy metabolism 122. Although liver and kidney contain the highest concentrations of AA, the (white) muscles provide by far the largest reservoir of amino acids on the basis of total body weight. In addition, substantial proteinase activity can be found in muscle tissues 4'64'68. Therefore this huge amount of energy storage may be important in times of starvation or migration (see below).

I4. Mobilization of endogenous fuels Energy metabolism is fuelled by a mixture of substrates which can be provided by two sources: (1) dietary intake and absorption from the intestine; and (2) endogenous, stored fuels. When dietary intake is absent (starting about 48 h after the last meal) the animal must rely on its fuel stores. In the following section we will discuss the mobilization of endogenous fuels as occurs during a number of conditions (hypoxia-anoxia, exercise, starvation, migration) which may all be encountered by fish in their natural environment.

G. van den Thillan and M. van Raaij

50 TABLE 3

Protein and amino acid contents of red and white muscle in teleostean fishes Species

Muscle type

Protein (% wet weight)

Carp

W h i t e 19 W h i t e 11

4 14

6.3 -

W h i t e 99 R e d 99

20 17

-

W h i t e 119 R e d 119

-

30 25

W h i t e 117 R e d 117

-

36 30

W h i t e 6s R e d 6s

-

10 8.8

Goldfish

Sockeye

salmon

T ot al amino (~tmol g - l )

Chum salmon

White 4

15-20

-

Rainbow trout

White ~ D a r k 99

19 16

-

W h i t e 98 D a r k 9s

-

40 36

European eel

W h i t e 21

27

-

eel

W h i t e 69 R e d 69

15 19

-

Cod

W h i t e 13~

10

-

Char

W h i t e 99 D a r k 99

21 18

-

Tilapia

W h i t e 99 R e d 99

19 17

-

American

See

text for

acids

species names.

1. Hypoxia and anoxia

Since fish live in a medium which essentially contains a low amount of dissolved oxygen, they can easily encounter hypoxic or even anoxic environments. Environmental hypoxia or anoxia can be caused by high temperature in tropical stagnant waters, by excessive algal growth/decay or by ice covering water ponds during winter time. The primary physiological reactions associated with hypoxia are hyperventilation, bradycardia, increased stroke volume of the heart, increased gill peffusion, redirection of blood flows and improved oxygen affinity of hemoglobin 43 ,44 , 105 . The metabolic changes during moderate hypoxia are often transient since most fish are able to adapt to environmental hypoxia 1~ Therefore, we will focus our attention on the effects on metabolic energy stores during acute severe hypoxia and anoxia. When the oxygen delivery to any tissue is hampered or inadequate, due to ischemia, hypoxia or anoxia, the rate of oxidative ATP generation cannot keep pace

Endogenous fuels; non-invasive versus invasive approaches

51

with the rate by which it is consumed. To provide the cell of a supplementary amount of ATP two possibilities are present: (1) Stabilization of the ATP concentration by PCr stores and (2) anaerobic glycolysis. Although ATP levels may vary between different tissues or species, its concentration is quite stable during normal oxidative metabolism. The rates of ATP consumption and regeneration are tightly regulated at the cellular level by redox and energy state and at the level of tissues by neural and hormonal input. Therefore, the concentration of ATP will not be affected to any great extent. During the absence of oxygen, PCr will transfer its high-energy phosphate to ADP which can proceed at very high velocity and is regulated primarily by the concentrations of H + and ADP 110,125. Using in vivo 31P-NMR, Van den Thillart and colleagues 115 demonstrated that ATP levels in the white muscle of anoxic carp (Cyprinus carpio) were maintained at control levels until PCr dropped to <20% of the control value. Interestingly, PCr in the same tissue of the anoxic goldfish was only reduced to 55% of the normoxic value and ATP levels were unaffected. Similarly, ATP levels in the white muscle of trout (O. mykiss) exercised to exhaustion are significantly decreased which is accompanied by a depletion of PCr to <20% of the control value 89. However, in the white muscle of submaximally exercised tuna, PCr decreased to values of about 15% of those in resting fish, while ATP was almost unaffected 5. The recovery of PCr is a rapid process and may be complete even when ATP levels are still depressed 89,115. The rationale for this observation is the low activity of the purine nucleotide cycle, which converts the accumulated IMP into ATP 115 (see also Chapter 16 of this volume). Thus, PCr is a high-phosphate energy-store which is rapidly depleted when cellular energy requirements cannot be fully met by oxidative and anaerobic metabolism which occurs during acute hypoxia, anoxia or intensive exercise 110. During oxygen shortage, the oxidative phosphorylation and electron transport chain is inhibited resulting in a change of the redox state of the tissue and an impaired mitochondrial ATP synthesis. One possibility to generate ATP apart from the electron transport chain is anaerobic glycolysis. Compared to normal oxidative metabolism this pathway is very inefficient since only 2 mol ATP are synthesized per mole glucose fermented whereas 38 mol ATP are obtained when glucose would be fully oxidated. Therefore, the flux through the glycolytic chain either has to be increased (Pasteur effect) or the metabolic activity of the tissue has to be depressed in order to adjust ATP consuming and producing processes. Since the efficiency of the glycolysis is rather low with respect to ATP to glucose ratio, the pathway proceeds at relatively high velocities. A prerequisite for proper cellular function is the maintenance of redox balance. Thus, pathways have evolved in which dehydrogenating reactions are coupled to hydrogenating reactions. The most common end product of anaerobic glycolysis is lactate. As discussed above, carbohydrates become the major if not only substrate for energy metabolism during anaerobiosis, whereas catabolism of fatty acids and amino acids becomes inhibited due to the absence of an appropriate coupling between dehydrogenating and hydrogenating reactions. The importance of anaerobic glycolysis is indicated by the occurrence of high levels of lactate dehydrogenase in most fish

52

G. ,an de,, Thillan and M. van Raaij

tissues 32'39'107. Glycogen mobilization during severe hypoxia or anoxia is observed in rainbow trout ~, flounder (Platichthys flesus) 52, cutthroat trout (Oncorhynchus clarki) and sunfish (Lepomis sp.) 4~ eel (,4. anguilla) n4, carp (Cyprinus carpio) 95, goldfish (Carassius auratus) n1.132 and Crucian carp (Carassius carassius) 45. The white muscle is the first tissue to become hypoxic because of its relatively low perfusion and utilizes mainly endogenous glycogen; the uptake of exogenous glucose is of minor importance since fish tissues contain only low activities of inducible hexokinase 57. The lactate diffuses into the circulation where it becomes a suitable substrate for other tissues (e.g. heart and red muscle). At more severe hypoxia, glycogen depletion also occurs in heart, brain and red muscle 2.52.124. Generally, glycogen in white and red muscle, heart and brain is mobilized during hypoxia or anoxia for metabolism in situ. In contrast, glycogenolysis in the liver is initiated mostly to release glucose to the blood which results in hyperglycemia. This phenomenon is observed in anoxia tolerant cyprinids 65,93. Depletion of liver glycogen is the major energy source during long term anoxia in goldfish and crucian carp which is associated with an increased glucose concentration in liver and blood 45,93,111. However, in hypoxic trout such a mobilization of liver glycogen and hyperglycemia is not always observed ~. At present, it is not known whether this can be attributed to the moderate hypoxic conditions which may be insufficient to induce the necessary stimuli for glycogenolysis or to a difference in metabolic organization. Beside the production of lactate as an anaerobic end product, alanine and succinate may accumulate during oxygen deprivation in several tissues43.105. Since anaerobic metabolism generally results in the accumulation of acidic end products, this way of energy production is only suitable for a limited period. A very special strategy, however, is employed by cyprinids of the genus Carassius and to some extent by Rutilus rutilus 1~ These fish are able to couple anaerobic glycolysis to the production of the neutral end product ethanol which is easily excreted to the environment 5~ and, in addition, they are capable of a considerable depression of their metabolic rates 128. Although in terms of energy equivalents this pathway may be 'wasteful' since the carbon skeletons of ethanol are lost, this strategy enables the animals to survive long periods of environmental anoxia especially at low temperatures 45'113. The catabolism of fatty acids is severely impaired during anoxia as was demonstrated in several mammalian tissues 55'71. As a result, FFA levels and their intermediates accumulate due to inhibition of the/~-oxidation ~1. In addition, the release of stress-related hormones such as catecholamines and cortisol stimulate lipolytic activity which augments the FFA increase 55. However, in fish this effect is completely absent and a significant decrease of plasma FFA by 50% may be observed, which can be attributed to a decrease of lipolytic activity26,65. Although the exact mechanism of this process is not resolved, experiments of Farkas 26 and recent studies in our laboratory suggest a special regulatory role for catecholamines and in particular for norepinephrine during anoxia (Van Raaij, thesis 1994). Although the catabolism of lipids is blocked during anoxia, lipogenesis may proceed. During the last decade, it has been frequently proposed that fatty acids may serve as anaerobic end products

Endogenous fuels; non-invasive versus invasive approaches

53

in anoxia tolerant cyprinids 43'1~ At present, the quantitative importance of such a process is unknown. However, recently we succeeded to demonstrate the presence of 'low-flux' fatty acid synthesis in anoxic goldfish using radiolabeled substrates (Van Raaij, thesis 1994). Amino acid metabolism in general is depressed during oxygen restriction because the flux through the citric acid cycle is diminished. Nevertheless, some metabolic turnover of AA is observed during anoxia (e.g. conversion of aspartate to succinate) which is mostly related to coupling of redox reactions. It has been suggested that AA catabolism may be of some significance in anoxic goldfish 1~ Interestingly, Van der Boon and coworkers 1~8 demonstrated that some constituents of the myofibriUar proteins in the white muscle of goldfish are depleted during anoxia which indicates protein breakdown. Hence, the role of protein and amino acid metabolism in hypoxic and anoxic fish has to be evaluated in future research. 2. Exercise

During exercise, the metabolic turnover in the muscles increases, sometimes by more than two orders of magnitude. Metabolic fuels have to be mobilized rapidly in order to provide the necessary substrates for energy production. The intensity of the exercise and the oxygen availability in the tissue determine to a great extent the preference of substrate utilization whereas white and red muscles show different metabolic strategies with respect to exercise. During sustained swimming and submaximal exercise, lipids and proteins are the preferred fuels. The bulk of energy production is thought to be derived from AA catabolism although surprisingly few quantitative studies have been performed. Already in 1968, Krueger et al. 58 concluded that coho salmon swimming at 6 BL s -1 derived 55% of their energy from protein catabolism whereas this contribution was estimated to amount 80% at higher speeds. Van den Thillart 1~ demonstrated that protein oxidation contributed 80% to energy metabolism in resting rainbow trout and even increased to 90% during sustained swimming. Ultimately, Kutty 59 noted that the level of NH + excretion by exercised Tilapia mossambica (now: Oreochromis mossambicus) in relation to oxygen consumption was high enough to propose that the energy production of these fish was totally supported by protein catabolism. In exercised rainbow trout, the total pool of free AA was significantly increased in liver, brain, and white and red muscle 98. These data indicate the quantitative importance of protein oxidation during exercise and call for further investigation. Lipid catabolism is thought to be an important contributor to aerobic metabolism at rest and during sustained exercise but again only few quantitative data are available. The importance of lipid oxidation especially in the red muscle (see above), was proposed already in the studies of Alexander 1. Histochemical studies later demonstrated the excellent capacity of red muscle for lipid catabolism 33.34. It was demonstrated that muscle lipid content of coho salmon was decreased after several hours of intensive exercise 58. The greatest decreases were found for the fatty acids 18 : 1, 16:0 and 16:1 which indicates the mobilization of triglycerides. At

54

G. van den Thillart and M. van Raaij

higher speeds (>7 BL s-1) the fish were rapidly exhausted and specific losses were observed for the fatty acids 18:2, 20:4 and 22:6. It is now well known that these fatty acids are associated with phospholipid hydrolysis and since these lipid classes arc normally hardly mobilized 41, this observation suggests that some tissue damage did occur. Nevertheless, it was estimated that during submaximal exercise, coho salmon would derive 45% of its energy from lipid catabolism whereas this value was decreased to about 15% during exhaustive exercise. The latter value however, may be of less significance because the authors did not account for glycogen utilization which was certainly present during exhaustive exercise. In mackerel (Trachuna symme~cus), it was observed that red muscle used endogenous triglycerides for sustained swimmings2 while this was not noted in white muscle. In resting rainbow trout, lipid oxidation may contribute about 20% to the energy metabolism whereas this value was about 10% during sustained swimming ~~ From the kinetics of arterially infused radiolabeled substrates, Van den Thillart 1~ concluded that a preferential oxidation of endogenous substrates occurred during the first hours of sustained aerobic exercise. Recently, S~ingerss demonstrated that the lipid content and the fine structure of red muscle showed considerable species-specific variation. The red muscle of Danube bleak, a cyprinid which is known to be a sustained swimmer, contained more then 10% lipid on a wet weight basis whereas in the Asp, a piscivorous predator which performs mainly burst type exercise, lipid contents of only 2% were observed. From these observations, S~ingerss proposed that these variations in lipid content are associated with a difference in swimming behavior. Interestingly, a positive statistical correlation between the amount of red muscle and mobility was reported ~. During intensive exhausting exercise or during burst type exercise, as can occur frequently in the white muscle of active pelagic or piscivorous species, the pattern of substrate utilization is different. Since oxygen supply is inadequate during these conditions, the changes in fuel mobilization resemble those during hypoxia with the result that metabolic flux is increased by several-fold. Thus, endogenous PCr and glycogen are the preferred substratcs during intensive exercise (see Hypoxia section). However, also during submaximal exercise, the white muscle may use endogenous glycogen as a substrate for anaerobic glycolysis resulting in the production of lactate. This area of fish physiology is well-documented 5'2~176176176 and we will only give an outline describing the general findings. The degree of glycogen depletion and the accumulation of lactate arc positively correlated with the intensity and the duration of the exercise24. Mobilization of glycogen may be extremely rapid and values of about 40 ~mol g-1 s-1 have been reported 24. Glycogenolysis in the muscle tissues is probably initiated by increased levels of Ca 2+ which is released from the sarcoplasmatic reticulum. These ions act upon protein kinase which subsequently activates glycogen phosphorylasc 44. The lactate formed in the white muscle diffuses to some extent to the blood, although the degree of this process seems to be species specific. Active pelagic fish, are sometimes called 'lactate-releasers' since their blood lactate levels increase significantly during exercise while in benthic inactive species blood lactate is elevated only modestly. The latter species have therefore been called , lactate-non-releasers ,79 ' 104 . As stated

Endogenous fuels; non-invasive versus invasive approaches

55

above, lactate released into the circulation is a preferred substrate for aerobic metabolism in other tissues or for gluconeogenesis in the liver ~~

3. Starvation and migration A number of fish species will encounter prolonged periods of starvation during their life, often seasonally dependent and associated with migration and reproduction. The utilization of endogenous fuels is dependent on the length of the starvation period and the species under investigation. A considerable number of papers have been published on this area during the last 30 years. In this section we will describe the generalities that have arisen from this research emphasizing the importance of mobilizing endogenous fuels for energy metabolism. With respect to the mobilization of glycogen stores, the fish species studied so far may be divided in two categories: species which do or do not mobilize glycogen during the initial phase of starvation. Glycogen utilization during the first stage of starvation was found in common carp (C. carpio), roach (R. rutilus), killifish (Fundulus heteroclitus), rainbow trout (O. mykiss) and brown trout (Salmo trutta). During wintering of common carp, Takeuchi and Ishii 1~176 found that liver and (white) muscle glycogen levels were reduced by 65 and 80% respectively. Glycogen contents of the carcass of juvenile roach were decreased from 5.1 to about 1.6 /zmol g-1 after about three weeks and remained at this level during the remainder of the starvation period (50 days) 67. Similarly, whole body glycogen was depleted by 50% after 5 days of starvation in juvenile rainbow trout ss while glucose availability was significantly enhanced. The liver glycogen pool of killifish was depleted by over 90% after fasting for five weeks 62. Recently, Navarro et al. 75 demonstrated glycogen depletion in the liver and muscle of brown trout of about 80 and 20% respectively already after eight days of starvation, although after 4 weeks the glycogen contents were partly restored (from gluconeogenesis). In common carp, starved for about one month, no significant mobilization of liver glycogen is observed whereas after three months, these stores were depleted to 20% of the initial value 73. Thus carp may display an intermediate response. Representatives of the second category are migrating sockeye salmon (Oncorhynchus nerka) and fasting European (Anguilla anguiUa) and American (A. rostrata) eels. During their migration to the spawning grounds, the liver and muscle glycogen levels of salmon are not significantly changed and are in fact increased just prior to spawning 3~ The glycogen content of liver and red muscle of American eel was not affected after six months of starvation while white muscle glycogen was decreased by 40% (ref. 69). Similarly, Larsson and Lewander 61 showed that liver glycogen of European eels was not decreased during the first three months of starvation but was decreased by 40% after about five months. Muscle glycogen was not changed at all. However, these observations do not rule out the possibility of carbohydrate catabolism since liver glycogen may be continuously replaced by gluconeogenesis as was proposed by Larsson and Lewander 61. Increased gluconeogenetic activity from lactate and alanine was observed in migrating salmon 3~ Liver glycogen contents of fasting cod were extremely low 54 (Table 2) and although these levels were reduced during

56

G. van den ThiUan and M. van Raaij

starvation, liver glycogen is probably of minor quantitative importance in this species. In the second category, glycogen is only used as energy source at prolonged starvation when the availability of other fuels (e.g. lipids) is reduced. In the first category, glycogen utilization may be of transient importance during the first stage of starvation until the mechanisms for utilization of other substrates are activated. Lipids and protein are then the major fuels for energy metabolism. Lipid mobilization is a common strategy in fasting fish although there appears to be some variation with respect to target tissues. Visceral lipid depots are easily mobilized and decline almost immediately after cessation of feeding 4s. Especially in salmonids, the mesenteric fat deposits are the first to be depleted 4s,75. The visceral index of brown trout decreased from 8% of total body weight to about 5% after starvation for one week 7s. A similar mechanism may be operative in fasting American eel (tt. rostrata) as well since a significantly increased [FFA] was observed in the plasma while liver and muscle lipids were not affected 69. Similarly, increased plasma [FFA] was also found in fasting plaice (Pleuronectus platessa) 13s. Lipid mobilization from liver and red muscle was observed in European eel (,4. anguilla) 21'61, rainbow trout 48,84, and carp 19,73. The lipid depletion of these tissues is much more gradual then of visceral depots and will contribute to the energy metabolism during the whole starvation period. In general, there appears to a preferential mobilization of saturated fatty acids from visceral depots while saturates and monounsaturates are derived from liver and muscle lipids. These fatty acids indicate the mobilization of triglycerides, the polyunsaturated fatty acids of the phospholipid fraction are usually retained 41'134. It is generally believed that during prolonged starvation, proteins are the major source of energy. The nitrogen loss of several tissues of common carp was found to decrease in the following order: muscle > spleen > kidney > liver > intestine 19. In particular the large mass of white muscle is believed to be a huge reservoir of energy equivalents. Protein utilization was observed in the muscles of starving eel (A. rostrata) 69 and in migrating sockeye salmon (O. nerka) 68. In both species, the 'insoluble' myofibrillar proteins were mostly affected, whereas the 'soluble' fraction was relatively unaffected. Based on changes in enzyme activities (especially alanine aminotransferase) M6ndez and Wieser 67 concluded that protein catabolism was enhanced also in fasting juvenile roach (Rutilus rutilus). Thus, amino acids may be mobilized from tissues proteins, especially in white muscle, and serve as substrates for energy metabolism either directly via catabolism in situ or via gluconeogenesis. The latter process requires inter-organ transport, since the highest gluconeogenetic activities are found in the liver. It has indeed be observed that the white muscle of migrating salmon releases relatively large amounts of alanine 68 and, moreover, gluconeogenesis from alanine was increased in hepatocytes from migrating salmon 3~ as well as from fasted rainbow trout 14. Increased protein catabolism during starvation is enabled by increased activities of proteolytic enzymes like cathepsin and other acid and neutral proteinases during migration of salmonids 3'4'68. In addition, it was found that the rate of protein synthesis in the white muscles of fasting rainbow trout and carp was decreasedl 1,53.

Endogenous fuels; non-invasive versus invasive approaches

57

In conclusion, the energy metabolism in fasting and migrating fish is fuelled primarily by amino acids and fatty acids. However, glycogen may be an additional substrate either during the first stage of starvation or only during prolonged periods of starvation.

VI. Summary Fuels are compounds that act as substrates for ATP producing pathways and can be stored to a certain extent. Fuels for anaerobic processes are ATP, PCr and glycogen. These anaerobic processes have a limited capacity, however, their survival value is very high, since they allow either a very high energy flux (exercise) for a short period, or a low energy flux during hypoxia/anoxia for a long period. Fuels for aerobic processes are sugars, lipids and proteins. During catabolism they produce NADH and FADH2 which are the ultimate substrates for the electron transport chain and mitochondrial ATP synthesis. The quantitation of fuels is fundamental to the understanding of energy metabolism. Most methods are of a destructive nature and cause as such a series of possible artifacts: stress due to handling and sampling, tissue damage, and incomplete metabolite extraction and denaturation of proteins. Most problems occur with those pathways where a high energy flux can be generated such as in muscle, i.e. it is very difficult to obtain low levels for lactate, and a high PCr/total creatine ratio. Another source of interference is where low metabolite levels occur together with high fluxes; this is found with FFA: low levels are easily disturbed due to lipolysis. Non-destructive techniques preclude most but not all problems. The most promising technique is in vivo NMR, which allows metabolite measurements without invasive or destructive actions. Particularly important is the finding that under resting conditions the phosphorylation potential of muscle is very high, resulting in >90% phosphorylation of creatine. Furthermore a review is given of the range of different fuels occurring in different tissues and different species. Obviously glycogen is quantitatively of minor importance in fish. The major energy source is protein, although lipids are in some species of equal importance. Fuel mobilization under hypoxia, exercise and starvation is discussed. Under conditions where anaerobic metabolism is activated, PCr and glycogen are the major fuels, while for long term exercise and starvation, both lipids and proteins are the predominant source of energy.

VII. References I. Alexander, K.M. A comparison of the gross chemical composition of the red and white muscles in two fishes, Scatophagus argus and Labeo rohita. I. Anim. Morph. Physiol. I: 58-61, 1955. 2. Alexis, M.N. and E. Papapareskeva-Papostoglou. Aminotransferase activity in the liver and white muscle of Mugil capito fed diets containing different levels of protein and carbohydrate. Comp. Biochem. Physiol. 83B: 245-249, 1986. 3. Ando, S., M. Hatano and K. Zama. Deterioration of chum salmon muscle during spawning migration - VI. Changes in serum protease inhibitory activity during spawning migration of chum salmon (Oncorhynchus keta). Comp. Biochem. Physiol. 82B: 11 I - I 15, 1985.

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4. Ando, S., M. Hatano and K. Zama. Protein degradation and protease activity of chum salmon (Oncorhynchus keta) muscle during spawning migration. Fish Physiol. Biochem. 1: 17-26, 1986. 5. Arthur, P.G., T.G. West, R.W. Brill, P.M. Schuite and P.W. Hochachka. Recovery metabolism of skipjack tuna (Katsuwonus pelamis) white muscle: rapid and parallel changes in lactate and phosphocreatine after exercise. Can. J. Zool. 70: 1230-1239, 1992. 6. Beamish, EW.H., J.W. Hilton, E. Niimi and S.J. Slinger. Dietary carbohydrate and growth, body composition and heat increment in rainbow trout (Salmo gairdneri). Fish Physiol. Biochem. 1: 85-91, 1986. 7. Beis, I. and E.A. Newsholme. The contents of adenine nucleotides, phosphagens and some glycolytic intermediates in resting muscle from vertebrates and invertebrates. Biochem. J. 152: 23-32, 1975. 8. Bessman, S.P. and P.J. Geiger. "l~ansport of energy in muscle: the phosphorylcreatine shuttle. Science 211: 448-452, 1981. 9. Bito, M. and K. Amano. Significance of the decomposition of adenosinetriphosphate in fish muscle around -2~ Bull. Tokai Reg. Fish. Res. Lab. 32: 149-153, 1962. 10. B6rjeson, H. and E. Fellenius. Towards a valid technique of sampling fish muscle to determine redox substrates. Acta PhysioL Scand. 96: 202-206, 1976. 11. Bouche, G. and E Vellas. Les vitesses de renouvellement des prot(~ines h~.patiques, musculaires et plasmatiques de la carpe (Cyprinus carpio) soumise a un jeflne total et prolongS. Comp. Biochem. Physiol. 51: 185-193, 1975. 12. Braley, H. and T.A. Anderson. Changes in blood metabolite concentrations in response to repeated capture, anaesthesia and blood sampling in the golden perch, Macquaria arnbigua. Comp. Biochem. Physiol. 103A: 445-450, 1992. 13. Cameron, J.N. and J.J. Cech. Lactate kinetics in exercised channel catfish, lctalurus punctatus. Physiol. Zool. 63: 909-920, 1990. 14. Canals, P., M.A~ Gallardo, J. Blasco and J. Sanchez. Uptake and metabolism of L-alanine by freshly isolated trout (Salmo trutta) hepatocytes: the effect of fasting. J. Exp. Biol. 169: 37-52, 1992. 15. Chiba, A., M. Hamaguchi, T. Tokuno, Asai, T. and S. Chichibu. Changes in high-energy phosphate metabolites in loaches (Cobitis biwae) during 2-phenoxyethanol anesthesia. Comp. Biochem. Physiol. 97C: 183-186, 1990. 16. Claireaux, G. and J.-D. Dutil. Physiological response of the Atlantic cod (Gadus morhua) to hypoxia at various environmental salinities. J. Exp. Biol 163: 97-118, 1992. 17. Connett, R.J., T.E.J. Gayeski and C.R. Honig. Lactate accumulation in fully aerobic, working, dog gracilis muscle. Am. J. Physiol. 246: 120-128, 1984. 18. Cowey, C.B. and M.J. Walton. Intermediary metabolism. In: Fish Nutrition, London, Academic Press, 1989. 19. Creach, Y. and A. Serfaty. Le je0ne et ia r~alimentation chez la carpe (Cyprinus carpio L.). J. Physiol. (Paris) 68: 245-260, 1974. 20. Dalla Via, J., M. Huber, W. Wieser and R. Lackner. Temperature-related responses of intermediary metabolism to forced exercise and recovery in juvenile Rutilus rutUis (L.) (Cyprinidae: Teleostei). Physiol. Zool. 62: 964-976, 1989. 21. Dave, G., M.L. Johansson-Sj6beck, ~ Larsson, K. Lewander and U. Lidman. Metabolic and hematological effects of starvation in the European eel, Anguilla anguiUa L., I. Carbohydrate, lipid, protein and inorganic ion metabolism. Comp. Biocher~ Physiol. 52: 423-430, 1975. 22. De Zwaan, A. and G. Van den Thillart. Low and high power output modes of anaerobic metabolism: invertebrate and vertebrate strategies. In: Circulation, Respiration, and Metabolism, edited by R. Gilles, Berlin, Springer-Verlag, pp. 167-192, 1985. 23. Diangelo, C.R. and A.G. Heath. Comparison of in vivo energy metabolism in the brain of rainbow trout, Salmo gairdneri, and bullhead catfish, lctalurus nebulosus during anoxia. Comp. Biochem. Physiol. 88: 297-303, 1987. 24. Driedzic, W.R. and P.W. Hochachka. Metabolism in fish during excercise. In: Fish Physiology Vol. VII, edited by W.S. Hoar and D.J. Randall, Academic Press, London, pp. 503-543, 1978. 25. Dunn, J.F. and P.W. Hochachka. Metabolic responses of trout (Salmo gairdneri) to acute environmental hypoxia. J. Exp. Biol. 123: 229-242, 1986. 26. Farkas, T. Examinations on the fat metabolism in fresh-water fishes, the sympathic nervous system and the mobilization of fatty acids. Ann. Inst. Biol. (Tihany), Hung. Acad. Sci. 34: 129-138, 1967. 27. Fauconneau, B. The measurement of whole body protein synthesis in larval and juvenile carp (Cyprinus carpio). Comp. Biochem. Physiol. 78A: 845--850, 1984.

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28. Fernandez-Reiriz, M.J., L. Pastoriza and G. Sampedro. Lipid changes in muscle tissue of ray (Raja clavata) during processing and frozen storage. J. Age. Food Chem. 40: 484-488, 1992. 29. Fraser, D.I., W.J. Dyer, H.M. Weinstein, J.R. Dingle and J.A. Hines. Glycolytic metabolites and their distribution at death in the white and red muscle of cod following various degrees of antemortem muscular activity. Can. J. Biochem. 44: 1015-1033, 1966. 30. French, C.J., P.W. Hochachka and T.P. Mommsen. Metabolic organization of liver during spawning migration of sockeye salmon. Am. J. Physiol. 245: R827-830, 1983. 31. From, A.H.L, S.D. Zimmer, S.P. Michurski, P. Mohanakrishnan, V.K. Ulstad, W.J. Thoma and K. Ugurbil. Regulation of the oxidative phosphorylation rate in the intact cell. Biochemistry 29: 3731-3743, 1990. 32. Gaudet, M., J.G. Racicot and C. Leray. Enzyme activities of plasma and selected tissues in rainbow trout. J. Fish. Biol. 7: 505-512, 1975. 33. George, J.C. A histophysiological study of the red and white muscle of the mackerel. Am. Midland Naturalist 68: 487-494, 1962. 34. George, J.C. and ED. Bokdawala. Cellular organization and fat utilization in fish muscle. J. Anita. Morphol. Physiol. 11: 124-132, 1964. 35. Gleeson, M. Effect of heparin and storage on human plasma free fatty acid concentration. Clin. Chim. Acta 169: 315-318, 1987. 36. Gnaiger, E. Calculation of energetic and biochemical equivalents of respiratory oxygen consumption. In: Polarographic Oxygen Sensors - Aquatic and Physiological Applications, edited by E. Gnaiger and H. Forstner, Springer-Verlag, Berlin, 337-345, 1983. 37. Gnaiger, E. Heat dissipation and energy efficiency in animal anoxibiosis: economy contra power. J. Exp. Zool. 60: 659-677, 1983. 38. Gnaiger, E. and H. Forstner (Eds.). Polarographic Oxygen Sensors - Aquatic and Physiological Applications. Springer-Verlag, Berlin, pp. 1-370, 1983. 39. Guppy, M. and P.W. Hochachka. Controlling the highest lactate dehydrogenase activity known in nature. Am. J. Physiol. 234: R136-R140, 1978. 40. Heath, A.G. and A.W. Pritchard. Effects of severe hypoxia in carbohydrate energy stores and metabolism in two species of fresh-water fish. Physiol. Zool. 38: 326-333, 1965. 41. Henderson, R.J. and D.R. Tocher. The lipid composition and biochemistry of fresh water fish. Prog. Lipid Res. 26: 281-347, 1987. 42. Hiltz, D.E, L.J. Bishop and W.J. Dyer. Accelerated nucleotide degradation and glycolysis during warming to and subsequent storage at -5~ of prerigor, quick-frozen adductor muscle of the sea scallop (Placopecten magellanicus). J. Fish. Res. Bd. Can. 31: 1181-1187, 1974. 43. Hochachka, P.W. Living Without Oxygen. Cambridge, MA, Harvard University, Press, 1980. 44. Hochachka, P.W. and G.N. Somero. Biochemical Adaptation, Princeton, NJ, Princeton University Press, 1984. 45. Hyv~irinen, H., I.J. Holopainen and J. Piironen. Anaerobic wintering of Crucian carp (Carassius carassius L.) - I. Annual dynamics of glycogen reserves in nature. Comp. Biochem. Physiol. 82: 797-803, 1985. 46. Ince, B.W. and A. Thorpe. The in vivo metabolism of 14C-glucose and 14C-glycine in insulin-treated northern pike (Esox lucius L.). Gen. Comp. Endocrinol. 28: 481-486, 1976. 47. Jayaram, M.G. and EW.H. Beamish. Influence of dietary protein and lipid on nitrogen and energy losses in lake trout, Salvelinus namaycush. Can. J. Fish. Aquat. Sci. 49: 2267-2272, 1992. 48. Jezierska, B., J.R. Hazel and S.D. Gerking. J. Fish Biol. 21: 681-692. 1982. 49. Johnston, I.A. Studies on the swimming musculature of the rainbow trout. II. Muscle metabolism during severe hypoxia. J. Fish Biol. 7: 459-467, 1975. 50. Johnston, I.A. and L.M. Bernard. Utilization of the ethanol pathway in crucian carp following exposure to anoxia. J. Exp. Biol. 104: 73-78, 1983. 51. Jonas, R.E.E. and E. Bilinski. Utilization of lipids by fish; III. Fatty acid oxidation by various tissues from sockeye salmon. J. Fish. Res. Bd. Can. 21: 653-656, 1964. 52. J6rgensen, J.B. and T. Mustafa. The effect of hypoxia on carbohydrate metabolism in flounder (Platichthys flesus L.) I. Utilization of glycogen and accumulation of glycolytic endproducts in various tissues. Comp. Biochem. Physiol. 67: 243-248, 1980. 53. Jiirss, K., I. Junghahn and R. Bastrop. The role of elongation factors in protein synthesis rate variation in white teleost muscle. Z Comp. Physiol. 162: 345-350, 1992. 54. Kamra, S.K. Effects of starvation and refeeding on some liver and blood constituents of Atlantic cod (Gadus morhua L.). J. Fish. Res. Bd. Can. 23: 975-982, 1966.

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55. Katz, A.M. and EC. Messineo. Lipid-membrane interactions and the pathogenesis of ischemic damage in the myocardium. Circ. Res. 48: 1-16, 1981. 56. Kenyon, A.J. The role of the liver in the maintainance of plasma proteins and amino acids in the eel with reference to amino acid degradation. Comp. Biochem. Physiol. 22: 169-175, 1967. 57. Knox, D., M.J. Walton and C.B. Cowey. Distribution of enzymes of glycolysis and gluconeogenesis in fish tissues. Mar. Biol. 56: 7-10, 1980. 58. Krueger, H.M., J.B. Saddler, G.A. Chapman, l.J. Tinsley and R.R. Lowry. Bioenergetics, exercise, and fatty acids of fish. Am. Zool. 8: 119-129, 1968. 59. Kutty, M.N. Respiratory quotient and ammonia excretion in Tilapia mossambica. Mar. Biol. 16: 126-133, 1972. 60. Lackner, R., W. Wieser, M. Huber and J. Dalla Via. Responses of intermediary metabolism to acute handling stress and recovery in untrained and trained Leuciscus cephalus (Cyprinidae, Teleostei). J. Exp. Biol. 140: 393-404, 1988. 61. Larsson, A. and K. Lewander. Metabolic effects of starvation in the eel, Anguilla anguiUa L. Comp. Biochem. Physiol. 44: 367-374, 1973. 62. Leach, G.J. and M.H. Taylor. The effects of cortisol treatment on carbohydrate and protein metabolism in Fundulus heteroclitus. Gen. Comp. Endocrinol. 48: 76-83, 1982. 63. Lennard, R. and H. Huddart. The effects of hypoxic stress on the fine structure of the flounder heart (Platichthys flesus). Comp. Biochem. Physiol. 101: 723-732, 1992. 64. Makinodan, Y., H. Toyohara and S. Ikeda. Comparison of muscle proteinase activity among fish species. Comp. Biochem. PhysioL 79: 129-134, 1984. 65. Mazeaud, E Recherches sur la R~gulation des AcMes Gras Libres Plasmatiques et de la Glyc~mie chez lez Poissons. Ph.D. Thesis, Paris, 1973. 66. McLaughin, R.L. and D.L. Kramer. The association between amount of red muscle and mobility in fishes: a statistical evaluation. Env. BioL Fish. 30: 369-378, 1991. 67. M6ndez, G. and W. Wieser. Metabolic responses to food deprivation and refeeding in juveniles of Rutilus rutilus (Teleostei: Cyprinidae). Env. Biol. Fish. 36: 73--81, 1993. 68. Mommsen, T.P., C.J. French and P.W. Hochachka. Sites and patterns of protein and amino acid utilization during the spawning migration of salmon. Can. J. Zool. 58: 1785-1799, 1980. 69. Moon, T.W. Metabolic reserves and enzyme activities with food deprivation in immature American eels, Anguilla rostrata (LeSueur). Can. J. Zool. 61: 802-811, 1983. 70. Moon, T.W. and I.A. Johnston. Amino acid transport and interconversion in tissues of freshly caught and food-deprived plaice, Pleuronectus platessa L. J. Fish BioL 19: 653-663, 1981. 71. Moore, K.H. Fatty acid oxidation in ischemic heart. Mol. Physiol. 8: 549-563, 1985. 72. Murgatroyd, ER., B.J. Sonko, A. Wittekind, G.R. Goldberg, S.M. Ceesay and A.M. Prentice. Non-invasive techniques for assessing carbohydrate flux: I. Measurement of depletion by indirect calorimetry.Acta Physiol. Scand. 147: 91-98, 1993. 73. Nagai, M. and S. Ikeda. Carbohydrate metabolism in fish I. - Effects of starvation and dietary composition on the blood glucose level and the hepatopancreatic glycogen and lipid contents in carp. Bull Jap. Soc. Sc. Fish. 37: 404-409, 1971. 74. Nagai, M. and S. Ikeda. Carbohydrate metabolism in fish. IV. - Effect of dietary composition on metabolism of acetate-U-14C and l-alanine-U-14C in carp. Bull. Jap. Soc. Sc. Fish. 39: 633-643, 1973. 75. Navarro, I., J. Guti~.rrez and J. Planas. Changes in plasma glucagon, insulin and tissue metabolites associated with prolonged fasting in brown trout (Salmo trutta fario) during two different seasons of the year. Comp. Biocher~ Physiol. 102A: 401--407, 1992. 76. Nelson, J.A. Muscle metabolite response to exercise and recovery in yellow perch (Percaflavescens): comparison of populations from naturally acidic and neutral waters. Physiol. Zool. 63: 886-908, 1990. 77. Nowlan, S.S. and W.J. Dyer. Glycolytic and nucleotide changes in the critical freezing zone, -0.8 to -5~ in prerigor cod muscle frozen at various rates. J. Fish. Res. Bd. Can. 26: 2621-2632, 1969. 78. Ottolenghi, C., A.C. Puviani and L. Brighenti. Glycogen in liver and other organs of catfish (Ictalurus melas): seasonal changes and fasting effects. Comp. Biochem. Physiol. 68: 313-321, 1981. 79. Pagnotta, A. and C.L. Milligan. The role of blood glucose in the restoration of muscle glycogen during recovery from exhaustive exercise in rainbow trout (Oncorhynchus mykiss) and winter flounder (Pseudopleuronectes americanus). J. Exp. Biol. 161: 489-508, 1991. 80. Partmann, W. Post-mortem changes in chilled and frozen muscle. J. Food Sci. 28: 15-27, 1963. 81. Pequin, L. and A. Serfaty. Eexcretion ammoniacale chez un t61(~ost6en dulcicole Cyprinus carpio. Comp. Biochem. Physiol. 10: 315-324, 1963.

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82. Pritchard, A.W., J.R. Hunter and R. Lasker. The relation bewteen exercise and biochemical changes in red and white muscle and liver in the jack mackerel, Trachurus symmetricus. Fish. Bull. 69: 379-386, 1971. 83. Reddy, EK.R., P.V. Reddy and N. Chari. Metabolic differentiation of cardiac, pectoral and caudal muscles of the fish Channa striatus (Bloch). Ind. J. Exp. Biol. 18: 420-421, 1980. 84. Robinson, J.S. and J.E Mead. Lipid absorption and deposition in rainbow trout (Salmo gairdneri). Can. J. Biochem. 51: 1050-1058, 1973. 85. Singer, A.M. Quantitative fine structural diversification of red and white muscle fibres in cyprinids. Env. Biol. Fish. 33: 97-104, 1992. 86. Savabi, E Free creatine available to the creatine phosphate energy shuttle in isolated rat atria. Proc. Natl. Acad. Sci. USA 85: 7476-7480, 1988. 87. Scarabello, M., G.J.F Heigenhauser and C.M Wood. Gas exchange, metabolite status and excess post-exercise oxygen consumption after repetitive bouts of exhaustive exercise in juvenile rainbow trout. J. Exp. Biol. 167: 155-169, 1992. 88. Scarabello, M., C.M. Wood and G.J.E Heigenhauser. Glycogen depletion in juvenile rainbow trout as an experimental test of the oxygen debt hypothesis. Can. J. Zool. 69: 2562-2568, 1991. 89. Schulte, P.M., C.D. Moyes and P.W. Hochachka. Integrating metabolic pathways in post-exercise recovery of white muscle. J. Exp. Biol. 166: 181-195, 1992. 90. Sen, EC., A. Ghosh and J.J. Durra. Sci. Food Agric. 27: 811-818, 1976. 91. Sheridan, M.A. Lipid dynamics in fish: aspects of absorption, transportation, deposition and mobilization. Comp. Biochem. Physiol. 90: 679-690, 1988. 92. Shoubridge, E.A. and P.W. Hochachka. Ethanol: a novel end-product of vertebrate anaerobic metabolism. Science 209: 308-309, 1980. 93. Shoubridge, E.A. and E W. Hochachka. The integration and control of metabolism in the anoxic goldfish. Mol. Physiol. 4: 165-195, 1983. 94. Sidell, B.C., D.B. Stowe and C.A. Hansen. Carbohydrate is the preferred metabolic fuel of the hagfish (Myxine glutinosa) heart. Physiol. Zool. 57: 266-273, 1984. 95. Smith, M.J. and A.G. Heath. Responses to acute anoxia and prolonged hypoxia by rainbow trout (Salmo gairdneri) and mirror carp (Cyprinus carpio) red and white muscle: use of conventional and modified metabolic pathways. Comp. Biochem. Physiol. 66B: 267-272, 1980. 96. Soivio, A., K. Nyholm and M. Huhti. Effects of anaesthesia with MS 222, neutralized MS 222 and benzocaine on the blood constituents of rainbow trout, Salmo gairdneri. J. Fish Biol. 10: 91-101, 1977. 97. Soivio, A., K. Nyholm and K. Westman. A technique for repeated sampling of blood of individual resting fish. J. Exp. Biol. 63: 207-217, 1975. 98. Storey, K.B. Metabolic consequences of exercise in organs of rainbow trout. J. E~. Zool. 260: 157-164, 1991. 99. Suzuki, T, T Hirano and T Shirai. Distribution of extractive nitrogenous constituents in white and dark muscles of fresh-water fish. Comp. Biochem. Physiol. 96:107-111, 1990. 100. Takeuchi, M. and S. Ishii. Biochemical studies on wintering of culture carp - II. Changes of oil content, fatty acid composition and glycogen content in the muscle and hepatopancreas. Bull. Tokai Reg. Fish. Res. Lab. 59: 19-27, 1969. 101. Tang, Y. and R.G. Boutilier. White muscle intracellular acid-base and lactate status following exhaustive exercise: a comparison between freshwater- and seawater adapted rainbow trout. J. Exp. Biol. 156: 153-171, 1991. 102. Tesch, P.A., A. Thorsson and N. Fujitsuka. Creatine phosphate in fiber types of skeletal muscle before and after exhaustive exercise. J. Appl. Physiol. 66: 1756-1759, 1989. 103. Tomlinson, N. and S.E. Geiger. Glycogen concentration and post mortem loss of adenosine triphosphate in fish and mammalian skeletal muscle. A Review. J. Fish. Res. Bd. Can. 19: 9971003, 1962. 104. Turner, J.D. and C.M. Wood. Physiological consequences of severe exercise in the inactive benthic flathead sole (Hippoglossoides elassodon): a comparison with the active pelagic rainbow trout (Salmo gairdneri). J. EXp. Biol. 104: 269-288, 1983. 105. Van den Thillart, G. Adaptations of fish energy metabolism to hypoxia and anoxia. Mol. Physiol. 2: 49-61, 1982. 106. Van den Thillart, G. Energy metabolism of swimming trout (Salmo gairdneri); oxidation rates of palmitate, lactate, alanine, leucine and glutamate. J. Comp. Physiol. 156B: 511-520, 1986. 107. Van den Thillart, G. and Smit, H. Carbohydrate metabolism of goldfish (Carassius auratus L.); Effects of long-term hypoxia-acclimation on enzyme patterns of red muscle, white muscle and

62

G. van den Thillart and M. van Raaij

liver. J. Comp. Physiol. !54B: 477-486, 1984. 108. Van den Thillart, G. and A. Van Waarde. Teleosts in hypoxia: aspects of anaerobic metabolism. Mol. Physiol. 8: 393--409, 1985. 109. Van den Thillart, G. and A. Van Waarde. pH changes in fish during environmental anoxia and recovery: the advantages of the ethanol pathway. In: Physiological Strategies for Gas Exchange and Metabolism, edited by A.J. Woakes, M.K. Grieshaber and C.R Bridges, Cambridge, Cambridge University Press, pp. 173-190, 1991. 110. Van den Thillart, G. and A. Van Waarde. The role of metabolic acidosis in the buffering of ATP by phosphagen stores in fish: an in vivo NMR study. In: Surviving Hypoxia: Mechanisms of Control and Adaptation, edited by P.W. Hochachka, P.L. Lutz, M. Rosenthal and G. van den Thillart, CRC Press, Boca Raton, 1993. 111. Van den ThiUart, G., E Kesbeke and A. Van Waarde. Anaerobic energy-metabolism of goldfish, (Carassius auratus L.); Influence of hypoxia and anoxia on phosphorylated compounds and glycogen. J. Comp. Physiol. 136: 45-52, 1980. 112. Van den Thillart, G., A. Van Waarde, E Dobbe and E Kesbeke. Anaerobic energy metabolism of goldfish, Carassius auratus (L.) Effects of anoxia on the measured and calculated NAD+/NADH ratios in muscle and liver. J. Comp. Physiol. 146: 41--49, 1982. 113. Van den Thillart, G., M. Van Berge-Henegouwen and E Kesbeke. Anaerobic metabolism of goldfish, (Carassius auratus L.): ethanol and CO2-excretion rates and anoxia tolerance at 20, 10 and 5~ Comp. Biochem. Physiol. 76: 295-300, 1983. 114. Van den Thillart, G., E K6rner, A. Van Waarde, C. Erkelens and J. Lugtenburg. A flow-through probe for in vivo 31p NMR spectroscopy of unanesthetized aquatic vertebrates at 9.4 Tesla. jr. MagrL Reson. 84: 573-579, 1989. 115. Van den ThiUart, G., A. Van Waarde, H.J. Muller, C. Erkelens, A. Addink and J. Lugtenburg. Fish muscle energy metabolism measured by in vivo 31p-NMR during anoxia and recovery. Am. J. Physiol. 256: R922-929, 1989. 116. Van den Thillart, G., A. Van Waarde, H.J. Muller, C. Erkelens and J. Lugtenburg. Determination of high-energy phosphate compounds in fish muscle: 31p.NMR spectroscopy and enzymatic methods. Comp. Biochem. Physiol. 95B: 789-795, 1990. 117. Van der Boon, J., G.E.E.J.M. Van den Thillart and A.D.E Addink. Free amino acid profiles of aerobic (red) and anaerobic (white) skeleta; muscle of the cyprinid fish, Carassius auratus L. (goldfish). Comp. Biochem. Physiol. 94B: 809-812, 1989. 118. Van der Boon, J., O.P.A. Robertus, A. Staal, G.E.E.J.M. Van den ThiUart and A.D.E Addink. SDS-polyacrylamide gel electrophoresis of sarcoplasmic and myofibrillar protein from the white skeletal muscle of the anoxic goldfish, Carassius auratus L. Comp. Biochem. Physiol. 99B: 693-697, 1991. 119. Van der Boon, J., EA. Eelkema, G.E.E.J.M. Van den Thillart and A.D.E Addink. Influence of anoxia on free amino acid levels in blood, liver, and skeletal muscles of the goldfish, Carassius auratus L. Comp. Biochem. Physiol. 101B: 193-198, 1992. 120. Van der Vusse, G.J. and R.S. Reneman. The myocardial non-esterified fatty acid controversy. J. Mol. Cell. Cardiol. 16: 677-682, 1984. 121. Van Dijk, P.L.M, G. Van den Thillart, P. Balm and S.E. Wendelaar Bonga. The influence of gradual water acidification on the acid/base status and plasma hormone levels in carp. J. Fish Biol. 1-29, 1993. 122. Van Waarde, A. Biochemistry of non-protein nitrogen compounds in fish including the use of amino acids for anaerobic energy production. Comp. Biochem. Physiol. 91B: 207-228, 1988. 123. Van Waarde, A. and M. De Wilde-Van Berge Henegouwen. Nitrogen metabolism in goldfish, (Carassius auratus L). Pathway of aerobic and anaerobic glutamate oxidation in goldfish liver and muscle mitochondria. Comp. Biochem. Physiol. 72B: 133-136, 1982. 124. Van Waarde, A., G. Van den Thillart and E Kesbeke. Anaerobic energy metabolism of the European eel, AnguUla anguilla L.J. Comp. Physiol. 149B: 469-475, 1983. 125. Van Waarde, A., G. Van den Thillart, C. Erkelens, A. Addink and J. Lugtenburg. Functional coupling of glycolysis and phosphocreatine utilization in anoxic fish muscle. J. BIOL Chem. 265: 914-923, 1990. 126. Van Waversveld, J., A.D.F Addink, G. Van den Thillart and H. Smit. Anaerobic heat production measurements: a new perspective. J. Erp. Biol. 138: 529-533, 1988. 127. Van Waversveld, J., A.D.F Addink, G. Van den ThiUart and H. Smit. Heat production of fish: a literature review. Comp. Biochem. Physiol. 92A: 159-162, 1989.

Endogenous fuels; non-invasive versus invasive approaches

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128. Van Waversveld, J., A.D.F Addink and G. Van den Thillart. Simultaneous direct and indirect calorimetry on normoxic and anoxic goldfish. J. Exp. Biol. 142: 325-335, 1989. 129. Veech, R.L., J.W.R Lawson, N.W. Cornell and H.A. Krebs. Cytosolic phosphorylation potential. I. Biol. Chem. 254: 6538-6547, 1979. 130. Von der Decken, A. and E. Lied. Dietary protein levels affect growth and protein metabolism in trunk muscle of cod, Gadus morhua. J. Comp. Physiol. 162B: 351-357, 1992. 131. Waiwood, B.A., K. Haya and L. Van Eeckhaute. Energy metabolism of hatchery-reared juvenile salmon (Salmo Salar) exposed to low pH. Comp. Biochem. Physiol. 101C: 49-56, 1992. 132. Walker, R.M. and P.H. Johansen. Anaerobic metabolism in goldfish (Carassius auratus). Can. J. Zool. 55: 1304-1311, 1977. 133. Walton, M.J. and C.B. Cowey. Aspects of intermediary metabolism in salmonid fish. Comp. Biochem. Physiol. 73B: 59-79, 1982. 134. Watanabe, T Lipid nutrition in fish. Comp. Biochem. Physiol. 73: 3-15, 1982. 135. White, A. and TC. Fletcher. Serum cortisol, glucose and lipids in plaice (Pleuronectus platessa L.) exposed to starvation and aquarium stress. Comp. Biochem. Physiol. 84: 649-653, 1986. 136. Wieser, W., U. Platzer and S. Hinterleitner. Anaerobic and aerobic energy production of young rainbow trout (Salmo gairdneri) during and after bursts of activity. J. Comp. Physiol. 155B: 485-492, 1985. 137. Williamson, J.R. and B. Corkey. Assays of intermediates in the citric acid cycle and related compounds by fluorometric enzyme methods. In: Meth. Enzymol., edited by J.H. Lowenstein, New York, Academic Press, pp. 435-512, 1969. 138. Wollenberger, A., O. Ristau and G. Schoffa. Eine einfache Technik der extrem schnellen Abk0hlung gr6sserer Gewebestiicke. Pfluegers Arch. Eur. I. Physiol. 270: 399-412, 1960.

Hochachka and Mommsen (eds.), Biochemistryand molecularbiology of fishes, vol. 4 9 1995 Elsevier Science B.V. All rights reserved. CHAPTER 4

Tissue carbohydrate metabolism, gluconeogenesis and hormonal and environmental influences THOMAS W. MOON AND GLEN D. FOSTER

Department of Biology, Ottawa-Carleton Institute of Biology, University of Ottawa, Ottawa, Ontario KIN 6N5, Canada

I. II.

Introduction Liver 1. The general organization of hepatic metabolism 2. Environmental adaptations 2.1. Fasting 2.2. Hypoxia 2.3. Temperature 2.4. Stress 2.5. Seasonality 3. Hormone signal transduction pathways III. Kidney 1. The organization of kidney metabolism 2. Environmental adaptations IV. Skeletal muscle and heart 1. Myotomal muscle 2. Cardiac muscle V. Brain VI. Red blood cells VII. Conclusions VIII. References

I. Introduction Carbohydrates are key to the metabolism of all vertebrates, including fish species. The diversity of lifestyles and habitats selected by fish has resulted in significant differences in the way species handle and partition dietary carbohydrates within their bodies. Few generalities can be presented, and the reader is directed to Love 9~ for a review of some of the issues which may be involved. Early studies by Leibson 87, Plisetskaya and Kuz'mina 134, and Palmer and Ryman 123 introduced the ideas of motor activities affecting carbohydrate disposition and glucose intolerance, but papers since have generally lost the comparative perspective which is critical to our understanding these species differences. The idea that at least carnivorous fishes (e.g. salmonids) have a poor tolerance to carbohydrate has been revisited

66

T.W. Moon and G.D. Foster

recently 133,177. It is clear that this is an issue in the metabolic biochemistry of fishes, but more information is needed. In particular, the sensitivities of hormonal release to circulating carbohydrate and the specific tissue hormone receptors and species with non-carnivorous life styles need to be examined. Although this review will mention a variety of species, many have similar nutrient requirements and a strong comparative approach is needed before an understanding of this issue will be possible. It remains unclear whether any fish tissues require or use preferentially carbohydrate as a source of energy. The red blood cell and kidney cortex of mammals have a strict glucose requirement based upon their anaerobic metabolic profile. Fish red cells are nucleated and may contain a few mitochondria, and it is reported that 90% of the resting nucleoside triphosphate is produced aerobically 33. Red cells of the sea raven (Hemitripterus americanus) also have an aerobic metabolism fueled by exogenous glucose x46. Lamprey (Petromyzon marinus) brain tissue minces do utilize glucose 4s and the trout brain is thought to use primarily exogenous glucose to supplement its low endogenous glycogen reserves 27. Glucose/glycogen is critical for the maintenance of cardiac performance in the Atlantic hagfish (Myxfne glutinosa) 151. Yet in no case is the quantitative significance of carbohydrates in overall tissue metabolism clearly defined. The ability of tissues to utilize a particular nutrient is dependent on the permeability of the cell to it and its cellular metabolism to ensure adequate membrane gradients. Carbohydrates such as glucose and lactate poorly penetrate the hydrophobic cell membrane, and transport is generally carrier-mediated. Fish tissues such as red cells and liver, with few exceptions, show non-saturable uptake kinetics with respect to these metabolites (red cells 16~ liver171). Hexokinase (HK) activities, the enzyme responsible for glucose phosphorylation and the maintenance of the membrane gradient for glucose, are generally low in fish tissues (see Tables 2-8), and although there is some evidence for its modulation by diet 37, there is no evidence for a mammalian-type glucokinase in fish. Unfortunately, our knowledge of glucose transporters in fish tissues is poor, with the possible exception of red cells 3s,lss, an area which could help identify the mechanism of 'glucose intolerance' in fish. Fish tissues do contain glycogen in varying quantities (Table 1) indicating that the inability of tissues to take-up glucose or maintain plasma glucose content is compensated for by other metabolic pathways. Gluconeogenesis is the pathway responsible for de novo glucose and glycogen synthesis (glyconeogenesis) from precursors including lactate, amino acids, glycerol, and fructose 1~ The importance of this pathway to tissue carbohydrate homeostasis in fish has been the subject of several recent reviews97,1~ The high dietary protein requirements of fish9~ should provide adequate substrate for this pathway and there is extensive evidence for the modulation of gluconeogenic rates by intrinsic and extrinsic factors. The prevalence of proteins in the carnivorous diet and their use as an energy source together with the generally lowered energy demands of fish n4, may have alleviated the strict need for carbohydrate as a key energy source to many fish species. The

Tissue carbohydrate metabolism, gluconeogenesis and hormonal and environmental influences

67

TABLE 1 Glycogen contents of various fish tissues Species/Condition

Liver

Hagfish fed fasted; 1 month fasted; 4 months anoxia

15.039 1.8 4.7 -

Skate

6311~

Crucian carp winter/normoxic summer Catfish winter/fed fasted; 60 days spring anoxic

Kidney

3039 0.72 36

Red muscle -

Heart

Brain

2257 0.9557

9.011~

150066,117 5566 600118 8012~ 67 31

White muscle

167117 22 _ 3.7 2.4 n

19118 4.2120 1.7 3.3 D

140118 12.912o 7.3 7.9 _

211118 4212o 30 23 _

1627 _

627

Perch fed fasted; 7 weeks Flounder normoxic hypoxic Tuna American eel fed fasted, 6 months Rainbow trout normoxic anoxic

62145 182 6476 18 4178 49 l~ 53 3526, 2000 u

15.345 10.8 45 TM

16TM 4

36

93178

24178

121o6 7

141o6 11

5.126 _

_

_

_

_

_

10136 3.027 1.527

Values are given in/zmol glucosyl units g-I tissue. These are representative values and not intended to be comprehensive. Superscript numbers represent reference numbers. If values are not followed by superscript numbers, reference is identical to that located above in the same column.

role of carbohydrates may, therefore, be for short-term responses to acute stress situations 19 and/or as a last resort. The purpose of this review, therefore, is to provide an overview of recent studies on carbohydrate metabolism in fish using a tissue approach. An overriding thesis in the review is that carbohydrate metabolism has a significant role in fish, and that this function becomes more understandable when considered in light of perturbations of the system. The question of whole fish carbohydrate homeostasis will be examined only in terms of its importance to the individual tissue. An attempt will be made to update those previous reviews on fish metabolism (e.g. refs. 19, 61, 176), and by doing so, to identify those areas which need further research.

68

T.W.Moon and G.D. Foster

II. Liver The liver is a central organ of metabolism and is key to the regulation of carbohydrate metabolism in all vertebrate classes. A huge literature is available and it is not our intent to be all-inclusive, but to select those areas of recent interest.

1. The general organization of hepatic metabolism The ability of fish to metabolize carbohydrate and the enzymes required for this purpose are clearly understood. A general listing of recently published enzyme activities in fish livers is provided on Table 2. A complete enzyme profile is not available for each fish, but as methods become more available, such holes will disappear. The overall importance of carbohydrate metabolism in the fish liver as well as the relative importance of glycolysis and gluconeogenesis, however, are less well defined. For instance, hepatectomy in the fiver lamprey (Lampetra fluviatilis) s6 and the Pacific hagfish (Eptatretus stouti) 69 affected neither survival nor basal blood glucose concentrations, leading to the suggestion that liver carbohydrate metabolism may not be critical to whole body carbohydrate homeostasis. These studies, however, did not consider the ability of these species to tolerate environmental stress, such as hypoxia, exercise, fasting, or temperature changes, nor do they explain the ubiquity of carbohydrate-metabolizing pathways in fish livers. We suggest that while the importance of carbohydrate metabolism in the whole animal energy budget may be less than in other vertebrate classes, carbohydrate metabolism is critical and becomes so during adaptive responses to the environment. Liver glycogen contents are extremely variable in fish (Table 1). Agnathans generally have levels below 20/zmol g-l, while elasmobraneh and teleost values range from 20 to 2000/zmol g-1. Measured glycogen values for fed rainbow trout (Oncorhynchus mykiss) range from 35 (ref. 26) to 2000 (ref. 84)/zmol g-1. This variability may reflect sampling procedures, strain and/or life history differences, or even the method of analysis. The Crucian carp (Carassius carassius) has liver glycogen concentrations above 1500/zmol g-1 liver, and during periods of high glycogen content, the liver may reach 15% of body weight66. This gives a total liver carbohydrate store of over 20,000/zmol 100 g-1 body weight, or 4% of body weight! A similar analysis using other teleosts gives values no higher than 1500/zmol g-1 body weight (0.3%). While other teleosts do not demonstrate these high amounts of liver glycogen, the liver still contains as much as 50% of the whole animal glycogen stores, assuming skeletal muscle contains the rest. The hormones glucagon, glucagon-like peptide (GLP), and catecholamines all increase gluconeogenesis and glycogenolysis in a variety of species by inhibition of pyruvate kinase (PK) and phosphofructokinase-1 (PFK-1) activities and increasing phosphoenolpyruvate earboxykinase (PEPCK) and glycogen phosphorylase (GPase) activities 23'42'44'47'56'98'100'102'103'118'119'130'167.These enzymes have all been reported in most species studied (Table 2), and most are controlled by reversible phosphorylation-dephosphorylation mechanisms. The vasoactive peptides vasotocin and isotocin also increase glycogenolysis and glueoneogenesis in three species

Tissue carbohydrate metabolism, gluconeogenesis and hormonal and environmental influences

69

where they have been tested, with the effects very pronounced in the American eel (Anguilla rostrata) 111. Elevated plasma cortisol concentrations stimulate gluconeogenesis in some teleosts, possibly by increasing the availability of gluconeogenic substrates (amino acids) and activation of specific liver enzymes 1~8. It is not clear to this point whether this is a direct affect of cortisol or it is mediated by other glucoregulatory hormones 169. Insulin, the only hypoglycemic hormone in mammals, acts to counteract glucagonstimulated gluconeogenesis and glucose production 65, with little or no effect on basal (no hormone) rates. This situation with insulin-counteracting the effects of glucagon on gluconeogenesis and glycogenolysis has been reported for American eel 42 and sea raven 41'46 hepatocytes. Petersen et al. 130, however, reported an inhibition of basal (no hormone) gluconeogenic flux by insulin in trout hepatocytes, with a concurrent inhibition of PK activity. This inhibitory effect of insulin in the absence of glucagon has been found in no other teleost species nor in mammals where PK is unaffected 65. In fact, insulin actually increased gluconeogenic flux in hepatocytes isolated from sea raven 41,46, American eel 42, and hagfish 49. These actions of insulin occurred through an enhanced inhibitory effect of ATP on PFK-1 in all three species. No effects of insulin on PK and PEPCK were found in sea raven hepatocytes. Insulin also decreased glucagon-stimulated glycogenolysis and decreased glycogenolysis below basal (no hormone) levels in both sea raven and American eel hepatocytes 41,42. It is apparent that the actions of insulin differ between species and with respect to the carbohydrate pathways in the liver. Mammalian hepatocytes show metabolic heterogeneity such that periportal cells catalyze glucose release and gluconeogenesis while perivenous cells preferentially take up glucose for glycogenesis and lipogenesis 77. No evidence for this mammalian form of heterogeneity has been found in the catfish 121 or the trout 99 liver. However, density gradient separation of cell types demonstrated more oxidative and gluconeogenic scope existed in the less dense cells of trout liver 99, and greater overall enzyme activities in the less dense cells of the Gulf toadfish (Opsanus beta) 1~ An interesting finding in the toadfish study was that although enzyme activities were higher in the less dense cells, the heavier cells exhibited 2.5-4 times more metabolic activity than the less dense population. Similarly, the distinctively large lobe of the hagfish liver was found to be more metabolically active than the small lobe 49. Metabolic heterogeneity may exist in fish livers, although rather than a segregation of metabolic pathways existing, as in the mammal, the heterogeneity may be more subtle or simply reflect differential overall metabolic activities of the cells. A review of substrate utilization has been published previously 1~ and the general principles will not be covered here. This section will concentrate on recent findings that relate to an understanding of the fate of certain substrates, as well take a comparative look at some interesting differences found in some species. Lactate is generally the most readily utilized substrate in fish liver 1~ leading to the suggestion that the liver is important in the Cori cycle 157 (Cori cycle is: muscle lactate ~ liver glucose ~ muscle glycogen). However, in starry flounder (Platichthys stellatus) 96 , American eel 21 and skipjack tuna (Katsuwonus

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Tissue carbohydrate metabolism, gluconeogenesis and hormonal and environmental influences

71

pelamis) 178, in vivo lactate turnover studies showed no more than 5% of the lactate label was incorporated into blood glucose, indicating low Cori cycle activity. The fate and function of lactate and other C-3 molecules in the blood remains unclear, other than their use as oxidative substrates for immediate energy production in peripheral tissues. Recent studies of the hormonal regulation of hepatocyte metabolism may shed some light on this question. As discussed above, increased flux of C-3 precursors such as lactate and alanine to C-6 endproducts resulted when isolated hepatocytes of hagfish, sea raven, and American eel hepatocytes were exposed to insulin, and these observations led to the hypothesis that a major role of liver carbohydrate metabolism is to convert C-3 substrates available from the diet either to liver energy stores or to glucose for export to peripheral tissues for storage (see refs. 41, 46). Thus, rather than the liver functioning as an intermediate organ in maintaining carbon cycling, it may function more to provide a reservoir of energy stores, namely glycogen. As such, the use of exogenous substrates and the regulation of their use will be directed more towards energy storage than towards blood euglycemia. This issue is called the 'glucose paradox' or the indirect pathway for glycogen production and has recently been reviewed 14a. Unfortunately, this issue is complicated by isotope methodology, and it is not clear what the contribution of this pathway compared to the direct pathway (glucose to glycogen) is to glycogen production. The fish system may be a good model to test these ideas. Lactate use by hepatocytes is generally greater than other substrates, although there are some notable exceptions. In sea raven hepatocytes, lactate flux to glucose is lower, if present at all, than the flux of serine and alanine 41,174, and in skate (Raja erinacea) 1~ and skipjack tuna 16 hepatocytes, lactate conversion rates are lower than for alanine. Low lactate utilization in sea raven hepatocytes may be due to the cytoplasmic/mitochondrial localization of PEPCK ls7 or to low LDH titers in the liver 174 (Table 2). PEPCK activities in sea raven liver, however, are 100% mitochondria1188; thus, there are no significant redox problems and lactate oxidation is as high as alanine and serine oxidation 41, indicating active flux through LDH. Furthermore, the LDH isozyme pattern in sea raven liver is not different from other teleosts 188. The low use of lactate in skipjack tuna 16 and skate 1~ suggests: (1) an inactive Cori cycle, with the liver function directed towards the conversion of amino acids (dietary source or muscle catabolism) to endproducts in the liver rather than the liver functioning as an intermediate cycling organ; and (2) elasmobranchs may have a minimal reliance on carbohydrate metabolism, using fats and ketone bodies to a greater extent. As such, the use of lactate (carbohydrate) may be reduced. At this time, there is no clear understanding of why species have particular preferences for substrates, but experiments on transport characteristics may assist in a better understanding of these differences.

2. Environmental adaptations Liver metabolism not only demonstrates species differences and substrate preferences, but also responds to a variety of environmental perturbations. Again, this

72

T.W.Moon and G.D. Foster

review is not intended to cover all aspects of this topic, but to select those aspects which focus on carbohydrate changes and update previous reviews 11e.157.

2.1. Fasting Enzyme changes and flux studies with fasting generally reflect a stimulation of gluconeogenesis 39,45,46,54,1~ Increased glucose production in isolated hepatocytes from fasted fish is sometimes found 46, although not always 45. The decreased glucose production is probably due to the confounding fact of the negative glycogen balance found in isolated hepatocytes 97,112 and the general decrease in liver glycogen levels found in fish (e.g. refs. 45, 46, 54, 115, 147, 169) which will decrease glycogenolysis43,97. GPase activities are generally unaltered during a fast in eel, trout, or yellow perch 42,45,1~ although GSase activities decreased in fasted trout 169. The use of glycogen during a fast appears to be species-specific. There are different strategies for dealing with a fast, some of which include maintaining glycogen levels at the expense of protein and/or lipid9~ As noted on Table 1, some species demonstrated significant changes in liver glycogen with fasting (perch, hagfish), while others do not (American eel, catfish). Hansen and Abraham 5s showed that fasting shifted the pattern of metabolite incorporation in vivo, favoring gluconeogenesis from alanine and serine. These experiments indicate the importance of both in vivo and in vitro approaches to the study of fish liver metabolism during fasting. While enzyme activities and flux demonstrate stimulated gluconeogenic activity as a function of tissue weight, analysis of the data in terms of total liver potential shows a decreased metabolic activity in the livers of yellow perch and trout, thus indicating a hypometabolic response to fasting45. A study of sea raven metabolism, however, showed that following a 6 week 46 or 8 week 169 fast, hepatocyte flux was not lowered after taking into account changes in the hepatosomatic index. Therefore, the hypometabolic response to fasting is not a general feature of all teleosts, but has been shown in only a few species. Dormancy or metabolic depression have been shown in a few fish species as a function of low temperatures ee,173, but its precise mechanistic basis has yet to be identified. Titers of insulin, glucagon and glucagon-like peptide (GLP) generally decrease during a fast in a variety of fish species 54,55,6~176 although in some of these species the glucagon or GLP to insulin ratio(s) increased 54,1~ indicating a hormonal profile suitable for stimulated gluconeogenesis. Most studies have employed endpoint quantification of hormones, but it is clear that hormone titers vary considerably during the fasting period and depending upon the time of year the experiments are conducted 1~ Guti6rrez and Plisetskaya 55 showed that following a 40 day fast, insulin binding capacity to the salmon liver was increased, but the affinity to the membranes was actually decreased. They concluded, therefore, that specific binding of insulin to the liver cell is unchanged following a fast of this duration even though insulin titers decreased. The additional complication of annual cycles 53 and of diet and feeding times 127 on plasma insulin and glucose must also be considered when designing these experiments, but seldom are. Glucagon receptors have recently been identified in bullhead (Ictalurus nebulosus) and eel

Tissue carbohydrate metabolism, gluconeogenesis and hormonal and environmental influences

73

hepatocytes 189, but how these are affected by nutritional status is not known. GLP binding has yet to be demonstrated in any fish system. Foster and M o o n 46 showed that the sensitivity of sea raven hepatocyte carbohydrate metabolism to insulin was unchanged following a 6 week fast, although glucagon sensitivity was increased. They concluded that this increased sensitivity allowed the hepatocyte to respond to the lower levels of glucagon presumably present in the circulation, while the unchanged sensitivity to insulin reflected a reduced metabolic impact of insulin. This study is in contrast to that reported by Klee et a1.82 which showed that glycogenolysis could be induced by epinephrine but not glucagon in trout fasted for 3 weeks; both hormones stimulated glycogenolysis in 1 week fasted trout. These studies suggested that at least glucagon may be critical only at certain times in the fasting process, and this could be related to the binding of this hormone to sites on the hepatocyte. Further studies to identify receptor binding and the physiological consequences of fasting on these parameters is needed before the known physiological changes in hormone titers can be interpreted at the tissue level. The duration of the fast has an affect on the liver changes that are measured, but few studies have followed these changes. Rainbow trout exhibited an initial increase in PEPCK and FBPase activities in the early stages of a fast, while in the later stages activities of these enzymes declined ~3. Navarro and her colleagues ~5 showed an initial (3-5 day) attempt to maintain plasma glucose levels in brown trout (Salmo trutta), followed by hypoglycemia. These changes were accompanied by an initial increase in the glucagon" insulin ratio, which disappeared at the later stage of the fast. Other studies by the group of Guti6rrez 9,1~ have followed plasma hormone and metabolite concentrations during the fasting period. These carp and trout studies suggested at least 2 fasting stages; an initial stage with few changes followed by a later stage with major changes. Their studies showed that the timing of these stages are species dependent, but the experiments are key as they suggest that the length of the fast is critical in determining the physiological response. Canals et al. 17 showed an increased alanine oxidation in isolated trout hepatocytes following a 2 week fast, but at 3 weeks this parameter was decreased below initial values. It is probable that there is an initial hypermetabolism in the early stages of a fast followed by a hypometabolic response to longer term fasts (see above). The fact that glycogen levels decrease in a fast, but do not completely disappear suggest that glycogen may be liberated in the very first phase of the fast, when glucagon concentrations are elevated 115. The levels will then stabilize when the fast becomes extended and glucagon titers decrease. It may be expected that in the last phases of a long term fast carbohydrate stores will finally be liberated, after lipid and protein pools are exhausted. Re-feeding results in a number of changes in the liver, most of which reflect a repletion of glycogen reserves. For example, re-fed cod (Gadus morhua) showed an overshoot in glycogen stores 8, and catfish (Rhamdia hilarii) liver slices showed an increased flux of 14C-glucose to glycogen91. These anabolic effects are consistent with an increased specific binding of insulin to liver membranes in salmon 55. Tilapia (Oreochromis [Sarotherodon] mossambicus) showed a less pronounced recovery of

74

T.W. Moon and G.D. Foster

liver glycogen levels, with the reserves still below fed values even after 30 days of re-feeding 13s. The mechanisms involved in glycogen recovery are not clear. In tilapia liver, GPase a activities were reduced during the fast, and remained low at 15 days of re-feeding. However, activities of this enzyme had recovered to control values even though the glycogen stores had not fully recovered 13a. Re-feeding mudskipper showed no change in the GPase %a activities, although total activities increased 88. It is probable that more is involved than simple changes in enzyme activities in the recovery of glycogen reserves. Blasco and collaborators 9 have reported that the changes noted in plasma hormones during fasting are returned to normal by 12 days of re-feeding in carp, concomitant with a complete recovery of liver reserves. Certainly the re-establishment of hormone titers are involved in these progresses, and the study by Sheridan and Mommsen 147 demonstrated that at least insulin, glucagon and GLP returned to prefasted levels within 2 weeks of re-feeding. Given that plasma insulin and glucagon levels peak within 2 h post-feeding in brown trout ~4, it is clear that fish can respond rapidly to changes in their nutritional state. 2.2. Hypoxia Numerous fish species experience prolonged periods of low oxygen availability and/ or complete anoxia. Furthermore, specific tissues may experience hypoxia during periods of strenuous exercise. The response to low oxygen availability (hypoxia) is either metabolic depression (e.g. the mudskipper) is and/or the anaerobic use of carbohydrates, and the response can be tissue-specific ls4. Hypoxic plaice demonstrated reduced serum glucose 18~ but blood glucose was maintained in trout through catecholamine inhibition of PK and stimulation of GPase, effects that were blocked in the presence of propranolo1184. Claireaux and Dutil 2~ also implicated catecholamine release with hypoxia in cod. However, Dunn and Hochachka 29 found no increase in glucose turnover in 3 h hypoxie trout, although lactate turnover was increased. Anoxia did not change glucose production in liver pieces of trout or bullheads s9, suggesting the absence of a Pasteur effect and the existence of metabolic depression. Consistent with these effects, liver from anoxic goldfish showed decreased OPase activities and decreased fructose 2,6-bisphosphate levels 154, again suggesting metabolic depression and the absence of a Pasteur effect. As this metabolic depression was not apparent in all tissues 59,1s4, the whole animal does not necessarily go hypometabolic. Instead, the metabolic depression was specific to the fiver, implicating an active protection of liver glycogen stores during anoxia/hypoxia. The protection of liver glycogen stores has been reported in air-exposed American eels (Moon et al., in preparation), hypoxic cod 2~ the anoxic Crucian carp 117, and 1 h hypoxic mudskippers (Channa punctata) 1st, although liver glycogen decreased in the anoxic flounder 76 (see Table 1). Activities of liver GPase are key to these changes. GPase activities declined and glycogen was stable with air-exposure, but by 3 days of air exposure, glycogen levels in American eel fiver drop dramatically, GPase a activities rose, liver lactate levels increased and eels begin to die (Moon et al., in preparation). Death in the anoxia-tolerant Crucian carp (after 18 days of anoxia) was correlated with near zero content of liver glycogen 117. Again, there was a reciprocal relationship between liver glycogen content and GPase activities

Tissue carbohydrate metabolism, gluconeogenesis and hormonal and environmental influences

75

in carp 66. Long term hypoxia (3 days) in the mudskipper also resulted in decreased liver glycogen reserves, although GPase activities were not affected ls6. Furthermore, the survival time under anoxia is dependent on the initial liver glycogen reserves TM, and these levels showed seasonal fluctuations 66. At least in the carp and goldfish, extended anoxia is associated with altered glycolytic endproducts (ethanol, acetic acid) 63. The protection of glycogen may be necessary to maintain a readily utilizable energy source during prolonged hypoxia/anoxia (as was suggested for fasting, see above) in order to respond to the superimposition of another (secondary) stress. Re-submergence experiments (Moon et al., in preparation) showed a rapid rise in liver glycogen content in eels, further supporting the importance of liver glycogen protection and maintenance; similar studies have not apparently been done with the carp. van den Thillart and Verbeek 166 correlated an oxygen debt during recovery from anoxia to a replenishment of glycogen pools in trout. It may be postulated that liver glycogen is maintained as a last-resort fuel, and depletion of this reserve results in death. Work is required to understand why depletion of this reserve may result in death and why liver glycogen is protected during early stages of anoxia/hypoxia. 2.3. Temperature Some fish species thermally compensate metabolic activity at decreased temperatures by increasing enzyme activities, and this is thought to be a major aspect of temperature acclimation 62,78-a~ Furthermore temperature-dependent effects on specific enzymes, such as PK and PFK-1, reflect stimulated pathway flux1,62. Recent studies have examined effects of temperature on in vitro liver metabolism and how changes in in vivo hormone titers may affect liver metabolism. Total glucose production in trout liver pieces showed some thermal compensation, but not in bullheads 59. Seibert 143 found glucose production in the absence of glycogen depletion in trout hepatocytes at low temperatures, while as the incubation temperature increased glycogen depletion rates met and eventually exceeded glucose production rates. Thus, thermal compensation of trout hepatocyte glucose production may be through gluconeogenesis, as low incubation temperatures inhibit the in vitro depletion of glycogen. A similar study must be performed on non-compensating species to see if such changes are really adaptive. Sea raven hepatocytes showed an exceptionally large Q10 value (Q10=29) for alanine gluconeogenesis between 10 and 2"C, indicating an inhibited gluconeogenic potential at lower temperatures 174, which is inconsistent with the observations on trout hepatocytes 143, but this species may not compensate. A number of studies have examined the relationship between temperature and nutritional and seasonal effects. Walsh and colleagues 173 compared 15*C-fed eel liver enzymes to the same enzymes from eels adapted to 5, 10 and 15"C but fasted for 6 months. They reported that enzymes and metabolite reserves were similar between the 5*C-fasted and 15~ eels, but significantly lower in the 10 and 15*C-fasted fish. These studies concluded that torpor (metabolic depression) can have a significant metabolic impact on an animal's energetics and that thermal compensation need not occur, nor may it be relevant in all species. Studies with the golden ide (Leuciscus idus melanotus) found that liver glycogen stores increased at

76

T.;E.Moon and G.D. Foster

low temperatures compared to lipids if ide were fed a natural diet 137. The question raised by this study is that of natural versus artificial diets and this aspect has not been adequately addressed in fish. Blasco et al.ll reported in carp that circulating glucagon was elevated at high acclimation temperatures (28~ compared to 15~ controls. In association to this, liver (and brain) glycogen levels were reduced; no changes, however, were found with low acclimation temperatures. They concluded that glucagon is important in the mobilization of carbohydrates during a high temperature stress. Different resuits were found with the sea bream (Chrysophrys major), a warm-water species, where both liver glycogen and hepatosomatic index increased at high acclimation temperatures lsl. Prosser and coworkers 136attempted to identify the hormonal mechanisms involved in temperature adaptations in catfish hepatocytes. They found greater rates of protein synthesis at high temperatures were inhibited by thyroxin, but were unable to identify any other factor which might be responsible. More studies are required to elucidate the hormonal adjustments involved in temperature effects on carbohydrate metabolism, but such studies must consider the appropriate physiological hormone content, so more whole fish hormone studies will also be required. 2. 4. Stress Stress may increase the importance of carbohydrate metabolism in the whole animal energy budget, and blood glucose and lactate generally increase during stress 5. The precise mechanism(s) involved are speculative and complicated by secondary factors, some of which are difficult if not impossible to control. Cortisol is thought to be an important stress hormone, but its effects may be both direct and indirect through the actions of other glucoregulatory hormones. In general, cortisol does have a glucoregulatory action in some fish speciess but its actions on the isolated hepatocyte are minimal4~ Factors including nutritional state will alter the metabolic response of trout, presumably due to changes in substrate availability (see above) 17~ Studies on salmonids do suggest that cortisol alters liver enzyme profiles to enhance gluconeogenic potentials 168, but similar results are not seen in all cases. The actions of this hormone on liver cell metabolism need to be re-examined, and new experimental designs developed. Of all the glucoregulatory hormones found in fish, this is the most inconsistent. 2.5. Seasonality A full discussion of seasonality and carbohydrate metabolism is beyond the scope of this review, but a few comments are important for completeness. As discussed above, seasonal differences in glycogen contents in the Crucian carp are found, and these differences correlate with the ability of the carp to withstand anoxia~s,117. Glycogen content generally decrease through the winter, when food consumption is low~ or during the spawning season, as is seen in the catfish 12~ and increase when fish are actively feeding. But in many of these cases, glycogen content can be very high as the species enters the winter season (see Table 1). Seasonal changes in hormone responses are also common. Hormone responsiveness is found to decrease in cold seasons4~176 which may reflect an impaired receptor or

Tissue carbohydrate metabolism, gluconeogenesis and hormonal and environmental influences

77

transduction system. Mommsen and M o o n 103 found a decreased cAMP response to glucagon in March as compared with November in eel hepatocytes. Consistent with this observation, glycogenolysis was less responsive to glucagon in March. A consideration of seasonality is critical in both designing experiments and in interpreting their results. Environmental parameters seldom function in isolation, and it is critical that their interactive effects on liver carbohydrate metabolism be studied. As many of these parameters change with season, such as temperature, feeding, and possibly predatory stress, controlled seasonal studies would be very helpful in understanding these biochemical adaptations to the environment. Kent and colleagues 8~ have attempted to place temperature effects on liver characteristics into an ecological framework by studying naturally reared catfish. They found their study was confounded by other seasonal factors such as nutrition, as well as reproduction and photoperiod. More studies of this kind are required before the complexity of fish liver metabolism can be fully understood. 3. Hormone signal transduction pathways Recent work with fish liver has been focused on understanding the signal transduction pathways of hormones. To date the most interesting finding is the significant species differences in the quantitative and qualitative responses to hormones. Glucagon stimulates cAMP concentrations in fish livers, although there is not always a good correlation between the physiological response (e.g. glycogenolysis or gluconeogenesis) and the rise in cAMP 1~ The vasoactive peptides, vasotocin and isotocin, have also been found to stimulate glycogenolysis through the activation of cAMP 111. Catecholamines in mammals act through either al- or fl-adrenoceptors 32. The absence of classic a-antagonist binding and studies showing that ~l-antagonists were many-times more effective than a-antagonists in competitive displacement studies, suggested fl-adrenoceptors predominated in the liver of fish species 7~ Other studies, however, found that the phenylephrine (a classic a-adrenergic agonist)-induced glycogenolysis in isolated hepatocytes of eel and catfish could not be totally explained by this agonist acting in the fish liver as a ~l-agonist 15'111. More recent studies have found epinephrine induced an increase in intracellular free calcium concentrations ([Ca2+]i) (refs. 108, 187). Such changes in [Ca2+]i in mammalian hepatocytes result from the activation of a-adrenoceptors 32, and these are the first studies to support the existence of an t~-adrenoceptor system on fish hepatocytes. Moon et al. 108, however, were unable to link changes in [Ca2+]i with the activation of glycogenolysis, the classic mammalian metabolic effector of changes in [Ca2+]i (ref. 32). In addition, this change in [Ca2+]i is very species dependent 187, and more studies are needed to more completely identify the signalling processes occurring in these hepatocyte systems. The liver is critical to the carbohydrate status of fish as demonstrated by the patterns of response to environmental perturbations and hormones. There are significant species differences in these patterns, but whether these reflect

T.W. Moon and G.D. Foster

78

evolutionary differences or simply the prior history of the fish used is unclear. Future experiments must consider the plethora of factors which may affect the collected data, and carefully design experiments to eliminate as many factors as possible. Even so, papers must clearly identify the history of the fish being used and how the fish were held prior to and during the experiment. Without such information, it is difficult to interpret this huge literature. Certainly patterns are emerging, but as new species are explored, it is critical to provide such information.

III. Kidney 1. The organization of kidney metabolism Fish kidney carbohydrate metabolism has been poorly studied, as has its response to environmental conditions. Few generalizations are possible, although in all species examined, glycolytic enzymes are active (Table 3). For example, cod, trout, and flatfish HK and PK activities are greater than in the fiver, and PFK-1 activities are equivalent s3. A similar situation exists for the kidney of the salmon 1~ flounder 76, and the more primitive skate ~1~ Thus, the fish kidney appears to have an active glycolytic pathway. Kidney does have glycogen, but the content is much less than that found in liver in species where it has been analyzed (Table 1). The gluconeogenic pathway is present in this tissue, although its activity and contribution to whole animal glucose metabolism has often been questioned. TABLE

3

Kidney enzymes from selected fish species Enzyme

HK PFK-1 PK

Skate 11~

Sturgeon 152

Rainbow trout 83

IO~

20~

15~

0.39 0.37 15.0

LDH

6.9

PC

0.085

0.79

0.38

0.10

23.0

39.0

.

Cod s3 15~

IO~

15~ 0.12

16.1 110 .

Flounder s3

ND

0.59

-

.

Atlantic salmon 105

.

Marlin 156 25~

0.07

-

0.73

1.09

-

9.6

7.8

-

-

-

1.64 0.53

.

.

-

0.33

0.044

0.32

0.37

0.96

0.13

0.57

0.92

0.82

0.13

1.36

0.17

-

-

2.14

-

-

-

G6PDH

1.06

-

-

4.21

-

-

-

ME

0.55

-

-

0.28

-

-

-

0.33

-

5.54

-

-

-

-

PEPCK

FBPase GPase

HOAD MDH

ND

0.86 35.1

0.26

-

-

-

113

CS

2.05

2.9

-

0.35

-

GDH

-

2.19

-

2.14

-

-

-

GOT

-

16.8

-

5.84

-

-

-

GPT

-

1.1

-

-

-

-

23.5

Values are/tmol min -1 g-I wet tissue weight at the temperature noted. N D - none detected; - - - not assayed. Temperature refers to assay temperature.

12.1

-

Tissue carbohydrate metabolism, gluconeogenesis and hormonal and environmental influences

79

FBPase and PEPCK activities are found in salmon 1~ marlin (Makaira nigricans)156, cod, flatfish, and trout 83, and flounder 76. FBPase, but not PEPCK, was found in the skate 11~ In comparison to the liver, the gluconeogenic enzymes tend to be lower in activity (Table 3), which, together with the greater activities of the glycolytic enzymes, suggests a smaller gluconeogenic potential for this tissue. Glucose 6phosphatase (G6Pase) in agnathans is an exception; the activity of this enzyme is 3.3- and 1.1-times greater than its counterpart in the liver in the hagfish and the lamprey, respectively 132. This compares with values in the salmon of 0.08-times 132 and in the flounder of 0.54-times 76 that of the liver. Gluconeogenic flux in kidney slices has been estimated in flounder 76, salmon 1~ and skate 1~ Gluconeogenesis occurred in salmon kidney with rates from alanine being similar to those in liver (0.04/zmol g-1 h-i; 10oC); lactate gluconeogenesis was 0.46/zmol g-1 h-1 which is 50% of that in the liver, but a full 10-times above that for alanine. This tissue has significant gluconeogenic potential, even though, as the authors point out, PEPCK activities are only 3% of that in the liver. This discrepancy between rate and enzyme activity may reflect an assay problem rather than a real enzyme activity difference. Jorgensen and Mustafa 76 measured flounder PEPCK in the forward direction and found activities equivalent to those in the liver, and rates of lactate gluconeogenesis identical to the liver (0.36 /zmol g-1 h-i; at 10~ The kinetics of kidney PEPCK are unknown, but it is possible that measurement in the reverse direction will lead to an underestimation of activities. The skate kidney appears different from that of the teleosts with respect to gluconeogenesis. Mommsen and Moon 1~ report very low rates of gluconeogenesis (0.01 /zmol g-1 h-i; at 100C), with the majority of the alanine and lactate being oxidized (2.5 gmol g-1 h-i); no PEPCK activities could be detected in this tissue. They concluded that the kidney in this animal has no significant gluconeogenic function. Hexose monophosphate shunt enzymes were reported in the skate kidney 11~ but only G6PDH, and not 6PGDH, was found in the salmon kidney l~ (see Table 3). The glycogen metabolizing system in fish kidney has been poorly studied. In the skate, GPase activities are present, as are substantial quantities of glycogen 1~ Oxidation of lactate and alanine is no greater in the kidney of the salmon than the liver, and this is consistent with the relatively low activities of citrate synthase l~ Given the high glycolytic enzyme activities found in the teleost kidney, it may be postulated that anaerobic glycolysis is a significant source of energy for kidney function. The low gluconeogenic as compared with oxidative potential of the skate kidney, and the fact that citrate synthase (CS) activities are 7-times more active than the liver enzyme, all suggest that kidney metabolism is oriented towards oxidationl05, ll0. Various oxidative and gluconeogenic substrates have been examined in the flounder 76, salmon 1~ and skate 1~ kidney. Lactate, alanine, and serine were all metabolized to both CO2 and glucose (alanine and serine were not examined in flounder). The distribution of carbon between CO2 and glucose varies between substrates and species. In salmon, gluconeogenesis from lactate is approximately 10-times greater than from alanine (see above), whereas in the skate gluconeogenic

80

T.W. Moon and G.D. Foster

flux is the same for the two substrates. In terms of carbon distribution, 75% of the lactate carbon is converted to glucose in the salmon as compared to 2% in the skate. With respect to alanine, 28% of the carbon is converted to glucose in the salmon as compared to 3% in the skate. The substrate that showed the greatest relative conversion to glucose in the skate kidney slice was serine, with 12% of the carbons reaching glucose. Mammalian kidney utilizes lactate and glutamate but not alanine or serine for glucose production sl, indicating subtle differences exist in these pathways between these two groups. Few kidney carbohydrate metabolism studies have been performed on fish, and questions that remain to be answered include the use of various substrates, the glueoneogenie potential of the tissue and its importance in whole animal glucose production, the predominant pathways for energy supply in the tissue, and glycogen metabolism.

Z Environmental adaptations The impact of environmental factors on kidney metabolism has been poorly studied. Morata et al. 113 reported increased activities of PEPCK and G6Pase in trout kidney after 60 days of fasting, while FBPase activities were unaffected. A long term fast results in increased gluconeogenesis in the mammalian kidney, with glucose production from this tissue accounting for up to 50% of whole animal glucose production sl. The results of Morata et aL 113 are consistent with the mammalian situation, but no metabolic studies have been performed to corroborate the enzyme changes. JCrgensen and Mustafa 76 reported that the flounder kidney maintained a stable energy charge during extended hypoxia, and suggested that this tissue may replace the liver as the principal gluconeogenic organ during hypoxia. It is clear that many metabolic questions remain unanswered with regards to the fish kidney. Few species have been studied and in even fewer eases are changes in function related to a change in the condition of the fish. It does appear, however, that this tissue has a reduced metabolic potential compared to the liver, but that it can be gluconeogenic in at least a few species of teleosts.

II(. Skeletal muscle and heart 1. Myotomal muscle Johnston and Altringham 71 have reviewed the organization of fish myotomal muscle, the energetics of muscle contraction, and adaptations of fish muscle in Volume 1 of this series. The use of carbohydrate as an energy source for muscle contraction has not been directly reviewed and is complicated by the diversity of fish species and their lifestyles. In general, sustained swimming is powered by red muscle and burst swimming by white muscle and the oxidation of fatty acids and the anaerobic breakdown of glycogen, respectively. Glycogen is found in red muscle in quantities slightly less than white muscle (Table 1), and studies have reported glycogen breakdown and lactate accumulation during red muscle activities 12s'lss'164, but fatty

Tissue carbohydrate metabolism, gluconeogenesis and hormonal and environmental influences

81

TABLE 4 W h i t e m u s c l e e n z y m e s f r o m s e l e c t e d fish s p e c i e s Enzyme

HK PFK-1 PK LDH PC PEPCK FBPase GPase G6PDH ME IDH HOAD MDH CS

S k a t e 11~

H a g f i s h 39

R a i n b o w t r o u t 83,155

P e r c h 45

G o l d f i s h 165

M a r l i n 156

10oC '

10oc

15~

10~

iSoC

25~

0.058 17.4 49.8 110 0.032 ND 0.076 1.9 0.044 0.096 0.037 12.0 0.63

5.8 98.0 305 0.043 0.40 ND 0.08 1.74 2.0 31.4 0.63

0.06 9.8 110 ND 0.13 . -

2723 51.4 -

0.056 39.0 260 0.10 0.25 23.0 0.06 0.60

1105 2697 ND 0.90 2.39 0.60

20~

.

.

. -

115 -

0.70 562 2.71

V a l u e s a r e / t m o l m i n -1 g - 1 w e t t i s s u e w e i g h t a t t h e t e m p e r a t u r e n o t e d . ND = none detected; - = not assayed. Temperature refers to assay temperature.

acids and proteins (amino acids) are thought to be the principal metabolizable fuel for red m u s c l e 71'164'179'182. Increased swimming speed elevates blood flow to muscle la2 and endurance 25 and sprint 126 exercise training does increase the aerobic capacity of fish myotomal muscle, but the contribution of endogenous substrates to energy production are probably greater than blood-borne substrates during muscle con traction 164,179. Enzyme activities reported in red and white myotomal fish muscle are noted on Tables 4 and 5. Parkhouse and colleagues 12s reported that GPase and PFK-1 are the primary glycolytic flux regulating enzymes in both red and white muscle of rainbow trout under all levels of exercise when glycogen contents were high. GPase total and %a values increased with exhaustive exercise in trout white but not red muscles 155. At low glycogen contents, HK and glyceraldehyde 3-P dehydrogenase-phosphoglycerate kinase (GAPDH-PGK)/LDH became the primary control points 125, suggesting that recovery metabolism is quite distinct from that during exercise and requires exogenous glucose. This latter idea is contrary to the general belief that teleost glucose turnover rates are insufficient to account for glycogen synthesis 2 .6. .7.29 . 89 177,179. Anaerobic glycogen breakdown to lactate is the hallmark of exhaustive exercise in fishes. Glycogen content per g tissue in white muscle is generally below that found in the liver with some exceptions, the most notable being the tuna (Table 1). The principal question plaguing fish physiologists/biochemists over the years has been the fate of this lactate following exercise. Wood and Perry la2 discussed this issue and have shown two patterns of handling the 'metabolic acid load' (AH+m) and the lactate load (ALa-) generated during exhaustive exercise. The more active species (e.g. tuna, trout, dogfish) increase blood AH+m and ALa- immediately

T.W. Moon and G.D. Foster

82 TABLE 5 Red muscle enzymes from selected fish species .

Enzyme .

.

.

.

.

.

.

Goldfish 165

Marlin 156

10*C

20"C

15"C; 20"C

15"C

25"c

.

.

.

Rainbow trout s3,155

.

1.27 16.8 52.0 59.3 0.10 ND 0.054 0.079 0.095 94.7 15.9 .

.

Sturgeon 152

.

HK PFK-1 PK LDH PC PEPCK FBPase GPase G6PDH ME HOAD MDH CS

.

Skate n ~

.

.

.

"

.

0.008 0.49 40.0 0.094 0.34 3.15 -

0.14 3.76 110 0.09 ND 15.3 -

4.40 ...

61.0 400 0.38 -

0.20 24.0 0.18 1.30 -

330 -

-. .

316 617 ND ND 0.68 17.6 4.40 17.4 336 18.3

.

Values a r e / x m o l min -1 g - I wet tissue weight at the temperature noted. ND --- none detected; - ffi not assayed. Temperature refers to assay temperature.

following exercise, but ALa- exceeds AH+m and persists in the blood for a greater time period. Sluggish species (e.g. flounder, sole, skate, sea raven) demonstrate only a minor ALa-, always below AH+m. These latter species are thought to retain metabolically generated lactate within the muscle. In all cases except the skipjack tuna 17s, fish species require 12-24 h to clear a metabolic lactate load 6,96,122,142. The principal question addressed in each study is what processes are involved in glycogen repletion following exhaustive exercise. The classic mammalian pattern known as the Cori cycle which can account for 30-55% of the post-exercise glycogen repletion 92,178, apparently does not operate in f i s h 6'21'96. Lactate turnover is directly related to plasma lactate concentrations, but even in the skipjack tuna where values of turnover are similar to those reported in mammals, Cori cycle activity is estimated to account for <5% of glycogen repletion 178. Lactate utilization by isolated fish hepatoeytes is well below that required to explain muscle glycogen repletion 172, and even if this were not the ease, glucose uptake into trout white muscle 179 represents a small fraction of trout glucose turnover 29. These inconsistencies and the fact that a stoichiometry between lactate disappearance and glycogen repletion does exist in fish muscle, has led most investigators to propose that muscle glyeogenesis from lactate must account for glycogen repletion 2,6,96,142. The precise mechanism for muscle glyeogenesis from lactate is not dear, although the enzymes PEPCK and malie enzyme (ME) and/or pyruvate carboxylase (PC) are thought to be important 92. As Table 4 indicates, these three enzymes occur in few species and are present in low activities where measured/ present. Only in goldfish 165 and possibly marlin 156 have all enzymes required for glycogenesis been reported. Activities of PK are high relative to other glyeolytie enzymes (Table 4), and a recent study by Sehulte et at 142 has proposed that a high [ATP]/[ADP] ratio resulting from a substantial decrease in [ADP] during

Tissue carbohydrate metabolism, gluconeogenesis and hormonal and environmental influences

83

early recovery in trout could drive PK in the reverse direction thus eliminating the need for these other enzymes. This solution to glycogen repletion has been previously suggested by others for higher vertebrate muscle glycogenesis. In their recent review, however, McDermott and Bonen 92 concluded that the pathway for glyco(neo)genesis in vertebrate muscle is 'far from being delineated' (p. 174) and certainly need not be identical to gluconeogenesis in the liver. This is still an incomplete story and new approaches are required, but the separation of myotomal muscle into distinct red and white fibre groups makes the fish an ideal model for such studies. Endurance exercise training has been shown to modify myotomal muscle structure, enzyme activities and aerobic capacities (see refs. 25, 71). Pearson and collaborators 126 reported that sprint trained trout performed better, accumulated more lactate and depleted less ATP without differences in glycogen and phosphocreatine depletion compared to untrained trout. In addition, trained trout repleted glycogen more quickly. They concluded that trained trout increased exogenous glucose use to conserve endogenous energy reserves, implying changes both in muscle capillary density25 and enzyme activities71. Such studies do not suggest changes in metabolites used for exercise, but imply an increased efficiency of their use. Environmental conditions have been shown to modify swimming performance by remodelling the myotomal muscles of a number of species71. Low temperature acclimation increases the volume of the aerobic fiber type, recruitment patterns and improves swimming performance in goldfish, green sunfish, chain pickerel, carp, stripped base and flounder, but not in salmonids (reviewed in refs. 52, 73, 74). it has been argued by Guderley and Blier52 that these species differences may reflect the species thermal tolerance and temperatures for optimum locomotion. Changes in performance at low temperatures are also related to changes in enzyme activities and concentrations of energy stores in these muscles (glycogen, lipid), and an apparent switch of metabolism away from carbohydrate to fats 75. The response of goldfish muscle to hypoxia is modified by thermal acclimation, with weaker responses noted at low temperatures 165. Hypoxia in tenth (Tinca tinca) was associated with a decline in aerobic capacities of myotomal muscle and a decrease in the threshold for activating anaerobic glycolysis, but a reduction in the use of exogenous compared to endogenous carbohydrate sources 72. The metabolic system of fish myotomal muscle is an example of matching supply with demand 71, and as environmental conditions change, demands will change. Studies need to precisely identify the metabolic consequences of the adaptability of fish muscles, to better understand these supply-demand issues.

2. Cardiac muscle The fish heart has different metabolic demands, and its metabolism would be expected to differ from that of myotomal muscle. Enzymes of glycogen metabolism, glycolysis, and the pentose phosphate shunt are present in all fish hearts examined (Table 6). PEPCK activities have not been detected in any fish heart examined. Activities of PFK-1 and PK resemble those found in skeletal muscle, while activities

T.W. Moon and G.D. Foster

84 TABLE 6 C a r d i a c m u s c l e e n z y m e s f r o m selected fish species Enzyme

ilK PFK-1 PK LDH PC PEPCK FBPase GPase a 6PDH ME HOAD MDH CS

H a g f i s h s7

S k a t e 11~

Dogfish is~

S e a r a v e n 15~

E e l p o u t 150

Cod s7

M a r l i n 156

15"(2

10"C

15"C

15"C

15"C

15"C

25"C

1.70 18.8 36.0 114 3.48 8.28 6.92

1'13 " 3.74 46.7 67.8 0.10 ND 0.12 . 0.42 1.35 0.13 67.3 4.10

4.35 10.4 66.7 251 -

3.53 1.83 51.9 217 . 4.09 16.9

.

. 1.78 21.3

. . . . . 2.45 1.17 36.3 128 1.35 226 12.8

6.23 9.63 58.9 33O

m

5.54 12.1

m

104 708 ND ND ND

2.5 8.63 332 14.2

Values a r e / z m o l m i n -1 g - l w e t tissue w e i g h t at t h e t e m p e r a t u r e n o t e d . N D - n o n e d e t e c t e d ; - - n o t assayed. T e m p e r a t u r e refers to assay t e m p e r a t u r e . a R e p r e s e n t s glycogen p h o s p h o r y l a s e a, r a t h e r t h a n total activity.

of FBPase arc approximately an order of magnitude lower in the heart than the myotomal muscle (Tables 4-6). These observations and the absence of PEPCK and PC suggest that the fish heart is one of the most glycolytically directed of the fish tissues. Glycogen concentrations in fish heart are generally 30-90 ~tmol g-1 (Table 1), which are relatively high compared to higher vertebrates. This may reflect a less advanced system to maintain oxygen and substrate supply to the heart necessitating the use of endogenous substrates under stress (hypoxic) conditions, and a coronary circulation is not found in all fish species 24. Driedzic and Hart 2s showed that sea raven heart function can be maintained in the absence of substrates, but that iodoacetate damaged performance. They concluded, therefore, that glycogen is consumed through glycolysis in the absence of substrates; GPase is present in the fish heart (Table 6). The mammalian heart metabolizes glucose, lactate, free fatty acids and ketone bodies, but fatty acid is the principal fuel (see ref. 150). This reliance upon fatty acids is related to the high aerobic efficiency of ATP production from this substrate. In contrast, glucose may be as important or more so as a fuel for the fish heart. Driedzic and Hart 28 demonstrated that the addition of glucose was equally effective as palmitate in preventing contractile failure in the perfuscd sea raven heart. In the more primitive hagfish heart, glucose outcompctes palmitate oxidation, and the addition of palmitate alone to isolated hearts resulted in decreased performance and decreased tissue glycogen content sT,re. These authors concluded that carbohydrates were an obligatory aerobic substrate for hagfish heart metabolism, and presented two explanations. Either the dependence upon carbohydrate is an adaptation to hypoxia in this species, or it may reflect a primitive feature of the chordate heart; as animals evolved a more active lifestyle, the importance of glycolysis diminished.

Tissue carbohydrate metabolism, gluconeogenesis and hormonal and environmental influences

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Palmitate decreased cardiac performance in the skate, glucose was without effect and only ketone bodies had a significant positive impact on heart performance 28. While glucose did not affect skate heart performance, the addition of iodoacetate dramatically decreased heart performance, suggesting glycogen utilization is important in maintaining heart function in the absence of exogenous substrates 28. The presence of HK in the elasmobranch heart (Table 6) does provide the potential for glycogen synthesis. Sidell et al. 150 examined a variety of temperate teleost species and have found a positive correlation between maximal myofibriUar ATPase, an index of ATP demand, and both HK and carnitine palmitoyl transferase activities. Their analysis indicated that unlike mammals where fatty acid use increased as work capacities expand, teleosts have developed no preference for fatty acid or carbohydrate use depending on power requirements. Lactate, in addition to fatty acids and glucose, is also an important substrate for teleost heart function. Lanctin et al. 85 showed greater oxidation rates of lactate than glucose in the isolated trout heart, a fact confirmed by Milligan 94. Lactate supplementation was sufficient to maintain sea raven cardiac performance in the presence of oxaloacetate 28. Furthermore, Milligan and Farrel195 showed that under aerobic conditions lactate was capable of providing sufficient energy at high workloads, and its transport was carrier-mediated. In addition, both glucose and lactate oxidation, and glycogenolysis, in the trout heart are adrenergically regulated through fl-receptors 94. A negative correlation was reported in sea ravens between body size and both heart oxygen consumption and PK activities, while LDH activities scale positively 31 This has lead to the hypothesis that large individuals may utilize more lactate than smaller ones, but this has yet to be critically tested. Fish hearts and in particular contractile performance have been assessed under numerous environmental conditions. For example, hearts from different species have differing abilities to withstand hypoxic stress. Hagfish cardiac performance is not affected by a 3 h hypoxia stress 149, and sea raven hearts withstand anoxia better than ocean pout even though performance in both decrease by 1 h 163. The decline in performance in these two teleosts was found to be related to acidosis rather than ATP depletion, with the sea raven having a better buffering capacity possibly due to the low myoglobin content of pout hearts 15~ These studies demonstrated adequate endogenous fuel reserves to maintain ATP concentrations during hypoxia, and the importance of carbohydrate in hagfish cardiac metabolism. Driedzic and colleagues studied the effects of temperature transitions on heart energy metabolism in a variety of fish species (sea raven, pike, eel, white and yellow perch, and bass). They reported that HK activities are greatly decreased in acute high to low temperature transitions, whereas fatty acid metabolizing enzymes decreased to a lesser extent, or were completely protected 4,144,145. Furthermore, acclimation to low temperatures resulted in increased fatty acid metabolizing enzyme activities and oxygen consumption was increased with palmitoleate but compromised with glucose additions 144. These data support an increased fatty acid catabolism in cold temperatures. They did find in sea raven hearts, however, a decreased palmitate oxidation at 5~ as compared to 15~ in the absence of changes in glucose oxidation. Thus, the relative importance of these metabolic fuels at low

86

T.W.Moon and G.D. Foster

temperatures is not dear, and these inconsistencies may relate to the use of these substrates for other muscle functions including the maintenance of Ca 2+ levels rather than energy production per se. Milligan94 has reported that the response of trout myoeytes to #-adrenergie stimulation was enhanced at warm temperatures. Species specific responses of cardiac metabolism to temperature changes have been reported. Eel and bass heart function is compromised at low temperatures, while that of pike, yellow and white perch is not 4,145. Consistent with these results, cardiac ATPase and fatty acid metabolizing enzyme activities decreased to a greater extent with a temperature change from 15 to 5"C in the eel and bass heart as compared with the others. Thus, in the compensating species, carbohydrate as an energy source decreased in favor of fatty acids. It is interesting to note that while ATPase, HK and fatty acid metabolizing enzyme changes differed between species, CS activities were protected in all species. Even though carbohydrate and fatty acid pathways may be differentially affected, the Krebs cycle is in all cases protected from temperature effects. There are few common features with regard to teleost cardiac metabolism except that hearts can utilize either glucose or fatty acids. The primitive hagfish use only carbohydrates, while elasmobranchs are unique in their use of ketones, consistent with this ability in other tissues and their presence in the blood. All hearts examined contain glycogen, which probably relates to the relatively poor oxygenation/circulation conditions of the cardiac tissues of many fish hearts. The most important question is what factors are key to substrate selection. The recent preparation of isolated myocytes by Milligan94 may be a useful metabolic model to address these issues. In addition, work by Stewart et al. 153 with fatty acid binding proteins in heart may lead to a better understanding of fuel use in this tissue of fishes.

I4. B r a i n

Extrapolation from other vertebrates has assumed that the fish brain is a glucoseconsuming tissue. While there is some evidence for this, little attention has been given to other possible energy substrates and pathways. Indirect evidence for this glucose-consuming capability comes from in vivo studies. When active fish that contain relatively low levels of brain glycogen are made hypoglyeemie by insulin injection, convulsions and death resulted, similar to the mammalian situation sT. Also, Washburn et aL 177 have reported that rainbow trout brain has the highest glucose utilization rate per unit weight of all tissues examined and as high as reported for rat; these values were found to be higher in female than male trout. Direct in vitro studies have shown glucose uptake in brain tissue; incubation of trout and bullhead brain pieces in the presence of glucose resulted in a net uptake of glucose59, but in no ease was glucose released, except in the sea lamprey~. Glycogen levels in the brain of fish (Table 1) are generally higher than their

Tissue carbohydrate metabolism, gluconeogenesis and hormonal and environmental influences

87

mammalian counterparts, and it has been shown that under hypoglycemic, anoxic, and ischemic stress that this energy store is or may be mobilized. Glycogen values greater than 100 ~mol g-I have been reported for the e e l 36 and the lamprey4a,132, and values for most fish species are generally above mammalian levels93. A notable exception is the rainbow trout where levels are in the range reported for mammals 27. While the high glycogen levels suggest a greater importance of this compound in energy metabolism in most teleosts as compared with mammals, little attention has been focused on the regulation of its synthesis and degradation. Glycogen synthase (GSase) and GPase, the two enzymes of glycogen metabolism, have been reported in the brain of the adult lamprey4a. Total activities of GSase are at least 10-times higher than the total GPase activities (Table 7), in contrast to the rat brain where GPase activities are 20- to 50-times more active than GSase 93. The regulation of these enzymes in fish is unknown, although the a and b forms appear to exist in the lamprey brain 48. The catabolism of endogenous glycogen is confused by the complexity of the brain itself and the location of the glycogen in the brain. Rovainen 14~ reported that brain glycogen in the larval lamprey (P. marinus) is localized primarily to the meninges rather than the brain itself. In fact, the 'cleaned brain' (brain less meninges) contained 33% of the glycogen content of the whole brain, while the meninges contained 170% more. Rovainen ~4~ postulated that the meninges catabolize glycogen to glucose which is used by the neural tissues, as evidenced by activities of glucose 6-phosphatase (Table 7) and high levels of glucose production in the meninges compared with the whole brain. Similar studies have yet to be done in teleosts, although incubation of trout and catfish brain pieces did not exhibit any net glucose production 59. Studies with adult lampreys also demonstrate that whole TABLE 7 B r a i n e n z y m e s f r o m selected fish species Enzyme

HK PFK-1 PK LDH PEPCK FBPase G6Pase GSase GPase G6PDH HOAD CS

L a m p r e y 48

S t u r g e o n 39

R a i n b o w t r o u t 83

F l o u n d e r s3

C o d s3

Goldfish154

11~

20~

15~

15~

15~

15~

16.5 326 1.7 ND 0.25 a 0.05 0.55 ND -

0.66 0.08 60.0 0.068 . . . 0.04 0.05 3.3

0.54 2.42 35.2 . ND ND

0.01 2.96 27.9

0.12 2.68 54.0

7.0 -

ND ND

-

. . .

.

. ND ND

. . . . . .

.

. . . . . .

. . 45.0 . . .

. . .

Values a r e t t m o l m i n - l g-1 wet tissue w e i g h t at t h e t e m p e r a t u r e n o t e d . N D = n o n e d e t e c t e d ; - -- n o t assayed. T e m p e r a t u r e refers to assay t e m p e r a t u r e . a Larval l a m p r e y ( r e f e r e n c e 140).

88

T.W.Moon and G.D. Foster

brains liberate glucose when incubated without substrate r and this release is blocked by insulin 48. Activities of hexose monophosphate shunt enzymes are reported in the mammalian brain 93, possibly to provide an alternative oxidative route for glucose or to provide reducing equivalents for brain lipid metabolism. G6PDH was not detected in the lamprey brain ~ but was in the sturgeon (Acipenserfulvescens)152;it has been examined in very few species, so the existence of this pathway is uncertain. Adult lamprey brain pieces convert both glucose and lactate to glycogen~. The flux from glucose to glycogen is enhanced by insulin under certain conditions. The existence of a gluconeogenic pathway in mammalian brain tissue is unlikely given the absence of PEPCK activities64 although FBPase is expressed in these tissues93. PEPCK, but not FBPase, activities are found in adult lamprey brainsr no fish species has been reported to have both PEPCK and FBPase activities in the brain (see Table 7). It seems unlikely that the teleost brain is capable of gluconeogenesis, although more species and experiments need to be undertaken. While lactate is converted to glycogen in the lamprey brain, the majority of this substrate is converted to CO2 (ref. 48). The oxidation of lactate is 2- to 4-times greater than that of glucose in this system. Thus, lactate is a better oxidative substrate than glucose in this preparation, but its quantitative importance is not known. The use of other substrates including amino acids and ketone bodies have not been examined in fish, although ketone bodies have been shown to increase during anoxia in trout brain 2:. Few studies have examined the effects of environmental factors on brain carbohydrate metabolism. DiAngelo and Heath 27 reported a correlation between glycogen content and anoxia-tolerance in trout and bullhead, similar to studies on other vertebrates ~39. Using a minced brain preparation from these two species, Heath 59 further suggested that bullhead brain relies more on endogenous reserves (glycogen) and possibly a depressed metabolism to tolerate anoxia, while the trout brain uses exogenous glucose to supplement its limited glycogen reserves. Given the recent interest in brain and depressed metabolism in mammals, the fish brain appears to be an ideal organ to examine the mechanisms of anoxic-tolerance. Brain glycogen content in goldfish and sardines was maximal during the winter months and increased with cold acclimation 14. The explanation for this coldinduced increase in brain glycogen is far from clear. These authors suggest it may be related to a general decrease in brain metabolism during the cold, but this is not consistent with the relative thermal independence of brain metabolism reported by Heath 59. Certainly coordination is required regardless of temperature, so again further studies are needed to address this interesting paradox. In this same vein, the importance of pesticides and other toxicants on brain metabolism need to be investigated. One study using lindane and eels found significant changes in brain metabolites ~. This should be a fruitful future area for toxicology studies. Thus, the fish brain remains a poorly studied tissue although it offers many interesting comparative and environmental challenges. Certainly carbohydrates are important metabolic fuels, but little is known of their compartmentalization, pattern of use, or qualitative significance, all areas for further study.

Tissue carbohydrate metabolism, gluconeogenesis and hormonal and environmental influences

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111. Red blood cells The oxygen consumption of the nucleated, mature red blood cells of fish is considered high relative to that of the mature mammalian cell. Values range from 2 to 10 /zmol 02 l-lcells h -1 in fish, or 3- to 10-times that of mammalian red cells 13'116. In fact, Nikinmaa 116 has suggested that the fish red cell is metabolically more comparable to the reticulocyte, the stage in mammalian erythropoiesis just prior to the loss of regular cellular organelles. Although these rates are low compared to other tissues of fish, they account for 2-5% of the resting whole-trout oxygen consumption 3~ 175. The question of what fuels this oxygen consumption is far from clear. The mature red cell of most mammals has a high glucose transport capacity which does not limit glycolysis, and anaerobic glycolysis is considered to meet all of the energetic needs of the cell 116,185. Fish red cells aregenerally poorly permeable to glucose. Red cells from rainbow trout 12,161, paddyfield eel (Monopterus albus) 161 and carp (Cyprinus carpio) 160 are considered to be deficient of o-glucose transport. A study of Amazonian fish species by Kim and Isaacks 81 showed significant glucose uptake in only 2 of 5 species. Species which do show high glucose permeability (and in some cases cytochalasin-B-sensitivity) are the river lamprey (L. fluviatilis) 16~ the Pacific hagfish (E. stouti) 68, the sea perch (Embiotaca lateralis) 67, and the Japanese eel TM. Equilibration times for glucose across red cells in most studies are from a few minutes (e.g. lungfish) al to hours (e.g. trout) 129, with the exception being hagfish where they are a few seconds 68. Ferguson and Storey 34 showed that the transmembrane glucose gradient was 9, favoring extracellular concentrations. The Pacific hagfish glucose transporter has now been isolated and found to have close structural similarities with the human erythrocyte glucose transporter GLUT-1 (ref. 185). Studies such as this one should help to better understand differences between red cell transport functions. Nikinmaa 116 stated that with the possible exception of the hagfish, glucose transport limits glucose metabolism in fish nucleated red cells. Enzymes from fish red cells have been reported from five studies, and in most eases, all the glycolytic enzymes are present (Table 8). Two studies suggested that HK limits flux, but most studies support PFK as the rate limiting enzyme 34,5~ Unfortunately, only the study of Pesquero et al. 129 has examined glucose transport, oxygen consumption, glucose oxidation and enzymes simultaneously. This study reported 3-O-methylglucose (3O-MG) transport rates of 430 nmol g-1 hb h -1, oxygen consumption rates of 70 nmol g-1 hb h-1 and HK activities of 760 nmol g-lhb h-l; transport in brown trout red cells does appear to limit glucose oxidation. This species is considered to have poor glucose permeabilities, and unfortunately no studies have examined these parameters in cells considered to have greater permeabilities (e.g. Japanese eel red cells have a transport rate as much as 200-times that of trout) 161. The data with respect to oxidation is confusing and again experiments have used primarily trout red cells rather than a red cell system which is metabolically more active (e.g. eel) 61. Walsh et al. 175 found CO2 production rates from 14Clabeled substrates in the order glucose > lactate > > alanine > oleate. If the

90

T.W. Moon and G.D. Foster

TABLE 8 Red blood cell enzymes from selected fish species Enzyme

Rainbow trout34.175 15oc34 15oc175

Brown trout129 15oc

Perch3 25oc

-HK

2.7 1.6 4.3 22.4 32.1 50.5 ND ND ND 14.1 2.9 64.6

0.76 0.2 9.7 30.0 8.1 ND ND

0.08 1.4 5.2 27.3 13.2 233 20.6 -

PFK-1 Aldolase G3PDH PK LDH PEPCK FBPase GPase G6PDH IDH MDH CS

0.58

ND 29.6 27.2 ND ND ND 24.4 4.4 32.0 ND

-

Sea r a v e n 146 15"C 0.25 0.69 0.15 1.00 1.03 . . .

14.9 0.38

Values are/~mol min-1 g-I hemoglobin except for sea raven which is/zmol min-1 m1-1 RBC at the temperature noted. ND = none detected;- = not assayed.

rates of all 4 substrates were summed, they represented less than 15% of the total oxygen consumption rate. They suggested other substrates were involved, and as their experiments used whole trout blood, endogenous compounds could not be excluded. There was significant hexose monophosphate shunt activity in this preparation (Table 8), but this makes the oxygen consumption rates even more difficult to interpret. They did report a concentration-dependent increase in CO2 production with glucose. Pesquero et ai.129 reported that total oxygen consumption was independent of glucose or pyruvate addition, and at I mM glucose, CO2 production represented less than I% of total oxygen consumption. Significant hexose monophosphate shunt activities were found as a-cyano-3-hydroxycinnamate, a compound which blocks pyruvate transfer into the mitochondria, decreased CO2 production only by 33%. In neither study 129,17s could CS activities be detected (Table 8), although only one other study has reported CS activities. Activities of this enzyme are low relative to that of other measurable enzymes. Also, Pesquero et at. 129 reported mitochondria 'were practically absent' (p. 450) as shown by EM images. Only in the study by Sephton et al. 146 was there a direct correlation between oxygen consumption and CO2 production rates from glucose and this was in the sea raven. This strongly supports the aerobic metabolism of glucose as the principal source of energy in these cells. Washburn and colleagues 177 have also shown that glucose utilization rates of trout red cells are greater than muscle but well below those of brain and gonad. Certainly the oxidative capacities of fish red cells are far from clear and more studies must be done in this area. The reliance upon anaerobic glycolysis for energy production of the mature mammalian red cell means that lactate must represent a major metabolic endproduct (see ref. 116). Even though it has been estimated by Ferguson et ai.35 that over

Tissue carbohydrate metabolism, gluconeogenesis and hormonal and environmental influences

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90% of the energy for the trout red cell is generated aerobically, lactate does appear as a major metabolic endproduct in most studies. Walsh et al. 175 demonstrated that lactate production represented 28% of the total glucose metabolism, even with high blood oxygenation. Values reported by Pesquero et al. 129 exceeded 90% in brown trout. They showed convincingly that, as in mammalian red cells, older trout red cells are less metabolically active and that their preparation consisted primarily of 'old' cells; this could account for the lowered oxygen consumption and glucose oxidation rates compared to those of Walsh et al. 175. Sephton et al. 146 also reported that rinsed, resuspended red cells of sea raven had a lower oxygen consumption than whole blood, but under no aerobic condition did these cells produce lactate. Whether other sources of lactate such as glycogen 34, or possibly carbon recycling 146 are important are not clear. It seems, therefore, that the basic metabolic organization of the fish red cell remains to be clearly elucidated. A number of conflicting studies exist, but we must await studies on red cells which are more permeable to glucose than trout cells. Certainly gluconeogenesis does not occur, but even the presence of an active Krebs cycle is uncertain (Table 8), even though oxygen consumption rates are considered 'high '13,116. The sea raven red cell is the only clear story, but this is an unusual species in its own right 116. An extensive literature indicates that catecholamines modify the red cells of many fish species both in vivo and in vitro 128,159. The red cell response to epinephrine involves the activation of the Na+/H + exchanger with the ultimate effect of maintaining blood oxygen transport by increasing the affinity and/or capacity of hemoglobin to bind oxygen. This complex process involves an elevation of intraceUular pH (pHi), cell swelling and decreased intracellular nucleoside triphosphates resulting from increased ATP utilization by the Na+-pump. As plasma catecholamine levels rise under a variety of environmental disturbances including hypoxia and exercise 159, such changes in nucleoside triphosphate levels would be expected to change red cell metabolism. Under oxygenated conditions, Ferguson and Boutilier 33 reported a tight coupling between nucleoside triphosphate utilizing and producing processes in adrenergicstimulated trout red cells. Tufts and Boutilier 162 reported that at rest, 20% of cellular energy was used by the Na+-pump, while this value increased to 43% in the presence of isoproterenol. Given that adrenergic stimulation increased oxygen consumption in trout red cells and ATP levels did not fall, Boutilier and Ferguson 13 argued that oxidative phosphorylation is adequate to supply the needed energy. Only when oxygen was limiting did ATP levels fall, but the small decrease in ATP and the small increase in lactate suggested metabolism was depressed in these cells. Unfortunately, the substrate utilized by these cells under either condition was not identified. Pesquero et al. 129 found that isoproterenol increased 3-O-MG uptake in brown trout red cells, but glucose metabolism to CO2 and lactate increased by 2- and 4-fold, respectively. Thus, aerobic glucose metabolism does not appear to fuel increased energy requirements. Sephton et al. 146 reported that substrates other than glucose were izwolved in isoproterenol-stimulated sea raven red cells. In both of

92

Zig. Moon and (7.1). Foster

these studies, the in vitro incubation of red cells under hypoxie conditions resulted in increased lactate and decreased nueleoside triphosphate levels. During exercise, Wood et al. 183 showed that trout red cells increased the utilization of lactate as a result of higher plasma lactate concentrations and the effects of epinephrine on red cell metabolism. The consistency and physiological importance of the red cell response to adrenergic stimulation makes this system ideal to study the importance of substrate type, availability and metabolism in a cell system. Such studies are critical to unravel the complexities of what was thought to be a relatively simple metabolic system. Also, it is critical that systems other than trout red cells are used as experimental models, as these cells may have quite distinct membrane permeabilities relative to red cells of other species.

VII. C o n c l u s i o n s

This review was not meant to be all-inclusive, but to look at specific fish tissues, the use of carbohydrates by these tissues and how utilization rates and patterns may be altered by environmental and hormonal conditions. Much information is available, but there are significant deficiencies in many areas, especially the kidney and brain. Liver, and in particular hepatoeytes, are well studied, but virtually nothing is known of hepatic function in non-carnivorous species. Muscle and red cells represent good model systems, but the evidence for glyeogenesis and oxidative metabolism, respectively, are circumstantial at best. To address these deficiencies, the comparative approach is key and the use of species of different life histories and life-styles essential. When it comes to hormone-binding, trout hepatoeytes seem to have a dearth of specific binding sites, yet in many eases these cells respond metabolically; why? What accounts for the significant species differences raised in this paper, or should we expect even more differences than we actually see? Fish represent a diverse vertebrate class with a very long evolutionary history. As fish biochemists/physiologists we need to begin to use these differences to better understand this group of animals and vertebrates evolution as a whole.

I/Ill. R e f e r e n c e s

1. Alonso, M.D.E, M.L.P. Perez, M.R. Amil and M.J.H. Santos. Regulation of liver sea bass pyruvate kinase by temperature, substrates and some metabolic effectors. Comp. Biochem. Physiol. 8213: 841-848, 1985. 2. Arthur, P.G., T.G. West, R.W. Brill, P.M. Schulte and P.W. Hochachka. Recovery metabolism of skipjack tuna (Katsuwonus pelamis) white muscle; rapid and parallel changes in lactate and phosphocreatine after exercise. Can. ]. Zool. 70: 1230-1239, 1992. 3. Bachand, L. and C. Leray. Erythrocytemetabolism in the yellowperch (Percaflavescens Mitchill)1. Glycolyticenzymes. Comp. Biochem. Physiol. 50B: 567-570, 1975. 4. Bailey,J., D. Sephton and W.R. Driedzic. Impact of an acute temperature change on performance and metabolism of pickerel (Esox niger) and eel (Anguilla rostrata) hearts. Physiol. Zool. 64: 697716, 1991. 5. Barton, B.A. and G.IC lwama. Physiological changes in fish from stress in aquaculture with

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1985. 33. Ferguson, R.A. and R.G. Boutilier. Metabolic-membrane coupling in red blood cells of trout: the effects of anoxia and adrenergic stimulation. J. Exp. BIOL 143: 149-164, 1989. 34. Ferguson, R.A. and ICB. Storey. Glycolytic and associated enzymes of rainbow trout (On. corhynchus mykiss) red cells: In vitro and in vivo studies. Jr. Er.p. BioL 155: 469-485, 1991. 35. Ferguson, R.A., B.L. Tufts and R.O. Boutilier. Energy metabolism in trout red cells: consequences of adrenergic stimulation/n vivo and in vitro. J. Exp. Biol. 143: 133-147, 1989. 36. Ferrando, M.D. and A. Andreu-Moliner. Changes in selected biochemical parameters in the brain of the fish, Anguilla anguilla (L.), exposed to lindane. Bull. Environ. Contam. Toxicol. 47: 459-464, 1991. 37. Fideu, M.D., G. Soler and M. Ruiz-Amil. Nutritional regulation of glycolysis in rainbow trout (Salmo gairdneri R.). Comp. Biochem. Physiol. 74B: 795-799, 1983. 38. Fincham, D.A., M.W. Wolowyk and J.D. Young. Characterization of amino acid transport in red blood cells of a primitive vertebrate, the Pacific hagfish (Eptatretus stouti). J. Exp. Biol. 154: 355370, 1990. 39. Foster, G.D. and T.W. Moon. Enzyme activities in the Atlantic hagfish, Myxine glutinosa: changes with Captivity and food deprivation. Can. J. Zool. 64: 1080-1085, 1986. 40. Foster, G.D. and T.W. Moon. Cortisol and liver metabolism of immature American eels, Anguilla rostrata (LeSueur). Fish Physiol. Biochem. 1: 113-124, 1986. 41. Foster, G.D. and T.W. Moon. Metabolism in sea raven (Hemitripterus americatms) hepatocytes: The effects of insulin and glucagon. Gen. Comp. Endocrinol. 66: 102-115, 1987. 42. Foster, G.D. and T.W. Moon. Insulin and the regulation of glycogen metabolism and gluconeogenesis in American eel hepatocytes. Gen. Comp. Endocrinol. 73: 374--381, 1989. 43. Foster, G.D. and T.W. Moon. Control of key carbohydrate-metabolizing enzymes by insulin and glucagon in freshly isolated hepatocytes of the marine teleost Hemitriptetus americanus. J. Exp. Zool. 254: 55-62, 1990. 44. Foster, G.D. and T.W. Moon. The role of glycogen phosphorylase in the regulation of glycogenolysis by insulin and glucagon in isolated eel (Anguilla rostrata) hepatocytes. Fish Physiol. Biochem. 8: 299-309, 1990. 45. Foster, G.D. and T.W. Moon. Hypometabolism with fasting in the yellow perch (Perca flavescens): a study of enzymes, hepatocyte metabolism and tissue size. Physiol. Zool. 64: 259-275, 1991. 46. Foster, G.D. and T.W. Moon. Hormonal sensitivity and responsiveness in sea raven hepatocytes: changes with fasting and collagenase exposure. Can. J. Zool. 71: 1755-1762, 1993. 47. Foster, G.D., K.B. Storey and T.W. Moon. The regulation of 6-phosphofructo-l-kinase by insulin and glucagon in isolated hepatocytes of the American eel. Gen. Comp. Endocrinol. 73: 382-389, 1989. 48. Foster, G.D., J.H. Youson and T.W. Moon. Carbohydrate metabolism in the brain of the adult lamprey. J. EW. Zool. 267: 27-32, 1993. 49. Foster, G.D., J. Zhang and T.W. Moon. Carbohydrate metabolism and hepatic zonation in the Atlantic hagfish, Myxine glutinosa liver: effects of hormones. Fish Physiol. Biochem. 12: 211-219, 1993. 50. Gaitan, S., E. Cuenllas, M. Ruiz-Amil and C. Tejero. Regulation of phosphofructokinase during haemopoiesis of rainbow trout (Salmo gairdneri R.) - Kinetic studies. Comp. Biochem. Physiol. 95B: 641-646, 1990. 51. Garcia-Salguero, L~ and J.A. Lupianez. Long term control of renal carbohydrate metabolism - 1. Effect of starve-feed cycles on renal tubules glycolysis. Int. J. Biochem. 20: 943-950, 1988. 52. Guderley, H. and P. Blier. Thermal acclimation in fish: conservative and labile properties of swimming muscle. Can. J. Zool. 66: 1105-1115, 1988. 53. Outi~rrez, J., J. Fern~mdez, M. Carrillo, S. Zanuy and J. Planas. Annual cycle of plasma insulin and glucose of sea bass, Dicentrarchus labrax L. Fish Physiol. Biochem. 4: 137-141, 1987. 54. Guti~rrez, J., J. Perez, I. Navarro, S. Zanuy and M. Carillo. Changes in plasma glucagon and insulin associated with fasting in sea bass (Dicentrarchus labrax). Fish Physiol. Biochem. 9: 107-112, 1991. 55. Guti~rrez, J. and E.M. Plisetskaya. Insulin binding to liver plasma membranes in salmonids with modified plasma insulin levels. Can. J. ZooL 69: 2745-2750, 1990. 56. Hanke, W. and P.A. Janssens. The role of hormones in regulation of carbohydrate metabolism in the Australian lungfish, Neoceratodus forsteri. GetL Comp. Endocrinol. 51: 364-369, 1983. 57. Hansen, C.A. and B.D. Sidell. Atlantic hagfish cardiac muscle: metabolic basis of tolerance to anoxia. Am. J. Physiol. 244: R356-R362, 1983.

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84. Laidley, C.W. and J.E Leatherland. Circadian studies of plasma cortisol, thyroid hormone, protein, glucose and ion concentration and liver and spleen weight in rainbow trout, Salmo gairdneri Richardson. Comp. Biochem. Physiot 89A: 495-502, 1988. 85. Lanctin, H.P., L.E. McMorran and W.R. Driedzic. Rates of glucose and lactate oxidation by the perfused isolated trout (Salvelinus fontinalis) heart. Can. J. Zool. 58: 1708-1711, 1980. 86. Larsen, L.O. Subtotal hepatectomy in intact or hypophysectomized river lampreys (Lampetra fluviatilis L.): effects on regeneration, blood glucose regulation and vitellogenesis. Gen. Comp. Endoctinol. 35: 197-204, 1978. 87. Leibson, L.G. Features of the metabolism and its endocrine regulation in fish with different motor activities. EvoL Physiol. Biochem. 8: 248-255, 1973. 88. Lim, A.L.L. and Y.K. lp. Effect of fasting on glycogen metabolism and activities of glycolytic and gluconeogenic enzymes in the mudskipper Boleophthalmus boddae~. I. Fish Biol. 34: 349-367, 1989. 89. Lin, H., D.R. Romsos, P.I. Tack and G.A. Leveille. Determination of glucose utilization in coho salmon [Oncorhynchus kisutch (Walbaum)] with (6-3H)- and (U-14C)-glucose. Comp. Biochem. PhysioL 59A: 189-191, 1978. 90. Love, R.M. The Chemical Biology of Fishes, London, Academic Press, pp. 1-943, 1980. 91. Machado, C.R., M.A.R. Garofalo, J.E.S. Roselino, I.C. Kettelhut and R.H. Migliorini. Effects of starvation, refeeding and insulin on energy-linked metabolic processes in catfish (Rhamdia hilarii) adapted to a carbohydrate-rich diet. Gen. Comp. Endocrinol. 71: 429-437, 1988. 92. McDermott, J.C. and A. Bonen. Glyconeogenic and oxidative lactate utilization in skeletal muscle. Can. ]. Physiol. Pharmacol. 70: 142-149, 1992. 93. Mcllwain, H. and H.S. Bachelard. Biochemistry and the Central Nervous System, London, Churchill Livingstone, 1985. 94. Milligan, C.L. Adrenergic stimulation of substrate utilization by cardiac myocytes isolated from rainbow trout./. Exp. Biol. 159: 185-202, 1991. 95. Milligan, C.L. and A.P. Farrell. Lactate utilization by an in situ perfused trout heart: effects of workload and blockers of lactate transport. I. Exp. Biol. 155: 357-373, 1991. 96. Milligan, C.L. and D.G. McDonald. In vivo lactate kinetics at rest and during recovery from exhaustive exercise in coho salmon (Oncothynchus kisutch) and starry flounder (Platichthys stellatus). J. Exp. Biol. 135: 119-131, 1988. 97. Mommsen, T.P. Comparative gluconeogenesis in hepatocytes from salmonid fishes. Can. ]. ZooL 64: 1110-1115, 1986. 98. Mommsen, T.P., P.C. Andrews and E.M. Plisetskaya. Glucagon-like peptides activate hepatic gluconeogenesis. FEBS Left. 219: 227-232, 1987. 99. Mommsen, T.P., E. Danulat, M.E. Gavioli, G.D. Foster and TW. Moon. Separation of enzymatically distinct populations of trout hepatocytes. Can. J. Zool. 69: 420--426, 1991. 100. Mommsen, T.R, E. Danulat and P.J. Walsh. Metabolic actions of glucagon and dexamethasone in liver of the ureogenic teleost Opsanus beta. Gen. Comp. Endocrinol. 85: 316-326, 1992. 101. Mommsen, T.E and T.W. Moon. The metabolic potential of hepatocytes and kidney tissue in the little skate, Raja erinacea. ]. Exp. Zool. 244: 1-8, 1987. 102. Mommsen, T.P. and T.W. Moon. Metabolic actions of glucagon family hormones in liver. Fish Physiol. Biochem. 7: 279-288, 1989. 103. Mommsen, T.P. and T.W. Moon. Metabolic response of teleost hepatocytes to glucagon-like peptide and glucagon. J. Endocrinol. 126: 109-118, 1990. 104. Mommsen, T.P. and P.J. Walsh. Metabolic and enzymatic heterogeneity in the liver of the ureogenic teleost Opsanus beta. J. Er~. Biol. 156: 407-418, 1991. 105. Mommsen, T.P., P.J. Walsh and T.W. Moon. Gluconeogenesis in hepatocytes and kidney of Atlantic salmon. Mol. Physiol. 8: 89-100, 1985. 106. Moon, T.W. Metabolic reserves and enzyme activities with food deprivation in immature American eels AnguiUa rostrata (LeSueur). Can. J. Zool. 61: 802-811, 1983. 107. Moon, T.W. Adaptation, constraint and the function of the gluconeogenic pathway. Can. I. Zool. 66: 1059-1068, 1988. 108. Moon, T.W., A. Capuzzo, A.C. Puviani, C. Ottolenghi and E. Fabbri. Alpha-mediated changes in hepatocyte intracellular calcium in the catfish, lctalurus melas. Am. .l. Physiol. 264: E735-E740, 1993. 109. Moon, T.W., G.D. Foster and E. Plisetskaya. Changes in peptide hormones and liver enzymes in the rainbow trout deprived of food for 6 weeks. Can. J. Zool. 67: 2189-2193, 1988. 110. Moon, T.W. and T.P. Mommsen. Enzymes of intermediary metabolism in tissues of the little skate,

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135. Plisetskaya, E.M., C. Ottolenghi, M.A. Sheridan, T.P. Mommsen and A. Gorbman. Metabolic effects of salmon glucagon and glucagon-like peptide in Coho and Chinook salmon. Gen, Comp. EndocrinoL 73: 205-216, 1989. 136. Prosser, C.L., G. Graham and V. Galton. Hormonal regulation of temperature acclimation in catfish hepatocytes. J. Comp. Physiol. B 161: 117-124, 1991. 137. Rafael, J. and T. Braunbeck. Interacting effects of diet and environmental temperature on biochemical parameters in the liver of Leuciscus idus melanotus (Cyprinidae: Teleostei). Fish Physiol. Biochem. 5: 9-19, 1988. 138. Reddy, V.D., M. Bhaskar and S. Govindappa. Influence of starvation and refeeding on hepatic tissue glycogen metabolism of freshwater fish Sarotherodon mossambicus. J. Environ. Biol. 9: 15-20, 1988. 139. Robin, E.D., N. Lewiston, A. Newman, L.M. Simon and J. Theodore. Bioenergetic pattern of turtle brain and resistance to profound loss of mitochondrial ATP generation. Proc. Natl. Acad. Sci. USA 76: 3922-3926, 1979. 140. Rovainen, C.M. Glucose production by lamprey meninges. Science 167: 889-890, 1970. 141. Rovainen, C.M., O.H. Lowry and J.V. Passonneau. Levels of metabolites and production of glucose in lamprey brain../. Neurochem. 16: 1451-1458, 1969. 142. Schulte, P.M., C.D. Moyes and P.W. Hochachka. Integrating metabolic pathways in post-exercise recovery of white muscle. I. Exp. Biol. 166: 181-196, 1992. 143. Seibert, H. Effects of temperature on glucose release and glycogen metabolism in isolated hepatocytes from rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. 81B: 877-883, 1985. 144. Sephton, D., J. Bailey and W.R. Driedzic. Impact of acute temperature transition on enzyme activity levels, oxygen consumption and exogenous fuel utilization in sea raven (Hemitripterus americanus) hearts. J. Comp. Physiol. B 160: 511-518, 1990. 145. Sephton, D.H. and W.R. Driedzic. Effect of acute chronic temperature transition enzymes of cardiac metabolism in white perch (Morone americana), yellow perch (Perca flavescens)and small mouth bass (Micropterus dolomieui). Can. J. Zool. 69: 258-262, 1991. 146. Sephton, D.H., W.L. Macphee and W.R. Driedzic. Metabolic enzyme activities, oxygen consumption and glucose utilization in sea raven (Hemitripterus americanus) erythrocytes. I. Exp. Biol. 159: 407-418, 1991. 147. Sheridan, M.A. and T.P. Mommsen. Effects of nutritional state on in vivo lipid and carbohydrate metabolism of coho salmon, Oncothynchus kisutch. Gen. Comp. Endocrinol. 81: 473-483, 1991. 148. Shulman, G.I. and B.R. Landau. Pathways of glycogen repletion. PhysioL Rev. 72: 1019-1035, 1992. 149. Sidell, B.D. Cardiac metabolism in the myxinidae: Physiological and phylogenetic considerations. Comp. Biochem. Physiol. 76A: 495-505, 1983. 150. Sideli, B.D., W.R. Driedzic, D.B. Stowe and I.A. Johnston. Biochemical correlations of power development and metabolic fuel preferenda in fish hearts. Physiol. ZooL 60: 221-232, 1987. 151. Sidell, B.D., D.B. Stowe and C.A. Hansen. Carbohydrate is the preferred metabolic fuel of the hagfish (Myxine glutinosa) heart. Physiol. Zool. 57: 266-273, 1984. 152. Singer, T.D., V.G. Mahadevappa and J.S. BaUantyne. Aspects of the energy metabolism of lake sturgeon, Acipenserfulvescens, with special emphasis on lipid and ketone body metabolism. Can. I. Fish. Aquat. Sci. 47: 873-881, 1990. 153. Stewart, ,I.M., W.R. Driedzic and J.A.M. Berkelaar. Fatty-acid-binding protein facilitates the diffusion of oleate in a model cytosol system. Biochem. I. 275: 569-573, 1991. 154. Storey, K.B. Tissue-specific controls on carbohydrate catabolism during anoxia in goldfish. Physiol. Zool. 60: 601-607, 1987. 155. Storey, K~B. Metabolic consequences of exercise in organs of rainbow trout. Jr. E~. Zool. 260: 157-164, 1991. 156. Suarez, R.K, M.D. Mallet, C. Daxboeck and P.W. Hochachka. Enzymes of energy metabolism and gluconeogenesis in the Pacific blue marlin, Makaira nigricans. Can. J. Zool. 64: 694-697, 1986. 157. Suarez, R.IL and T.P. Mommsen. Gluconeogenesis in teleost fishes. Can../. Zool. 65: 1869-1882, 1987. 158. Sundby, A., G.-I. Hemre, B. Borrebaek, B. Christophersen and A.K. Blom. Insulin and glucagon family peptides in relation to activities of hepatic hexokinase and other enzymes in fed and starved Atlantic salmon (Salmo salar) and cod (Gadus morhua). Comp. Biochem. Physiol. 100B: 467-470, 1991. 159. Thomas, S. and S.E Perry. Control and consequences of adrenergic activation of red blood cell Na+/H + exchange on blood oxygen and carbon dioxide transport in fish. I. Exp. Zool. 263: 160175, 1992.

Tissue carbohydrate metabolism, gluconeogenesis and hormonal and environmental influences

99

160. Tiihonen, K. and M. Nikinmaa. Substrate utilization by carp (Cyprinus carpio) erythrocytes. J. Exp. BioL 161: 509-514, 1991. 161. Tse, C.M. and J.D. Young. Glucose transport in fish erythrocytes-variable cytochalasin-B-sensitive hexose transport activity in the common eel (AnguiUa japonica) and transport deficiency in the paddyfield eel (Monopterus albus) and rainbow trout (Salmo gairdneri). J. Exp. Biol. 148: 367-383, 1990. 162. "IhRs, B.L. and R.G. Boutilier. Interactions between ion exchange and metabolism in erythrocytes of the rainbow trout Oncorhynchus mykiss. J. Exp. Biol. 156: 139-151, 1991. 163. "lhrner, J.D. and W.R. Driedzic. Mechanical and metabolic response of the perfused isolated fish heart to anoxia and acidosis. Can. J. Zool. 58: 886-889, 1980. 164. van den ThiUart, G. Energy metabolism of swimming trout (Salmo gairdneri). Oxidation rates of palmitate, glucose, lactate, alanine, leucine and glutamate. J. Comp. Physiol. B 156: 511-520, 1986. 165. van den Thillart, G. and H. Smit. Carbohydrate metabolism in the goldfish (Camssius aumtus L.). Effects of long-term hypoxia-acclimation on enzyme patterns of red muscle, white muscle and liver. J. Comp. Physiol. B 154: 477-486, 1984. 166. van den Thillart, G. and R. Verbeek. Anoxia-induced oxygen debt of goldfish (Camssius aumtus L.). Physiol. Zool. 64: 525-540, 1991. 167. Vernier, J.M. and M.E Sire. In vitro study of hepatic glycogen phosphorylase in rainbow trout: its control by glucose, corticoids, adrenaline and glucagon. Gen. Comp. Endocrinol. 34: 360-369, 1978. 168. Vijayan, M.M., J.S. Ballantyne and J.ELeatherland. Cortisol-induced changes in some aspects of the intermediary metabolism of Salvelinus fontinalis. Gen. Comp. Endocrinol. 82: 476-486, 1991. 169. Vijayan, M.M., G.D. Foster and T.W. Moon. Effects of cortisol on hepatic carbohydrate metabolism and responsiveness to hormones in the sea raven Hemitripterus americanus. Fish Physiol. Biochem. 12: 327-335, 1993. 170. Vijayan, M.M. and TW. Moon. Acute handling stress alters hepatic glycogen metabolism in food-deprived rainbow trout (Oncorhynchus mykiss). Can. ]. Fish. Aquat. Sci. 49: 2260-2266, 1992. 171. Walsh, P.J. Lactate uptake by toadfish hepatocytes: passive diffusion is sufficient. ]. Exp. Biol. 130: 295-304, 1987. 172. Walsh, P.J. An in vitro model of post-exercise hepatic gluconeogenesis in the gulf toadfish Opsanus beta. ]. Exp. Biol. 147: 393-406, 1989. 173. Walsh, P.J., G.D. Foster and TW. Moon. The effects of temperature on metabolism of the American eel Anguilla rostrata (LeSueur): compensation in the summer and torpor in the winter. Physiol. Zool. 56: 532-540, 1983. 174. Walsh, P.J., TW. Moon and T.P. Mommsen. Interactive effects of acute changes in temperature and pH on metabolism hepatocytes from the sea raven Hemitripterus americanus. Physiol. Zool. 58: 727-735, 1985. 175. Walsh, P.J., C.M. Wood, S. Thomas and S.E Perry. Characterization of red blood cell metabolism in rainbow trout. ]. Exp. Biol. 154: 475-489, 1990. 176. Walton, M.J. and C.B. Cowey. Aspects of intermediary metabolism in salmonid fish. Comp. Biochem. Physiol. 73B: 59-79, 1982. 177. Washburn, B.S., M.L. Bruss, E.H. Avery and R.A. Freedland. Effects of estrogen on whole animal and tissue glucose use in female and male rainbow trout. Am. J. Physiol. 263: R1241-R1247, 1992. 178. Weber, J.M., R.W. Briil and P.W. Hochachka. Mammalian metabolite flux rates in a teleost: lactate and glucose turnover in tuna. Am. ]. Physiol. 250: R452-R458, 1986. 179. West, T.G., P.G. Arthur, R.K. Suarez, C.J. Doll and P.W. Hochachka. In vivo utilization of glucose by heart and locomotory muscles of exercising rainbow trout (Oncorhynchus mykiss). J. E~. Biol. 177: 63-79, 1993. 180. White, A. and TC. Fletcher. The effect of physical disturbance, hypoxia and stress hormones on serum components of the plaice, Pleuronectes platessa L. Comp. Biochem. Physiol. 93A: 455-461, 1989. 181. Woo, N.Y.S. Metabolic and osmoregulatory changes during temperature acclimation in the red sea bream, Chrysophrys major, implications for its culture in the subtropics. Aquaculture 87: 197-208, 1990. 182. Wood, C.M. and S.E Perry. Respiratory, circulatory and metabolic adjustments to exercise in fish. In: Circulation, Respiration and Metabolism, edited by R. Gilles, Berlin, Springer-Verlag, pp. 2-22, 1985. 183. Wood, C.M., P.J. Walsh, S. Thomas and S.E Perry. Control of red blood cell metabolism in rainbow trout after exhaustive exercise. ]. Exp. Biol. 154: 491-507, 1990.

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184. Wright, RA., S.E Perry and T.W. Moon. Regulation of hepatic gluconeogenesis and glycogenolysis by catecholamines in rainbow trout during environmental hypoxia. J. Exp. Biol. 147: 169-188, 1989. 185. Young, J.D., S.Y.-M. Yao, C.M. Tse, A. Davies and S.A. Baldwin. Functional and molecular characteristics of a primitive vertebrate glucose transporter. Studies of glucose transport by erythrocytes from the Pacific hagfish (Eptatretus stouti). J. Erp. BioL 186: 23-41, 1993. 186. Yu, K.L. and N.Y.S. Woo. Metabolic adjustments of an air-breathing teleost, Channa punctata, to acute and prolonged exposure to hypoxic water. J. Fish BioL 31: 165-175, 1987. 187. Zhang, J., M. D~ilets and T.W. Moon. Evidence for the modulation of cell calcium by epinephrine in fish hepatocytes. Am. J. PhysioL 263: E512-E519, 1992. 188. Foster, G.D., J. Zhang and T.W. Moon. Are cell redox or lactate dehydrogenase kinetics responsible for the absence of gluconeogenesis from lactate in sea raven hepatocytes? Fish PhysioL Biochem. 13: 59-67, 1994. 189. Navarro, I. and T.W. Moon. Glucagon binding to hepatocytes isolated from two teleost fishes, the American eel and the brown bullhead. J. EndocrinoL 140: 217-227, 1994.

Hochachka and Mommsen (eds.), Biochemistry and molecular biology of fishes, vol. 4 9 1995 Elsevier Science B.V. All rights reserved. CHAPTER 5

Metabolism of the swimbladder tissue BERND PELSTER Institut far Physiologic, Ruhr-Universiti~t Bochum, D-44780 aochum, Germany

I. II. III.

Introduction Histology of the swimbladder Energy metabolism 1. Aerobic metabolism 2. Anaerobic glycolysis 3. Pentose phosphate shunt 4. Synopsis IV. Metabolism and gas deposition: the single concentrating effect V. Control of swimbladder metabolism VI. Role of carbonic anhydrase VII. Metabolism of rete capillaries VIII. Antioxidants IX. References

I. Introduction Many fish posses a gas-filled swimbladder as a hydrostatic organ in order to achieve neutral buoyancy. As a compliant bladder it obeys Boyle's law and changes pressure and volume with changes in hydrostatic pressure, i.e. with water depth. The hydrostatic pressure increases by about 1 atmosphere for every 10 m of water depth. To keep the bladder volume constant and thus to remain neutrally buoyant during vertical migrations a descending fish has to deposit gas into the bladder to compensate for the compression of the bladder, an ascending fish has to resorb gas to compensate for the expansion of the bladder caused by the changes in hydrostatic pressure. Neutral buoyancy usually is achieved with the bladder making up some 5% or 8% of the body volume in marine and freshwater fish, respectively. The swimbladder is filled only by diffusion of gases from the blood or from the swimbladder epithelium into the lumen 19,49. Therefore, at great depths very high gas partial pressures must be generated in the blood in order to maintain a positive pressure gradient towards the lumen, although the pressure of gases dissolved in water remains close to 1 atmosphere, irrespective of the water depth is. The initial step in the generation of very high partial pressures is the secretion of metabolites into the blood which decrease the physical solubility of gases in the blood or liberate gases from a chemical binding site (the single concentrating effect) 34. According to Henry's law the decrease in solubility results in an increase

102

B. Pelster

in gas partial pressures. In a counter-current system, the rete mirabile of the swimbladder, the venous blood is then brought into close contact to its own arterial supply, in which gas partial pressures are not yet elevated. Along the partial pressure gradients gas molecules diffuse back to the arterial side and the initial increase in gas partial pressure (single concentrating effect) is further enhanced by back-diffusion and counter-current concentration. Depending on the magnitude of the single concentrating effect and on the properties of the counter-current exchanger finally gas pressures of several hundred atmospheres can be achieved, sufficient to explain the occurrence of fishes with a gas-filled swimbladder at a depth of several thousand meters 31034,62. A crucial step in the functioning of the swimbladder thus is the production and release of metabolites that modify the gas carrying properties of blood passing the tissue. The morphological arrangement of the various layers of swimbladder tissue provides close contact of the cells producing these metabolites, the gas gland cells, to the vascular system, and also ensures accumulation of gas molecules in the bladder, and not in the surrounding tissues. A brief discourse on the morphology and histology of the bladder, therefore, appears to be worthwhile before concentrating on the metabolism of the swimbladder tissue and especially of the gas gland cells.

II. Histology of the swimbladder The swimbladder originates as an unpaired dorsal outgrowth of the foregut. During development the connection to the gut may persist (physostome fishes), but in many species it is completely lost and the swimbladder is a closed gas cavity (physoclist fishes). In adult teleosts the structural diversity in general swimbladder morphology is remarkable 61. Talking about swimbladders, usually, the secretory part of the bladder is envisaged, although it should be kept in mind that in most species either a separate resorbing bladder is present, or a special section of the secretory bladder can be closed off by muscular activity and is designed to allow for the resorption of gas. As the resorption of gases occurs by diffusion along the diffusion gradients and does not include any special metabolic features, I will focus my attention on the secretory part of the swimbladder. The wall of the secretory bladder consists of a number of thin tissue layers (Fig. 1), usually including layers of smooth muscle cells. The terminology of Fange 17 distinguishes between the inner epithelium, muscularis mucosae, submucosa and tunica externa. The tunica externa represents a dense connective tissue capsule. The submucosa may be impregnated with guanine crystals29,36 or include layered lipid membranes 6, providing a low gas permeability of the swimbladder wall and thus reducing diffusional loss of gases. In physostome fishes the muscularis mucosae mainly consists of smooth muscle cells, but muscle cells may also be present in the swimbladder wall of physoclist fishes. In the innermost layer, the swimbladder epithelium, the so-called gas gland cells are found. While in the eel (Anguilla) gas gland cells are spread over the whole

Metabolism of the swimbladder tissue

103

Fig. 1. Tissue layers of the swimbladder wall (secretory part) of the European eel, AnguiUa anguilla, s = serosa; s m = submucosa; m = museularis mucosae; ep = epithelium; c = capillary. (from Dornl3; with permission).

internal epithelium of the secretory bladder, in many species (Perca, Gadus) gas gland cells are clustered together, forming a massive complex of several cell layers. In some species the compact gas gland is the result of an extensive secondary folding of the single-layered epithelium (Gobius, Syngnathus) 19,65. Gas gland cells usually are in intimate contact to the vascular system, either by an extensive capillary network underlying the epithelium, as in the eel, or by close proximity of a massive gas gland and the rete mirabile. Gas gland cells are cylindrical or cubical with a size ranging from 10-25 /zm to giant cells of 50-100/zm or even more, but the size of the cells appears not to be correlated to the water depth at which fish normally live 17,4~ The cells are polarized with some small microviUi on the luminal side, while the basal side often is more densely vacuolated and shows a number of infoldings, known from other secretory or resorbing tissues, which, however, always lack mitochondria. The meaning of these foldings is not yet understood 9,13,42. The variable density of the granulated plasma of gas gland cells may represent variable functional states, and not necessarily indicates presence of different cell types (Fig. 2) 13,42. Gas gland cells are characterized by the presence of only few filamentous or elongated mitochondria with few tubular cristae 9,13,26,42. The expression of further organelles such as Golgi apparatus, endoplasmatic reticulum and ribosomes is not consistent among different species, but they often are only poorly developed and may even be missing completely in some species, although Jasinski and Kilarski 26 and Morris and Albright 42 found a well developed Golgi complex in Perca fluviatilis

104

R Pelster

Fig. 2. Schematic drawing of gas gland cells of the swimbladder epithelium of the European eel; (from Dorn13; with permission).

and in Fundulus heteroclitus gas gland cells, respectively. The density of the Golgi complex and of related vesicular structures may increase during periods of gas deposition, as observed in Perca fluviatilis and in Acerina cemua 26. A general feature again is the presence of lipid droplets or even lipid-containing vacuoles. A comprehensive review on the significance of lipids for the achievement of neutral buoyancy has recently been published in this series s4.

III. Energy metabolism Studies on the metabolism of swimbladder tissue almost exclusively concentrate on the swimbladder epithelium and the gas gland complex. In gas gland cells the metabolites are produced that in blood provoke the reduction of gas solubility or release gases from their chemical binding sites and thus are crucial for the functioning of the swimbladder. Very little is known about the metabolism of muscular cells or the connective tissues. According to our present knowledge, however, they are not involved in the process of gas deposition and it can be assumed, that their metabolism is comparable to the metabolism known from similar cells in other tissues. Due to the morphology of the swimbladder, it is almost impossible to obtain a clean preparation of gas gland cells without contamination of smooth muscle cells, connective tissue or endothelial cells from the capillary network, which should be kept in mind when discussing the metabolism of gas gland cells.

Metabolism of the swimbladder tissue

105 TABLE 1

Activities of selected enzymes of aerobic metabolism in gas gland tissue Enzyme Citrate synthase Malate dehydrogenase Fumarase C~ochrome oxidase C~ochrome oxidase

Anguilla anguilla 4a (25~

Gadus morhua (10"C)16

1.1 4- 0.2 51.7 4. 3.9 ND ND 0.074 a

2.27 4. 0.21 159.55 4. 27.56 ND 1.06 4. 0.09

(23oc)5 ND 163 4- 5 1.47 + 0.1 21.5 4. 3.1 0.041 a

Opsanus beta 63 (24oc) 0.14 4- 0.05 9.69 4. 1.09 ND ND

Activities are given in ttmol min -1 g-1 fresh mass (4- S.E.). ND = not determined. a From reference 22.

1. Aerobic metabolism Mitochondria, representing the structural basis for aerobic metabolism, are not abundant in the swimbladder epithelium. Accordingly, activities of enzymes of the tricarboxylic acid cycle or the respiratory chain are very low (Table 1), even when compared with a purely non-oxidative tissue, the white muscle. In the cod, Gadus morhua, for example, cytochrome oxidase activity in gas gland cells represents only 20% of the activity found in white muscle tissue s . Somewhat surprising is, therefore, the result of Ball et al.2, who measured an oxygen uptake of about 0.3 ml g-1 h-1 at a temperature of 30~ for gas gland tissue, but also for slices of heart muscle, by employing the manometric Warburg procedure. On the other hand, oxygen uptake could not be stimulated by the addition of glucose and was several times lower than the lactate production in the same preparations. Similar results were obtained for cod gas gland, where catecholamines or acetylcholine stimulated lactate formation, but did not increase oxygen uptake of the tissue 16. In vitro oxygen uptake was sensitive to changes in the sodium concentration and a decrease in sodium or a replacement of NaCI by KCI in the saline solution significantly decreased oxygen consumption. Oxygen uptake has also been measured in saline-perfused swimbladder preparations 46 and in situ in anesthetized, immobilized European eels (Anguilla anguilla) 5~ In situ oxygen consumption of the swimbladder tissue of the European eel was about 3.8-times higher than in saline-perfused swimbladder preparations, indicating an increase in metabolic activity during periods of gas deposition. In saline-perfused swimbladders gas deposition was not measurable, while in the anesthetized eels gas was deposited at a rate of 0.48 ml h -1. Mass specific oxygen uptake of the active swimbladder tissue is in the same range as oxygen consumption of the whole organism under resting conditions. Oxygen uptake of resting fish at a temperature of 15-20~ varies between 15 and 65 nmol g-1 rain-1 (refs. 27, 53), and the weightspecific oxygen uptake of swimbladder tissue amounts to approximately 22-45 nmol g-~ min -1, assuming a mass of the swimbladder tissue of about 1-2 g for a 400-600 g eel. If the difference in oxygen consumption of saline-peffused and gas depositing swimbladders in situ represents the transition from resting to active state, oxygen

106

B. Pelster

uptake of the swimbladder tissue is lower than in other tissues, as suggested by the presence of only few mitochondria. The unsuccessful attempts to stimulate oxygen uptake of gas gland tissue in vitro 2.16 thus may indicate that gas gland cells are already stimulated maximally during preparation. Like the oxygen consumption, the rate of CO2 formation in the active gas depositing swimbladder in situ was about 3.9-times higher than the CO2 formation of saline-perfused swimbladders, and in both preparations the rate of oxygen consumption of the swimbladder tissue was much lower than the rate of CO2 formation 46.5~ Therefore, beside aerobic metabolism another metabolic pathway must be available for the formation of CO2 in the swimbladder tissue (see below). Furthermore, in vitro 2 and in situ 5~ lactate formation clearly surmounts the rate of oxygen consumption, indicating a predominant role of anaerobic glyeolysis in the swimbladder tissue. 2. Anaerobic glycolysis

Energy production via glycolysis may be fueled by glycogen stores or by extracellular glucose. Activity of phosphorylase, key enzyme for the breakdown of intracellular stores of glycogen, has not been measured, but several histological s.~3,17,26,42,Ss and biochemical studies 11'17 demonstrated the presence of glycogen in gas gland cells. Copeland s and F~inge17 reported a decrease in glycogen content of gas gland cells during swimbladder inflation, and it was originally believed that glycogen was the main source for the metabolism of these cells. Comparison of the measured rates of lactate production with the amount of glycogen stored in the cells, however, revealed that glycogen could fuel the anaerobic glycolysis only for a few minutes 11, and it is now generally accepted that gas gland metabolism is fueled mainly by blood glucose 19. In general, the enzyme activities of the anaerobic glycolytic pathway are quite high and in the range of activities measured in white muscle (Table 2), indicating a dominate role of this pathway. The production of acid by gas gland cells has been known for many years 23,37, and analyzing the kinetic properties of lactate dehydrogenase in nine species of marine fishes Gesser and Fange 22 concluded that the gas gland enzyme predominantly is the muscle type enzyme, suited for the formation of lactic acid. Gas gland tissue of various species incubated in vitro has indeed been shown to produce large amounts of lactate. In experiments using gas gland of scup, Stenotomus chrysops 2, 70-90% of the glucose taken up from the incubation medium was balanced by lactate formation, and in the study of D'Aoust 11 using gas gland of Sebastodes miniatus, glucose was almost completely converted to lactate. As already observed for the aerobic metabolism, a decrease in sodium concentration of the incubation medium also caused a decrease in lactate formation in Stenotomus chrysops gas gland2. It is also interesting to note that, although enzyme activities of the anaerobic glycolysis in gas gland are comparable to white muscle, the maximal rate of lactate formation observed in vitro is several times lower than in white muscle 16. In most tissues the rate of anaerobic glycolysis decreases very much with increasing oxygen

Metabolism of the swimbladder tissue

107 TABLE 2

Activities of selected enzymes of anaerobic glycolysis in gas gland tissue Enzyme

Anguilla anguilla48 Gadusmorhua (25~

Hexokinase Phosphofructokinase Phosphoglucose isomerase Glyceraldehydephosphate dehydrogenase Glycerolphosphate dehydrogenase Pyruvate kinase Lactate dehydrogenase Lactate dehydrogenase

(10~

1.2 4. 0.2 10.1 4- 1.3 ND 79.8 4- 1.7

16.13 4- 1.33 26.51 4- 1.44 ND ND

ND

ND

123 4- 22 190 4- 51 46 a

209.4 4- 23.9 1203 4. 99

Opsanus beta 63

(23oc)5

(24oc)

1.45 4. 0.11 ND 0.71 4- 0 . 0 5 23.964- 2.78 ND 29.15 4- 1.82 ND 29.97 4- 3.52 4.80+

0.48

345 4- 25 1154 4- 46 134 a

ND

ND 173.8 4. 18.4

Activities are given in/zmol min -1 g-I fresh mass (4- S.E.). ND = not determined. a From reference 22.

t e n s i o n ( P a s t e u r effect). I n c u b a t i o n of gas gland cells with 9 5 % oxygen a n d 5 % CO2 ( t e l 2) o r e v e n with h y p e r b a r i c oxygen p r e s s u r e (51 a t m o s p h e r e s ) 11, however, did n o t result in a r e d u c e d rate of lactate f o r m a t i o n , clearly indicating a b s e n c e of the P a s t e u r effect. P r o d u c t i o n o f lactate has b e e n shown in a R i n g e r - p e r f u s e d p r e p a r a t i o n of the E u r o p e a n eel, but, as o b s e r v e d for Stenotomus chrysops gas gland in vitro 2, it did n o t c o m p l e t e l y b a l a n c e the c o n c o m i t a n t u p t a k e of glucose, indicating that p a r t of the glucose was shifted to s o m e o t h e r m e t a b o l i c pathway 46. In vivo, lactate f o r m a t i o n has only b e e n d e m o n s t r a t e d in two species, n a m e l y the b a r r a c u d a a n d the E u r o p e a n eel, w h e r e b l o o d samples w e r e o b t a i n e d by b l o o d vessel p u n c t u r e , b u t b l o o d flow could not be m e a s u r e d . As s h o w n in Table 3 the v e n o - a r t e r i a l c o n c e n t r a t i o n difference varies b e t w e e n 1 a n d 5 m m o l 1-1, a n d t h e highest lactate c o n c e n t r a t i o n m e a s u r e d in a s w i m b l a d d e r b l o o d s a m p l e was TABLE 3 Lactate release from gas gland tissue into the blood measured as concentration difference between venous efltux from (re) and arterial influx into (ai) the swimbladder tissue, and the highest lactate concentration value measured in blood samples obtained by blood vessel puncture Species

A Lactate (re - ai) (/zmol m1-1)

Lactatemax (/tmol m1-1)

European eel 33 European eel6~ European eel 3~ Barracuda 14

1.3 4- 1.4 2.6 4- 1.8 2.0 4- 1.5 4.4 4- 1.0

15.1 20.9 a 11.4 a 12.6

Values are presented as means 4- SD. Indicates blood samples obtained from the swimbladder pole of the rete after passage of the gas gland (vi), where the highest lactate concentration is expected to occur. European eel = Anguilla anguilla L.; barracuda = Sphyraena barracuda. a

108

R Pdster

20.9 mmol 1-1. In a recent study, lactate and glucose balance of the active, gas depositing swimbladder of the eel could be measured in situ revealing that about 7580% of glucose taken up from the blood were converted to lactate 51. As aerobic metabolism cannot account for the difference in glucose uptake and lactate secretion, this study again indicates that part of the glucose is shifted to additional metabolic pathways. A likely candidate appears to be the pentose phosphate shunt 46. 3. Pentose phosphate shunt

Freshly deposited gas may contain remarkable fractions of CO2, sometimes even up to 30% (ref. 64). Therefore, the assumption that CO2 originates almost exclusively from the HCO 3 pool of the blood has been questioned, and as the respiratory exchange ratio of gas gland tissue usually exceeds 1 (see above) aerobic metabolism cannot be held responsible. Fringe 18 included the pentose phosphate shunt as a possible origin of CO2 in his schema of gas gland metabolism, but did not present experimental evidence except for activities of two enzymes of the shunt, measured in gas gland of the cod 5. In vitro traces of uniformly labeled, but not of C3,4 labeled, glucose were converted to CO2 by gas gland of Sebastodes miniatus, probably in the pentose phosphate shunt 11. Glucose uptake of the tissue, however, was completely balanced by the formation of lactate and the author concluded that the pentose phosphate shunt was of no significance for the tissue. Gas gland tissue of various fishes indeed is characterized by the presence of key enzymes of the pentose phosphate shunt (Table 4), and in European eel their activity is even comparable to the activity measured in liver tissue, which is known for the presence of this shunt 48. More direct evidence for the existence of metabolic pathways generating CO2 without concomitant oxygen consumption was provided by analyzing the gas exchange and glucose metabolism of saline-perfused eel swimbladder preparations 46 and of the active, gas depositing swimbladder in situ 5~ In both preparations, the production of CO2 by the tissue by far exceeded the rate of oxygen uptake and the glucose uptake could not be balanced by lactate formation, indicating that part of the glucose was metabolized in additional metabolic pathways, presumably the pentose phosphate shunt. In a recent study, the involvement of this pathway in gas gland glucose metabolism could be demonstrated for Opsanus beta 63. The ratio of CO2 formed from C1

TABLE 4 Activities of selected enzymes of the pentose phosphate shunt of gas gland tissue .

.

.

.

Enzyme

Anguilla anguilla 4s

Glucose-6-phosphate dehydrogenase 6-Phosphogluconate dehydrogenase Transaldolase Transketolase

2.7 4- 0.5 0.7 4- 0.2 ND ND

.

.

.

.

.

.

.

Gadus morhua 5 .

.

1.99 4- 0.11 0.92 4- 0.07 ND ND

Opsanus beta 63 .

2.22 1.89 0.29 0.10

+ 0.29 4- 0.12 4- 0.04 4- 0.02

Activities are given in/tmol min -1 fresh mass (4- S.E.), measured at 23-25"C. ND = not determined.

Metabolism of the swimbladder tissue

109

and C6 labeled glucose reached a value of 3.4 under normoxia and of 6.4 under hyperoxia, clearly indicating a significant contribution of the pentose phosphate shunt to glucose metabolism. 4. Synopsis

A summary of the metabolic pathways involved in gas gland metabolism is given in Fig. 3. Glucose, representing the main fuel, and oxygen are removed from the blood. The main fraction of glucose is converted to lactate, which in turn is released into the blood. Part of the glucose is shifted t o the pentose phosphate shunt and decarboxylated in the 6-phosphogluconate dehydrogenase reaction. Along the diffusion gradient, the CO2 diffuses into the swimbladder lumen, but also into the blood 46. Only a very small fraction of glucose is oxidized in the aerobic metabolism, also forming CO2.

Fig. 3. Present concept for pathways of glucose metabolism and CO2 formation in gas gland cells. AGI = anaerobic glycolysis; CA = carbonic anhydrase; GA-3-P = glyceraldehyde-3-phosphate; G-6-P = glucose-6-phosphate; PPS = pentose phosphate shunt; TCA = tricarboxylicacid cycle.

B. Pelster

110 TABLE 5

Contribution of various metabolic pathways to total glucose metabolism and total CO2 production of gas gland tissue of the European eel, Anguilla anguilla, at 22-24~

Glucose (/zmol min -1) CO2 (#mol min -1)

Total

Lactate formation

Aerobic metabolism

Pentose phosphate shunt

0.72 0.20

0.58 -

0.007 0.041

0.14 0.16

Glucose uptake and CO2 formation not accounted for by aerobic or anaerobic glycolysis are listed under pentose phosphate shunt, although it cannot be excluded that some glucose was converted to glycogen (see text for further explanations). Data from references 50 and 51.

A quantitative assessment of the contribution of these pathways to glucose metabolism in the eel swimbladder is given in Table 5. According to the oxygen consumption of swimbladder tissue only 1% of the glucose removed from the blood is completely oxidized in the aerobic metabolism, leaving 99% for anaerobic glycolysis and the pentose phosphate shunt. Aerobic metabolism can explain only 20% of the CO2 formation. To account for the additional CO2 about 22% of the glucose has to be decarboxylated in the pentose phosphate shunt, leaving the main fraction of glucose metabolism for anaerobic glycolysis. Depending on the reaction sequence, glucose shifted to the pentose phosphate shunt may, after decarboxylation in the 6phosphogluconate dehydrogenase reaction, finally also be converted to lactate if the pentose is metabolized to glyceraldehyde-3-phosphate and thus enter the glycolytic pathway (Fig. 3). Nevertheless, these results clearly demonstrate that the main fraction of glucose is fermented directly in the anaerobic glycolysis because glucose uptake from the blood by far exceeds the formation of CO2 in the tissue.

IV. Metabolism and gas deposition: the single concentrating effect The importance of gas gland metabolism for swimbladder function can be demonstrated by plotting the rate of lactate formation or the acidification of the blood during passage of the gas gland tissue versus the rate of gas deposition: gas deposition increases with increasing acidification of the blood as well as with increasing lactate formation (Fig. 4). Lactic acid and CO2 are released from gas gland cells and in blood initiate the single concentrating effect, the first step in the generation of high gas partial pressures and in gas deposition, for inert gases and also for oxygen and CO2. The increase in blood lactate concentration reduces the physical solubility of gases in blood via the salting-out effect, which is of special importance for inert gases. The increase in lactate concentrations measured in vivo ranges between 1 and 5 mmol 1-1 with maximum lactate levels of about 21 mmol 1-1 (Table 3), resulting in a solubility reduction of only 1% or even less45,47, but nevertheless may be sufficient to allow for a 10- to 15-fold increase in inert gas partial pressure in the counter-current system of the rete mirabile31.

Metabolism of the swimbladder tissue

111

Fig. 4. Correlation between blood acidification, represented by the ven0-arterial pH differenceS~ or lactate release by the gas gland cellssl (bottom) and the rate of gas deposition in the swimbladder of the European eel (Anguilla anguilla).

Oxygen in blood is bound to hemoglobin, and protons reduce the oxygen affinity of the hemoglobin (Bohr effect). Hemoglobin of fish with a swimbladder usually is characterized by the Root effect, that is by a reduction in oxygen-carrying capacity with increasing proton concentration 57. At low pH, Root hemoglobins are fixed in the deoxygenated state and do not bind oxygen even at high oxygen tensions 52 56 The release of lactic acid and of CO2 from gas gland cells acidifies the blood and thus provokes a release of oxygen from the hemoglobin via the Root effect. According to the pH values measured in swimbladder blood about 40% of the hemoglobin may be deoxygenated during passage of the swimbladder .

.

112

B. Pelster

tissue, resulting in a tremendous increase in gas partial pressure s2. In the rete mirabile this large single concentrating effect may be multiplied v/a back-diffusion and counter-current concentration, allowing for the generation of a gas pressure of several hundred atmospheres 31,34,62.

V. Control of swimbladder metabolism Gas gland metabolism is of pivotal importance for swimbladder function and with increasing acid production and acid release of the cells, the rate of gas deposition increased 5~ while inhibition of lactate production by injection of oxamie acid, an inhibitor of lactate dehydrogenase, into the bladder drastically decreased the rate of gas deposition 11. The description of 'active' and 'inactive' gas glands in the literature 17,1s,26 and the significantly reduced rate of acid secretion under hypoxic conditions in the eel swimbladders~ clearly indicate the presence of an effective control system. Cutting the vagus abolishes the deposition of gas, and the swimbladder metabolism thus appears to be under vagal control 4.zl,3s. The presence of aeetylcholine esterase in gas gland tissue and the inhibitory effect of atropine on gas deposition may indicate that cholinergic fibers are involved, adrenergic fibers could not be detected 1,2~ Attempts to stimulate gas deposition by parasympathicomimetic drugs or by electrical stimulation of the vagus, however, have been unsuccessful 17. Very little is known about control mechanisms at the cellular level. In gas gland of the bluegill sunfish (Lepomis macrochirus) presence of glucagon in the incubation medium stimulated the formation of lactate from internal glycogen stores 12. The concentration of glucagon applied, however, appears to be quite high (30 mg 1-1), and the main fuel for metabolism is blood borne glucose and not the internal glycogen stores (see above). Epinephrine has been shown to have n o effect on lactate production in bluegill sunfish 12, while a significant stimulation has been observed in cod gas gland in vitro 16. The possible importance of epinephrine for regulation of the metabolism is not obvious because adrenergic fibers have not been detected in gas gland tissue, and a stimulatory effect on metabolism would counteract the well known inhibitory effect of catecholamines on swimbladder perfusion 19. The important feature of the metabolism is the production of acid (lactic acid and CO2) and its release into the blood stream. An interesting question is the mechanism of acid release and its implications on the metabolism. Analysis of the acid base changes in blood during passage of the gas gland tissue revealed that proton extrusion mainly occurs via CO2 after dehydration of HCO~, probably with recycling of the HCO 3 mediated by an anion exchanger (Fig. 3). Only about 25% of the total acidification of the blood can be ascribed to proton release 32. If proton release occurs along a proton gradient, the intracellular pH must be low because pH values of 6.5-7.0 have been measured in the blood 32,6~ A high buffer capacity of gas gland cells63 will provide stability of the internal milieu, but will not change possible gradients for proton extrusion. At low pH, glycolytic activity is

Metabolism of the swimbladdertissue

113

depressed in most tissues, but Ewart and Driedzic ~6 reported a 3-times higher rate of lactate production at pH 6.5 compared to pH 7.8 at 10*C for cod gas gland, which may indicate special adaptations of glycolytic enzymes to Operate at low pH. A comparison of white muscle and gas gland phosphofructokinase, however, revealed no difference in the pH optimum 48. Beside the acid, the gas gland metabolism also has to cope with a high tension of oxygen. Presence of oxygen usually diminishes or even abolishes the production of lactate in the anaerobic glycolysis, which would impair gas deposition into the swimbladder. Accordingly, gas gland tissue does not show the Pasteur effect 11. Control site usually is the enzyme phosphofructokinase and one may speculate that this step is by-passed in glucose metabolism by shifting glucose towards the pentose phosphate shunt and entering the glycolytic pathway at the glyceraldehyde3-phosphate level (Fig. 3). The low activities of the enzymes transaldolase and transketolase are not in favor of this hypothesis63 (Table 4), and the dominating role of anaerobic glycolysis has been demonstrated by the comparison of glucose uptake and CO2 formation (see above). With the pentose phosphate shunt contributing no more than 20-25% to glucose metabolism, we need to look for control mechanisms in the anaerobic glycolytic pathway and one possibility appears to be that the decrease in phosphofructokinase activity typically observed on oxygenation is prevented by the action of some regulatory cofactor. The inhibitory effect of increasing ATP concentrations on the gas gland enzyme is not different from the effect on the white muscle enzyme48, but cellular ATP levels in vivo are unknown and there are other cofactors or metabolites, like citrate or ADP, which could be of importance.

VI. Role o f carbonic anhydrase Based on biochemical measurements, the presence of carbonic anhydrase in swimbladder epithelium has been known for a long time 11,17,39,59. Histological analysis also demonstrated the presence of carbonic anhydrase in the swimbladder epithelium of the eel, and the staining was especially intense in the basal region of the cells 13. More detailed studies on the localization are not yet available and it is unknown whether a membrane bound enzyme, perhaps facing the extracellular space, is present. Results demonstrating the presence of carbonic anhydrase in the rete mirabile are equivocal. Biochemical studies indicate presence of the enzyme in rete membranes 17,39,s9. The very high activity of carbonic anhydrase in red cells, however, renders the measurement of enzyme activities in tissue homogenates very sensitive to contaminations with erythrocytes, and histological studies failed to demonstrate the enzyme in rete membranes 13. The significance of carbonic anhydrase for swimbladder function has been verified in a number of studies: inhibition of the enzyme by injection of sulfonamide into the bladder decreases gas deposition 17'39.59. Kutchai 35 observed a decrease in lactate production after inhibition of carbonic anhydrase, but an interpretation

114

B. Pelster

Fig. 5. Possible pathways for proton transfer from gas gland cells to the erythrocytos and the role of carbonic anhydrase. CA = carbonic anhydrase; Hb = hemoglobin.

of his results is somewhat difficult because at a concentration of 10-4 mol 1-1, employed in this study, acetazolamide is known to inhibit anion exchange. Figure 5 presents a possible sequence of reactions elucidating the role of carbonic anhydrase for swimbladder function. To initiate the single concentrating effect, protons, generated in the anaerobic metabolism, must be excreted into the blood. Lactic acid formation clearly dominates the formation of CO2 in the pentose phosphate shunt, and an increase in proton concentration in the gas gland cells will shift the equilibrium of the reaction H + + HCO~ ~x - CO2 +

H20

(1)

towards formation of CO2. Uncatalyzed this reaction is very slow, but presence of carbonic anhydrase in the cell results in an immediate equilibration, providing the highly diffusive gas CO2. CO2 easily crosses cell membranes and enters the erythrocytes. In the red cells, the reverse reaction occurs, again catalyzed by carbonic anhydrase, liberating the H +, which in turn will liberate oxygen from the hemoglobin via the Root effect. HCO~ returns to the plasma in exchange for 121by the Band III anion exchanger. To avoid a HCO]" depletion of the gas gland cells, the molecule must be recovered from the plasma, closing the circle. Entry of the negatively charged HCO]" into the cell may at least partly compensate for the loss of

Metabolism of the swimbladder tissue

115

negative charges due to the extrusion of lactate into the blood. Another possibility for lactate release would be non-ionic diffusion as lactic acid, which appears to be not very likely24.

VII. Metabolism ofrete capillaries The common eel has a bipolar rete mirabile, allowing access to blood vessels proximal and distal to the rete as well as complete isolation of the rete mirabile. Rasio 55 took advantage of this morphology and analyzed the glucose metabolism of isolated rete mirabile preparations. Anaerobic glycolysis appears to be the main root for glucose metabolism and lactate formation accounted for more than 95% of the glucose uptake of about 0.4 mmol g-1 rain-l, 1.9% was converted to CO2, and less than 1% incorporated into glycogen or lipids. The ratio of 1.8 for CO2 formed from C1 and C6 labeled glucose indicates the presence of the pentose phosphate shunt in rete cells, although not as pronounced as in gas gland tissue. The main fraction of CO2 appears to originate in the aerobic metabolism as indicated by the decrease in CO2 formation under cyanide inhibition of cytochrome oxidase. Elevated glucose concentrations in the incubation medium, stimulated all pathways of glucose metabolism, and especially the incorporation into glycogen and lipids5s. The metabolism with predominance of anaerobic glycolysis and presence of the pentose phosphate shunt thus shows some similarities to gas gland metabolism, and enzyme activities measured in the rete mirabile indeed are similar to those reported for gas gland tissue 4a.

Fill. Antioxidants Oxygen being the main gas in the swimbladder of most fishes, tissues of this organ are exposed to almost constant hyperoxia and probably face the highest oxygen tensions ever encountered in nature. Most tissues are highly sensitive to hyperbaric oxygen and for lung tissue, for example, development of edema and proliferation of certain cells have been described during exposure to only one or two atmospheres of oxygen, finally resulting in a loss of the respiratory function 1~ The swimbladder epithelium obviously is not severely affected by hyperoxia and it would be interesting to know whether special metabolic adaptations avert the damage usually caused by oxygen radicals. Activities of enzymes involved in the breakdown of oxygen radicals like catalase, superoxide dismutase and glutathione peroxidase have been measured in the gas gland tissue of a number of fishes. Catalase and glutathione peroxidase activity were low and not elevated compared to other tissues, only superoxide dismutase activity was significantly higher than in other tissues and the activity increased during exposure to hyperoxic conditions 43 '44 . In eel swimbladder epithelium, glutathione reductase activity has also been detected, while it was not measurable in muscle tissue (B. Pelster and P. Scheid, unpublished result). The swimbladder of trout (Salmo

116

B. Pelster

trutta) also appears to contain antioxidant mechanisms which inhibit peroxidative oxidation of polyunsaturated fatty acids 7. The importance of the pentose phosphate shunt for the formation of CO2 has already been discussed, it may also be of great significance for the degradation of oxygen radicals. In some reactions of this pathway NADPH is formed, which has to be reoxidized so as not to stop the shunt. Radical-oxidizing enzymes like glutathione reductase, however, use NADPH as a coenzyme, and the presence of the pentose phosphate shunt thus may also be linked to the defense from oxygen toxicity. In lung tissue the activity of the pentose phosphate shunt increases under hyperoxia 3,2s. In gas gland of the toadfish Opsanus beta, the ratio of CO2 formed from C1 and C6 labeled glucose was about twice as high under hyperoxia than under normoxia, clearly indicating an increased contribution of the pentose phosphate shunt to glucose metabolism under hyperoxic conditions 63 and proving its dual role for swimbladder function.

IX. References 1. Augustinsson, ICB. and R. Ftnge. Innervation and acetylcholine splitting activity of the air-bladder of fishes. Acta Physiol. $cand. 22: 224-230, 1951. 2. Ball, E.G., C.E Strittmatter and O. Cooper. Metabolic studies on the gas gland of the swim bladder. Biol. Bull, 108: 1-17, 1955. 3. Bassett, D.J.P. and A.B. Fisher. Glucose metabolism in rat lung during exposure to hyperbaric 02. I. Appl. Physiol. 45: 943-949, 1979. 4. Bohr, C. The influence of section of the vagus nerve on the disengagement of gases in the airbladder of fishes../. Physiol. 15: 494-500, 1894. 5. Bostr6m, S.L., R. Fiinge and R.G. Johansson. Enzyme activity patterns in gas gland tissue of the swimbladder of the cod (Gadus morthua). Comp. Biochem. Physiol. 43B: 473-478, 1972. 6. Brown, D.S. and D.E. Copeland. Layered membranes: a diffusion barrier to gases in teleostean swimbladders. Tissue and Cell 10: 785-796, 1978. 7. Calabrese, V., E Guerrera, M. Avitabile, M. Fama and V. Rizza. Superoxide dismutase and reduced glutathione: possible defenses operating in hyperoxic swimbladder of fish. In: Toxins, Drugs, and Pollutants in Marine Animals, edited by L. Bolis, J. Zadunaisky and R. Gilles, Heidelberg, SpringerVerlag, pp. 130-136, 1984. 8. Copeland, D.E. The stimulus of the swimbladder reflex in physoclistous teleosts. J. Exp. Zool. 120: 203-212, 1952. 9. Copeland, D.E. Fine structural study of gas secretion in the physoclistous swim bladder of Fundulus heteroclitus and Gadus callarias and in the euphysoclistous swim bladder of Opsanus tau. Z. f. Zellforschung 93: 305-331, 1969. 10. Crapo, J.D. Morphologic changes in pulmonary oxygen toxicity. Annu. Rev. Physiol. 48: 721-731, 1986. 11. D'Aoust, B.G. The role of lactic acid in gas secretion in the teleost swimbladder. Comp. Biochem. Physiol. 32: 637-668, 1970. 12. Deck, J.E. Lactic acid production by the swimbladder gas gland in vitro as influenced by glucagon and epinephrine. Comp. Biochem. Physiol. 34: 317-324, 1970. 13. Dorn, E. Uber den Feinbau der Schwimmblase yon Anguilla vulgaris L. Licht- und elektronenmikroskopische Untersuchungen. Z. f. Zellforschung 55: 849-912, 1961. 14. Enns, T, E. Douglas and P.E Scholander. Role of the swimbladder fete of fish in secretion of inert gas and oxygen.Adv. Biol. Med. Phys. 11: 231-244, 1967. 15. Enns, T., P.E Scholander and E.D. Bradstreet. Effect of hydrostatic pressure on gases dissolved in water. J. Physic. Chem. 69: 389-391, 1965. 16. Ewart, H.S. and W.R. Driedzic. Enzyme activity levels underestimate lactate production rates in cod (Gadus morhua) gas gland. Can. J. Zool. 68: 193-197, 1990. 17. F~inge, R. The mechanisms of gas transport in the euphysoclist swimbladder. Acta Physiol. Scand.

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30: 1-133, 1953. 18. Finge, R. The physiology of the swimbladder. In: Comparative Physiology, edited by L. Bolis, K. Schmidt-Nielsen and S.H.P. Maddrell, Elsevier North-Holland Publishing Company, pp. 135-159, 1973. 19. Finge, R. Gas exchange in fish swim bladder. Rev. Physiol. Biochem. Pharmacol. 97: 111-158, 1983. 20. Finge, R. and S. Holmgren. Choline acetyltransferase activity in the fish swimbladder. J. Comp. Physiol. B146: 57-61, 1982. 21. Finge, R., S. Holmgren and S. Nilsson. Autonomic nerve control of the swimbladder of the goldsinny wrasse, Ctenolabrus rupestris. Acta Physiol. Scand. 97: 292-303, 1976. 22. Gesser, H. and R. F~inge. Lactate dehydrogenase and cytochrome oxidase in the swimbladder of fish. Int. J. Biochem. 2: 163-166, 1971. 23. Hall, EG. The functions of the swimbladder of fishes. Biol, Bull, 47: 79-124, 1924. 24. Heisler, N. Lactic acid elimination from muscle cells. In: Funktionsanalyse Biologischer Systeme, edited by J. Grote, Stuttgart, Gustav Fischer Verlag, pp. 241-251, 1988. 25. Huber, G.L. and D.B. Drath. Pulmonary oxygen toxicity. In: Topics in Environmental Physiology and Medicine. Oxygen and Living Processes: An Interdisciplinary Approach, edited by D.L. Gilbert, Heidelberg, Springer-Verlag, pp. 273-342, 1981. 26. Jasinski, A. and W. Kilarski. On the fine structure of the gas gland in some fishes. Z. Zellforschung 102: 333-356, 1969. 27. Johansen, K. Respiratory gas exchange of vertebrate gills. In: Gills (Society for Experimental Biology Seminar Series; 16), edited by D.E Houlihan, J.C. Rankin and TJ. Shuttleworth, Cambridge, Cambridge University Press, 1982, pp. 99-128. 28. Kimball, R.E., K. Reddy, TH. Peirce, L.W. Schwartz, M.G. Mustafa and C.E. Cross. Oxygen toxicity: augmentation of antioxidant defense mechanisms in rat lung. Am. J. Physiol. 230: 1425-1431, 1976. 29. Kleckner, R.C. Swimbladder wall guanine enhancement related to migratory depth in silver phase Anguilla rostrata. Comp. Biochem. Physiol. 65A: 351-354, 1980. 30. Kobayashi, H., B Pelster and P. Scheid. Water and lactate movement in the swimbladder of the eel, AnguiUa anguiUa. Respir. Physiol. 78: 45-57, 1989. 31. Kobayashi, H., B. Pelster and P. Scheid. Solute back-diffusion raises the gas concentrating efficiency in counter-current flow. Respir. Physiol. 78: 59-71, 1989. 32. Kobayashi, H., B. Pelster and P. Scheid. CO2 back-diffusion in the rete aids 02 secretion in the swimbladder of the eel. Respir. Physiol. 79: 231-242, 1990. 33. Kuhn, H.J., P. Moser and W. Kuhn. Haarnadelgegenstrom als Grundlage zur Erzeugung hoher Gasdriicke in der Schwimmblase yon Tiefseefischen. Nachweis der Sekretion kleiner Mengen yon Milchsiure am Scheitel der Haarnadel als Ursache des Einzeleffektes. Pfltigers Arch. 275: 231-237, 1962. 34. Kuhn, W., A. Ramel, H.J. Kuhn and E. Marti. The filling mechanism of the swimbladder. Generation of high gas pressures through hairpin countercurrent multiplication. Experientia 19:497-511, 1963. 35. Kutchai, H. Role of carbonic anhydrase in lactate secretion by the swimbladder. Comp. Biochem. Physiol. 39A: 357-359, 1971. 36. Lapennas, G.N. and K. Schmidt-Nielsen. Swimbladder permeability to oxygen. J. Exp. Biol. 67: 175-196, 1977. 37. Ledebur, J. v. Beitr~ige zur Physiologie der Schwimmblase der Fische. V. Ober die Beeinflussung des Sauerstoffbindungsverm~igen des Fischblutes dutch Kohlens~iure bei hohem Sauerstoffdruck. Z. Vergl. Physiol. 25: 156-169, 1937. 38. Lundin, K. and S. Holmgren. An x-ray study of the influence of vasoactive intestinal polypeptide and substance P on the secretion of gas into the swimbladder of a teleost Gadus morhua. J. Exp. Biol. 157: 287-298, 1991. 39. Maetz, J. Le role biologique de l'anhydrase carbonique chez quelques t~l~ost6ens. In: Supplements au Bulletin Biologique de France et de Belgique. Les Presses Universitaires de France, Paris, pp. 1-129, 1956. 40. Marshall, N.B. Swimbladder structure of deep-sea fishes in relation to their systematics and biology. Discovery Rep. 31: 1-122, 1960. 41. Mclean, J.R. and S. Nilsson. A histochemical study of the gas gland innervation in the Atlantic cod, Gadus morhua.Acta Zool. 62: 187-194, 1981. 42. Morris, S.M. and J.T. Albright. The ultrastructure of the swimbladder of the toadfish, Opsanus tau L. Cell Tissue Res. 164: 85-104, 1975. 43. Morris, S.M. and J.T Albright. Superoxide dismutase, catalase and glutathione peroxidase in the swim bladder of the physoclistous fish, Opsanus tau L. Cell Tissue Res. 220: 739-752, 1981.

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44. Morris, S.M. and J.T. Albright. Catalase, glutathione peroxidase and superoxide dismutase in the rete mirabile and gas gland epithelium of six species of marine fishes. J. Exp. Zool. 232: 29-39, 1984. 45. Pelster, B., H. Kobayashi and P. $cheid. Solubility of nitrogen and argon in eel whole blood and its relationship to pH. J. Exp. BioL 135: 243-252, 1988. 46. Pelster, B., H. Kobayashi and P. Scheid. Metabolism of the perfused swimbladder of European eel: oxygen, carbon dioxide, glucose and lactate balance. J. Exp. BioL 144: 495-506, 1989. 47. Pelster, B., H. Kobayashi and P. Scheid. Reduction of gas solubility in the fish swimbladder. In: Oxygen Transport to Tissue, edited by J. Piiper, T.K. Ooldstick and M. Meyer, New York, Plenum, pp. 725-733, 1990. 48. Pelster, B. and P. Scheid. Activities of enzymes for glucose catabolism in the swimbladder of the European eel, AnguiUa anguilla. J. Exp. Biol. 156: 207-213, 1991. 49. Pelster, B. and P. Scheid. Counter-current concentration and gas secretion in the fish swim bladder. Physiol. Zool. 65: 1-16, 1992. 50. Pelster, B. and P. Scheid. The influence of gas gland metabolism and blood flow on gas deposition into the swim bladder of the European eelAnguilla anguilla.Z Exp. Biol. 173: 205-216, 1992. 51. Pelster, B. and Scheid, P. Glucose metabolism of the swimbladder tissue of the European eel Anguilla anguilla.J. Exp. Biol., 185: 169-178, 1993. 52. Pelster, B. and R.E. Weber. The physiology of the Root effect. Adv. Comp. Environm. Physiol. 8: 51-77, 1991. 53. Peyraud-Waitzenegger, M. and P. Soulier. Ventilatory and circulatory adjustments in the European eel (Anguilla anguiUa L.) exposed to short term hypoxia. Exp. Biol. 48: 107-122, 1989. 54. Phleger, C.E Biochemical aspects of buoyancy in fishes. In: Biochemistry and Molecular Biology of Fishes. Phylogenetic and Biochemical Perspectives, edited by P.W. Hochachka and T.P. Mommsen, Amsterdam, Elsevier, pp. 209-248, 1991. 55. Rasio, E.A. Glucose metabolism in an isolated blood capillary preparation. Can. J. Biochem. 51: 701-708, 1973. 56. Riggs, A.E The Bohr effect.Annu. Rev. Physiol. 50: 181-204, 1988. 57. Root, R.W. The respiratory function of the blood of marine fishes. Biol. Bull. 61" 427--456, 1931. 58. Schwarz, A. Swimbladder development and function in the haddock, Melanogrammus aeglefinus L. Biol. Bull. 141: 176--188, 1971. 59. Skinazi, L. Eanhydrase carbonique dans deux T61(~ost6ens voisins. Inhibition de la sb.cr~.tion des gaz de la vessie natatoire chez la perche par les sulfamides. CR Soc. BioL Paris 147: 295-299, 1953. 60. Steen, J.B. The physiology of the swimbladder in the eel Anguilla vulgaris. III. The mechanism of gas secretion. Acta PhysioL Scand. 59: 221-241, 1963. 61. Steen, J.B. The swim bladder as a hydrostatic organ. In: Fish Physiology, edited by W.S. Hoar and D.J. Randall, New York, Academic Press, pp. 413-443, 1970. 62. Sund, T A mathematical model for counter-current multiplication in the swim-bladder. J. Physiol. 267: 679-696, 1977. 63. Walsh, P.J. and C.L Milligan. Roles of buffering capacity and pentose phosphate pathway activity in the gas gland of the gulf toadfish, Opsanus beta.J. Exp. Biol. 176: 311-316, 1993. 64. Wittenberg, J.B., M.J. Schwend and B.A. Wittenberg. The secretion of oxygen into the swim-bladder of fish. III. The role of carbon dioxide. J. Gen. Physiol. 48: 337-355, 1964. 65. Woodland, W.N.E On the structure and function of the gas glands and retia mirabilia associated with the gas bladder of some teleostean fishes. Proc. Zool. Soc. London 1: 183-248, 1911.

Hochachka and Mommsen (eds.), Biochemistryand molecularbiology offishes, vol. 4 9 1995 Elsevier Science B.V. All rights reserved. CHAPTER 6

Glycerophospholipid metabolism DOUGLAS R. TOCHER

NERC Unit of Aquatic Biochemistry, School of Natural Sciences, University of Stirling, Stirling FK9 4LA, Scotland, U.K.

I. Introduction II. Biosynthesis, turnover and catabolism III. Digestion, absorption and transport 1. Digestion and absorption 2. Transport 2.1. Transport from intestine to liver 2.2. Transport between liver and extra-hepatic tissues 2.3. Vitellogenin IV. Composition 1. Content 2. Head group composition 3. Fatty acyl composition 3.1. Total glycerophospholipids 3.2. Glycerophospholipid classes 4. Molecular species 5. Dietary effects 6. Adaptation to environmental factors 6.1. Temperature 6.2. Salinity and hydrostatic pressure V. Roles 1. Structural roles 2. Metabolic roles 2.1. Eicosanoid metabolism 2.1.1. Species and tissue distribution of eicosanoids in fish 2.1.2. Range of eicosanoids in fish 2.1.3. Stimuli for production of eicosanoids 2.1.4. Fatty acid precursors of eicosanoids 2.1.5. Glycerophospholipid sources of precursor fatty acids 2.1.6. Functions of eicosanoids 2.2. The phosphoinositide cycle 2.3. Other metabolism 2.3.1. Protein kinase C 2.3.2. Platelet-activating factor 3. Nutritional roles 3.1. Embryonic development 3.2. Larval diet VI. Conclusions and perspectives VII. References

120

D.R. Tocher

I. Introduction Glycerophospholipids are the major class of complex lipids characterized by a glycerol backbone with one of the primary hydroxyl groups (sn-3) esterified to phosphoric acid (Fig. 1). The secondary hydroxyl group in glycerophospholipids (sn2) is always esterified to a long-chain fatty acid, which in the majority of instances is monounsaturated or polyunsaturated. The sn-1 hydroxyl group is most commonly esterified to another fatty acid, generally saturated or monounsaturated, forming the diacyl glycerophospholipids (or phosphoglycerides). However, the sn-1 position can also contain a long aliphatic chain in c/s adS-unsaturated ether linkage in the case of the plasmalogens (alk-l-enyl acyl derivatives), or a saturated aliphatic chain in simple ether linkage (alkyl acyl derivatives) (Fig. 1). Phosphatidic acid (PtdA), a quantitatively minor glycerophospholipid, is nonetheless an important intermediate in the biosynthesis of glycerophospholipids and can be regarded as the simplest. In PtdA the glycerol backbone is esterified to two fatty acids and phosphoric acid (Fig. 1). The quantitatively important glyeerophospholipids contain nitrogenous bases, esterified to the phosphoric acid, such as choline (phosphatidylcholine, PtdCho), ethanolamine (phosphatidylethanolamine, PtdEtn) and serine (phosphatidylserine, PtdSer) or polyalcohols, such as inositol (phosphatidylinositol, Ptdlns) or glycerol (phosphatidylglycerol) (Fig. 1). Cardiolipin

CH2-O-H I CH-O-H I CH2-O-H Glycerol

CH2-O-CO-RI I CH-O-CO-R2 I CH2-O-H Diacylglycerol(DAG)

CH2-O-CO-RI I CH-O-CO-R2 I CH2-O-P-O-H Phosphatidic acid (PtdA)

CH2-O-CO-RI I CH-O-CO-R2 I CH2-O-P-O-X Diacyl glycerophospholipid

CH2-O-R1 I CH-O-CO-R2 I CH2-O-P--O-X 1-O-alkyl-2-acyl-glycerophospholipid

CH2-O-C-C-R1 I CH-O-CO-R2 I CH2-O-P-O-X 1-O-alk-1'-enyl-2-acyl-glycerophospholipid

R1 Long-chainaliphatic group, usuallysaturated or monounsaturated R2 - Long-chain aliphatic group, usuallypolyunsaturated or monounsaturated P -- PO2H X - choline (-CH2CH2N+(CH3)3), ethanolamine (-CH2CH2NH~), serine (-CH2CTI(NH+)COO-), myo-inositol(-C6H11O5), glycerol (-CH2CH(OH)CH2OH) or phosphatidylglycerol. - -

Fig. 6.1. Basic structures of the glycerophospholipids,their precursors and head groups.

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(diphosphatidylglycerol) is formed when the phosphate groups of two phosphatidic acid molecules are bridged by a third glycerol moiety esterified at the 1 and 3 positions. All the glycerophospholipids are important components of biological membranes and are not found in high concentrations elsewhere in the cell. Glycerophospholipids, phosphoglycerides and phospholipids are notsynonymous terms although the terminologies are still used in a confused manner today. Not all glycerophospholipids are phosphoglycerides, as the term phosphoglyceride should, strictly speaking, be reserved for the diacyl derivatives alone and should not include the ether-linked derivatives. Similarly, not all phospholipids are glycerophospholipids. For instance, sphingomyelin contains phosphorus and so is a phospholipid but the phosphoric acid is esterified to a sphingosine backbone and not glycerol and so is correctly termed a sphingolipid. This chapter deals exclusively with the metabolism of the major classes of glycerophospholipids (PtdCho, PtdEtn, PtdSer and Ptdlns), including the ether-linked derivatives, although, due to the dearth of information on the metabolism of the ether-linked derivatives in fish, the focus will be on the diacyl derivatives. I have endeavored to maintain this nomenclature throughout this chapter, but there are many instances where the use of phospholipid rather than glycerophospholipid has been more appropriate in the discussion of previous work and therefore for simplicity the term 'phospholipid' is often used.

II. Biosynthesis, turnover a n d catabolism The pathways of glycerophospholipid biosynthesis have not been extensively studied or elucidated in fish83. However, the existing evidence strongly suggests that the same pathways operate in fish as in mammals. Holub et al. 1~176 demonstrated the existence of glycerol-3-phosphate acyltransferase in the liver of rainbow trout (Oncorhynchus mykiss). When liver microsomes were incubated with sn-[U-14C]glycerol3-phosphate in the presence of activated fatty acid, palmitoyl-CoA, 77% of the radioactivity was recovered in total glycerophospholipids with the remainder recovered in neutral lipids. PtdA and lysoPtdA were also labeled, supporting the conclusion that glycerophospholipid and lipid biosynthesis in general proceeded via a PtdA intermediate in fish. The presence of cytidine diphosphate (CDP)-choline-l,2-diacylglycerol choline phosphotransferase has been demonstrated in the microsomes of trout liver 1~ and brain and liver from goldfish (Carassius auratus) 129. The synthesis of PtdCho from 14C-CDP-choline and 1,2-diacylglycerol (diolein) in the presence of Mg2+established that the CDP-choline pathway for the biosynthesis of PtdCho, as studied in detail in mammals, also operated in fish 101'129. There have been few studies in fish to fully characterize the biosynthetic pathways for PtdCho, PtdEtn, PtdSer, PtdIns and cardiolipin or the pathways, known in mammals, for interconversion between the glycerophospholipids. However, in a recent study, the de novo pathways of glycerophospholipid biosynthesis were investigated in trout hepatocytes and the activities of CDP-choline and CDP-ethanolamine phosphotransferases,

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PtdEtn-methyltransferase (PtdEtn ~ PtdCho) and PtdSer-decarboxylase (PtdSer PtdEtn) were demonstrated 9~ In mammals, ether-linked glycerophospholipids are formed solely via the dihydroxyacetonr phosphate pathway (see ref. 194). Briefly, fatty alcohol is formed by the NADPH reduction of fatty acyl-CoA. Fatty alcohol then reacts with fatty acyldihydroxyacetone phosphate to form alkyldihydroxyaeetone phosphate which is then reduced, specifically with NADPH, to form alkylglycerophosphate. The enzymes responsible for the synthesis and reduction of alkyldihydroxyacetone phosphate are located in the peroxisomes. Reaction with fatty acyl-CoA, removal of phosphate and reaction with CDP-base results in the formation of alkyl glycerophospholipid. Alk-l-enyl acylglycerophospholipids (plasmalogens) are formed by oxidation of the corresponding alkyacylglycerophospholipid by a microsomal enzyme requiring NADPH and molecular oxygen. Little of the above pathway has been characterized in fish, but the available evidence from studies with spiny dogfish (Squalus acanthias) appears to suggest that the biosynthesis of ether-linked glycerophospholipids in fish is via a pathway similar to that outlined above (see ref. 194). A review of muscle lipase activities in various fish species indicated that the catalytic hydrolysis of phospholipids was primarily under the control of phospholipases A1 and A2 205. Intracellular phospholipase A activities have been demonstrated in muscle tissue from rainbow trout 112, pollock (Gadus pollachius) 11, winter flounder (Pseudopleuronectes americanus) z~ and Atlantic cod (Gadus morhua) 48. Neas and Hazel 154-156 studied the activity of phospholipase A2 towards PtdCho in the microsomes of trout liver. Activity of phospholipase C has been demonstrated directly in isolated olfactory cilia from the channel catfish (Ictalurus punctatus) 39 and has been implicated indirectly in other tissues by the demonstration of a phosphoinositide cycle (see section V.2.2)1~176 but it appears that phospholipasr D activity has not been investigated in fish. Holub et al. ~02 showed that trout liver microsomes also contained acylCoA: 1-acyl-sn-glycero-3-phosphorylcholine acyltransferase activity. Therefore, enzymes required for partial catabolism of glycerophospholipids and for the reacylation of lyso-glycerophospholipids, and thus for the turnover of glycerophospholipids, have been demonstrated in fish. Catabolism of ether-linked glycerophospholipids hinges on the cleavage of the ether bond. Enzymic cleavage of the O-alkyl bond has been demonstrated in fish 194. The cleavage is considered to occur in two steps, whereby the alkyl bond is first oxidized to alk-l-enyl via a reaction involving NADPH, molecular oxygen and a pteridine cofactor, before cleavage to generate fatty aldehyde 194. The above enzyme system has yet to be directly studied in fish, and so it is not known if it will also cleave plasmalogens. Plasmalogenases, as described in mammalian brain, do not appear to have been studied in fish 194. The specificities of the enzymes involved in both de novo synthesis of the glycerophospholipids and in the turnover processes of deacylation/reacylation with respect to both head group and fatty acyl chains have important consequences in maintaining the normal glycerophospholipid class composition, the fatty acyl distribution among the glyeerophospholipids, and in the adaptation to environmental

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changes. Some of the more direct enzyme studies, discussed above, addressed this problem in relation to environmental temperature 1~176 However, there has been a considerable amount of data obtained from more indirect studies of the effects of environment on glycerophospholipid metabolism and this is summarized later (see section IV.6).

III. Digestion, absorption and transport I. Digestionand absorption Depending upon the precise nature of the diet, a significant and potentially large and consistent portion of the lipid component in the natural food of fishes will be biomembrane lipids, primarily glycerophospholipids. Unfortunately, the lack of a discrete pancreas in most teleost species has hampered studies on intestinal lipolysis in fish. In consequence, even less is known about the digestion and absorption of dietary glycerophospholipids than is known about the biosynthetic pathways. There are virtually no studies on the intestinal digestion of glycerophospholipids in fish, but it could be presumed that the mechanisms are similar to those in mammals. Therefore dietary glycerophospholipids are presumably digested by pancreatic or intestinal phospholipases resulting in the formation of 1-acyl lyso-glycerophospholipids and free fatty acids that are absorbed by the intestinal mucosal cells 96'198. Mankura et al. studied the hydrolysis of L-1palmitoyl-2-[1-14C]arachidonyl-3-sn-glycerophosphatidylcholine by carp hepatopancreas preparations TM. They found phospholipase A2 activity distributed in all the subcellular fractions, although the highest activity was located in the 10,000 g supernatant. The activity was dependent upon Ca 2+ and bile salt, consistent with a pancreatic enzyme, but had a conflicting acidic pH optimum of 5.0 TM.Whether this phospholipase activity reflects an intestinal activity or an intracellular phospholipase is, therefore, unclear. Recent work has suggested that cod pyloric caeca/pancreas contains a single, bile salt-activated, lipase activity with a wide substrate specificity including triacylglycerols, steryl esters, fatty acid methyl esters and carboxyl esters 79,8~ Whether this enzyme is also active towards phospholipid and whether cod intestine actually lacks phospholipase A2 activity is unclear. The concentration of lyso-glycerophospholipids is very low in fish plasma and so it has also been assumed that the majority of lysophospholipid is re-esterified within the intestinal mucosa before export into the circulatory system. However, studies on the incorporation of [1- 14C]palmitate and [U-14C]L-glycerol-3-phosphate into lipids in carp (Cyprinus carpio) intestinal homogenates in the presence of CTP, CDP-choline and CDP-ethanolamine showed that glycerophospholipid biosynthesis proceeded via PtdA and diacylglycerol (DAG) intermediates 11~ Therefore, mechanisms may exist in fish intestinal mucosa for the synthesis of glycerophospholipids from moieties more degraded than lyso-glycerophospholipids. Iijima and coworkers l~ have also studied the absorption of radioactivity from [l:4C]dioleoyl PtdCho force-fed to carp. At 20-28 h after dosing, radioactivity in the lipids of

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plasma lipoproteins was primarily associated with triacylglycerols followed by PtdCho and free fatty acids l~ The radioactivity associated with the sn-1 position of PtdCho was more than twice that associated with the sn-2 position 1~ These data support the hypothesis that the majority of glycerophospholipids are digested and absorbed v/a 1-acyl lyso-glycerophospholipid intermediates with re-estedfication before export from the intestinal cells. Z Transport

There are several reviews that between them cover the area of lipid transport in fish very thoroughly 16,66,n6,2~ This section will give an overview of the subject particularly focusing on the role of glycerophospholipids in lipoprotein structure and function without major consideration of the apoprotein constituents. 2.1. Transport from intestine to liver As in mammals, glycerophospholipids are transported from the intestine in the form of lipoproteins. The re-esterification reactions occur primarily in the endoplasmic reticulum leading to the production of chylomicron-like and very low density lipoprotein (VLDL)-like particles in the lumen as observed in carp 162, tench (Tinca tinca) 163 and trout 1s,2~176 Studies in trout showed that lipid load and degree of unsaturation affected the relative production of the lipoproteins, with high dietary lipid and polyunsaturated fatty acids (PUFA) leading to the production of larger chylomicrons, whereas high dietary saturated fatty acids resulted in the production of smaller VLDL particles 2~ The effects of glycerophospholipid content and composition on the production of intestinal lipoproteins has not been studied. However, as trout chylomicrons contain about 8% phospholipid and trout VLDL contains approximately 21% phospholipid ~,67,69,211, it is possible that variable proportions of dietary phospholipid could be accommodated by varying the relative proportions of the intestinal lipoproteins produced. In mammals, the intestinal lipoproteins are transported from the intestine almost exclusively v/a the lymphatic system. It appears that in fish such as trout and tench the majority of the intestinal lipoproteins are similarly transported via the lymphatic system31,32,163,21~before appearing in the circulatory system47'204,212 and delivery to the liver. In carp, however, it was shown that intestinal lipoproteins may be transported exclusively and directly to the liver v/a the portal system 162. This pathway may also operate for a portion of intestinal lipoproteins in other fish such as trout and tench. 2. 2. Transport between liver and extra-hepatic tissues Due to their amphipathic nature, glycerophosholipids are integral components of the plasma lipoproteins responsible for the transport of the neutral lipid classes such as triacylglycerol and steryl esters that are insoluble in aqueous solvents. As well as chylomicrons and VLDL, mammalian plasma also contains low density lipoproteins (LDL), intermediate density lipoproteins (IDL) and high density lipoproteins (HDL) 13~ The lipoproteins vary in size and structure, as well as in protein'lipid

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ratios and in the relative proportions of the different lipid classes leading to the density differences which have been used traditionally to separate and classify the different types 13~ Fish plasma contains a similar range of lipoproteins 16,66,2~ Although there are differences in detail, the general size, structure and composition of the plasma lipoproteins are comparable throughout the vertebrates, including fish 46. In most fish virtually all glycerophospholipid is transported in the plasma in the form of lipoprotein but analysis of an albumin-like protein from carp plasma showed that it contained 22% lipid of which 15% was phospholipid, predominantly PtdCho 151. This accounted for approximately 50% of the total plasma lipid in carp, with the remainder being transported via the lipoproteins ~5~ Total phospholipids account for 8, 21, 25 and 29% of the total weight of chylomicrons, VLDL, LDL and HDL, respectively47,67,69,211. However, as a percentage of the total lipids, the proportion of total phospholipids in trout lipoproteins ranges from under 9% in chylomicrons to 23% in VLDL, 35% in LDL and 53% in H D L 47'67'69'211. Similar levels of phospholipids were found in serum VLDL, LDL and HDL from Pacific sardine (Sardinops caerulea) 125, HDL from pink salmon (Oncorhynchus gorbusha) 158 and HDL from chum salmon (Oncorhynchus keta) 8. Most of the above studies were performed with fed fish. Iijima et al. 1~ compared the lipid composition of carp plasma lipoproteins under starved and fed conditions. The carp lipoproteins contained proportionally more phospholipid than trout, salmon or sardine lipoproteins with the total lipid from VLDL, LDL and HDL containing 30, 58 and 82% phospholipid, respectively, in fed fish 1~ The proportion of phospholipid in the total lipid was not significantly altered by starvation in the carp 1~ In a later study, lijima et al. l~ studied the absorption and transport of radioactivity from [1-14C]dioleoyl PtdCho fed to carp. At 20-28 h after dosing, radioactivity was primarily associated with HDL followed by LDL and VLDL 1~ Chylomicrons are produced exclusively in the intestine, but although some VLDL can also be synthesized in the gut as described above, the majority of VLDL in the plasma is synthesized in the liver, at least in rainbow trout ~aa,2~ The major enzymes of lipoprotein metabolism and remodelling, including lipoprotein lipase (LPL) and hepatic lipase (HL), have been shown in trout and cod tissues 34,36. Lecithin:cholesterol acyl transferase (LCAT), a plasma enzyme which catalyzes the esterification of cholesterol using fatty acid from PtdCho, has been demonstrated in the plasma of trout 35, carp 11a and char (Salvelinus alpinus) 57. Lecithin:alcohol acyltransferase, which catalyzes the transfer of an acyl group from PtdCho to longchain alcohols has been shown in carp plasma 139 and may be, at least partially, responsible for the surprisingly high level of circulating wax ester reported in that species 139,14~ In addition, intermediate density lipoprotein (IDL), a fraction with density between that of VLDL and LDL has been fractionated from trout serum 14. The presence of the whole spectrum of lipoproteins, including IDL, and the enzymes described above strongly suggests that lipoprotein remodelling processes, as characterized in mammals, also occur in fish. Therefore, triacylglycerols in chylomicrons and VLDL are hydrolyzed by LPL and HL at tissue sites with the hydrolysis products being absorbed. Excess surface constituents, including phospholipids, 'bud off' as nascent HDL particles (similar to HDL3), which can

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also be secreted by the liver. HDL3 can take up free cholesterol from peripheral tissues which is then esterified by the action of LCAT resulting in the production of mature HDL (HD~). Remnants of ehylomierons and VLDL hydrolysis can be taken up by the liver, but further action by LPL and HL leads to the formation of LDL via IDL. As in mammals, the relative proportions of the plasma lipoproteins in fish can vary from species to species, but is a constant characteristic of each species depending upon dietary status. In trout, HDL is the predominant class ranging from 0.5-2.3 g/dl, followed by LDL (0.2-1.1 g/dl) and then VLDL (0.1-0.7 g/dl) 47,67,69,211. HDL was also the main lipoprotein class in carp 6, sea bass (Dicentrarchus labrax) 193, pink salmon (O. gorbusha) 158, chum salmon ls2 and channel catfish 136. HDL appeared to be absent from the plasma of carp 15~ The relative amounts of HDL, LDL and VLDL vary with age, nutrition and sexual cycle66. In common with most vertebrates, PtdCho appears to be the predominant glycerophospholipid class in fish lipoproteins 46,158. However, the precise phospholipid class composition in fish plasma lipoproteins has been rarely reported in the above studies. Similarly, the precise fatty acid compositions of individual glycerophospholipid classes have not been extensively studied. The fatty acid compositions of total lipids and total phospholipids have been reported. Fish lipoproteins generally contain higher levels of PUFA, particularly (n-3)PUFA, than the corresponding mammalian lipoproteins ~i,nT.158,211,2n. In trout the phospholipid fractions from all lipoproteins were particularly rich in 16:0 and 22:6n-3, and total (n-3)PUFA was higher and total (n-6)PUFA lower, in phospholipids in comparison with triacylglycerols47'67'69'127. However, the levels of (n-3)PUFA in the cholesteryl ester fractions of LDL and HDL exceeded those of the phospholipids 47,67,69,127. The exact fatty acid composition of the plasma lipoproteins were affected by diet, both acutely, particularly with chylomierons and VLDL after a meal, and chronically as seen with essential fatty acid-deficiency in trout 7~ The apoprotein compositions of fish lipoproteins are similar to mammalian lipoproteins with the major apoproteins being apoprotein A (I and II) in HDL, apoprotein B in LDL and mixtures of apoproteins B, C, and E in VLDL and A, B and C in chylomicrons6,13,15,16,'ui'47,136,ls8,lsS'211.There are few direct studies on the functions of the different apoproteins in fish, but it is likely that they have the same metabolic functions such as receptor binding (apoproteins B and E) and enzyme activation (AI and LCAT; CII and LPL) as in mammalian systems~37. Consistent with this, it was shown that trout adipose tissue LPL was activated by the apoprotein fraction of trout HDL (mainly AI and C)ss. There are virtually no reports of studies on the uptake of phospholipid from the plasma lipoproteins in fish. However, by analogy with the system characterized in mammals, phospholipids can probably be taken up into the tissues by two or three main mechanisms 137. Probably the most important pathway, quantitatively, is receptor-mediated endocytosis via B/E and E receptors. These are important pathways for LDL (apo B), VLDL- and ehylomieron-remnants (apo B and E) and HDL (apo E, especially in HDL1, an apo E-rich variant). These receptors are found on various tissues including liver. In mammals the precise tissue distribution

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varies between different species. Probably all the lipoproteins, but particularly LDL and HDL, can be taken up by tissues via non-specific pinocytosis. For instance, in liver approximately 30% of LDL uptake is via a non-receptor-mediated pathway. Finally, it may be that surface components of VLDL and chylomicrons, including phospholipids, may be taken up or exchanged via direct interaction with the endothelial cell membranes in the tissues.

2.3. Vitellogenin (very high density lipoprotein) Another lipoprotein class in fish important in the transport of phospholipids is vitellogenin which is only found in mature oviparous females or estrogeninjected fish 43,55,166,217,248. Vitellogenin has a density higher than HDL and has been designated very high density lipoprotein I (VHDL 1)149 and contains about 80% protein and 20% lipid in rainbow trout 43,7~ sea trout (Salmo trutta) 166 and goldfish 61,1~ The lipid is predominantly phospholipid (about 65-70% of total lipid) 7~176 and rich in (n-3)PUFA 7~ particularly 22:6n-3, which accounts for 20% of total fatty acids in trout vitellogenin 127. There are few data on the detailed class compositions of fish vitellogenins. Vitellogenin is synthesized in the liver and is transported to the ovary during the first stage of oogenesis termed vitellogenesis 247,248. Vitellogenin is taken up intact by receptor-mediated micropinocytosis 2~ into the developing oocytes where it is cleaved into a phosphaterich protein, phosvitin and a lipid-rich protein, lipovitellin 111,2~ Lipovitellin in trout eggs was composed of 77% protein and 23% lipid with a lipid class composition similar to HDL 212. Cod roe lipovitellin had higher lipid content than the trout at over 40% with 70% of the lipid being glycerophospholipids 226. Of the total glycerophospholipids in cod lipovitellin, 67% was PtdCho and 22% was PtdEtn with 4% each of PtdSer and PtdIns 226. During the early stages of vitellogenesis, VLDL in the plasma may also be increased in response to estrogen 187, and may also be taken up into the developing oocytes by receptor-mediated endocytosis 248, at least in eggs with high triacylglycerol content and lipid droplets. Recently it has been confirmed that winter flounder contains a further high density lipoprotein (VHDL II), originally called Pk A as its density and relationship to vitellogenin was unknown 216, that is taken up in vivo by the ovary of viteUogenic females 149.

114 Composition The net result of the many metabolic pathways discussed in Sections II and III is the composition of glycerophospholipids in the tissues. The metabolism is a very dynamic situation, of course, but the glycerophospholipid composition is more stable provided the environmental conditions and diet are reasonably constant. There are many papers reporting the glycerophospholipid composition of fish tissues, and the effects of diet and environmental factors. A comprehensive review of these areas is beyond the scope of this article. The reader is directed to some other reviews that cover various aspects of glycerophospholipid composition in

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fish 1-5'96'194'198. This section contains some generalizations that can be made about fish glycerophospholipid composition focusing on some features that have been of particular interest in our own laboratory.

1. Content It is difficult to generalize about the glycerophospholipid content of fish tissues for several reasons. One of the major reasons is that studies have tended to report contents in a variety of ways, such as mg/g tissue or as percentage of weight of tissue, in both cases either wet or dry weights, or as percentage of total lipids. Irrespective of the method used, nutritional status of the fish will have an effect as variation in neutral lipid content will influence the results obtained. This is complicated by tissue differences. Other than adipose tissue, some fish, such as cod and halibut (Hippoglossus hippoglossus) store significant amounts of lipid in the liver, whereas in others, such as mackerel (Scomber scombrus) and capelin (Mallotus villosus), deposits of lipid between skin and muscle can account for a large proportion of the fish's total reserves 96. In snakehead (Channa sp.) fillet, eviscerated body of the guppy (Poecilia retie. ulata) and carp muscle, total phospholipids accounted for 21-26% of the total lipid s6,96. Phospholipids were present in goldfish muscle mitochondria at almost 2 mg/g muscle e42. In several studies on trout, phospholipids ranged from approximately 45-75% of the total lipid in liver 9~. In chum and coho (Oncorhynchus kisutch) salmon livers, phospholipids were consistently less than 40 and 30%, respectively, whereas in parr and smolt cherry salmon (Oncorhynchus masou) livers, phospholipids predominated 96. About 58% of the total lipid in goldfish intestinal tissue was phospholipid 144. It was reported in two studies on goldfish brain that phospholipids accounted for 3.9% of the tissue weight 129, and 47% of the total lipid 189. In contrast, total lipid from trout and cod brains was approximately 75% total polar lipids, predominantly glycerophospholipids 2~. In sea bass on a variety of diets, the proportion of phospholipids in the brain varied between 65 and 71% 17s. Phospholipids in coelacanth (Latimeria chalumnae) brain amounted to over 41 mmol/g wet weight of tissue 223. In a study of many marine fish including elasmobranchs and teleosts, total phosphorus in the brains varied between 460 and 2650 mg/g fresh weight 123. Total polar lipids, predominantly glycerophospholipids, made up 60 and 82% of the total lipid in retinal tissue from trout and cod 228. However in whole eyes from guppy, phospholipids were only 28% of the total lipid s6. Relatively high levels of polar lipids, predominantly glyeerophospholipids, are generally associated with fish eggs containing relatively low lipid contents 96. Therefore, the roe of some species of marine fish including cod, herring (Clupea harengus), haddock (Melanogrammus aeglefinus), whiting (Merlangus merlangus) and saithe (Pollachius virens) were relatively rich in phospholipids, which accounted for 61-72% of the total lipid 23~ However, in sand eel (Ammodytes lancea) roe which was richer in lipid and contained distinct oil globules, total lipid contained only 23% phospholipid 23o.

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2. Head group composition The class composition of glycerophospholipids in fish tissues can be far more easily generalized than content. PtdCho is almost invariably the major glycerophospholipid class with PtdEtn almost invariably being the second most abundant. PtdCho accounted for between 47 and 84% and PtdEtn accounted for between 16 and 34%, of the total phospholipids in a range of tissues including muscle, liver, gill and intestines from various fish species 96. This is a similar situation to that in mammals, and also like mammals, the main exception to this is brain tissue where the percentage of PtdEtn can exceed that of PtdCho, as reported for brains from cod and trout 228, coelacanth 223, rohu (Labio rohita)2~ and a range of elasmobranchs and teleosts 13s. However, other studies have found PtdCho exceeding PtdEtn in brain tissues from pike (Esox lucius) and carp 153, hake (Merluccius hubbsi) and sea bass (Acanthustius brasilianus)12 and a range of fishes from the Caribbean 123. PtdSer ranged from 2 to 9%, and Ptdlns ranged from 3 to 8% in several tissues from various species of fish 86,88,96,128,129. The levels of PtdSer exceed those of Ptdlns in fish neural tissues 96,123'129,153 and trout brush border membranes 12a, whereas the opposite was true in trout gills 88 and liver86, goldfish liver 129 and the roes from several marine fish 23~ Polyphosphoinositides, Ptdlns4P and Ptdlns4,5P2, constituted 1.0 and 0.9 mol% of the total phospholipids in the lesser spotted dogfish (Scyliorhinus canicula) rectal gland 29,2~ and were also found in cod gills3~ Cardiolipin ranged from 3 to 4% in trout liver and gills to over 8% in trout intestinal brush border membranes 86,88,128. As in mammals, relatively high levels (6-11%) of cardiolipin were associated with mitochondria in goldfish gill5a and muscle 242. Many aspects of ether-linked glycerides including the glycerophospholipid derivatives in marine animals, including fish, were the subject of recent reviews 45,194. Plasmalogens (alkenylacyl derivatives) were detected in small amounts in total hepatic lipid of the spiny dogfish 138. In the non-myelinated olfactory nerve of the garfish (Lepisosteus osseus), ethanolamine, choline and serine phospholipids contained 58, 1.4 and 2.5% of the totals as plasmalogens 44. In goldfish brains, ethanolamine and choline phospholipids contained 43 and 9% as plasmalogens 63. Bonito (Euthynnus pelamis) white muscle contained 3-6% and 4-8% of total choline and ethanolamine glycerophospholipids as plasmalogens 17~ Ethanolamine plasmalogen accounted for 12-13% of the total ethanolamine glycerophospholipids in trout spleen and gill, about 6% of the total in kidney and <1% of the total in liver241. Less than 1% choline plasmalogens were found in trout tissues 24~. Plasmalogens have also been reported in fish gills38'16~ carp muscle mitochondria 251, goldfish optic nerve 142 and smooth dogfish (Mustelus canis) erythrocyte membranes 25~ About 3% alkylacylphospholipids were detected in the total lipids of anchovy (Engraulis mordax) TM. Alkylacylphospholipids accounted for 8 and 12%, respectively, of total ethanolamine and choline glycerophospholipids from garfish olfactory nerve 44. Bonito white muscle contained 1.4 and 0.6% of total choline and ethanolamine glycerophospholipids as the alkyacyl derivatives 17~ 1-O-alkyl-2-acylsn-glycero-3-phosphocholine accounted for 10% of total choline glycerophospholipids in trout gill, kidney and spleen 241. In the trout, 1-O-alkyl-2-acyl-sn-glycero-3-

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phosphoethanolamine was only found in spleen where it accounted for 6% of total ethanolamine glycerophospholipids241.

3. Fatty acyl composition The fatty acyl composition of glycerophospholipids and the effects of diet, environment, pollutants, toxins and drugs etc. is one of the most studied areas in lipid metabolism in fish. Therefore, there is a considerable amount of data in the literature. However, as diet so markedly affects fatty acid compositions, only the compositions of fish caught in their natural habitats, rather than captive fish, are reviewed briefly in this section. Readers are directed to several review articles that more comprehensively cover fatty acid compositions in fish tissues2,3,5,96,198.

3.1. Totalglycerophospholipids Total phospholipids from tissues of wild fish are characteristically rich in PUFA which can account for almost 60% of the fatty acids present s4,96. Phospholipids contain higher proportions of PUFA, lower saturated fatty acids and similar amounts of monoenoic fatty acids compared with triacylglycerol. The PUFA of phospholipids are of longer chain length than those in neutral lipids with the ratio of C20 + C22 PUFA to C1a PUFA 4-10 times greater than in neutral lipida4,96. In most cases 22:6n-3 is the major PUFA of total phospholipid, followed by 20:5n-396. The proportion of 20:4n-6 in total phospholipid is variable and can be occasionally higher than 20:5n-364. Other common C20 and (222 PUFA include 22:5n-3, 20:4n-3, 20:3n-3, 20:2n-6, 20:3n-6, 22:4n-6 and 22:5n-6, present in variable amounts in phospholipids, but rarely exceeding 2-3% of the total fatty acids. The C1s PUFA are dominated by 18:3n-3 and 18:2n-6, with small percentages of 18:4n-3 and 18:3n-6. The level of 18:2n-6 can frequently exceed that of 18:3n-3s4,96. Furan fatty acids, a relatively common component of fish neutral lipids, are almost totally absent from phospholipids s4,174. The ether linked chains in alkenylacyl and alkylacyl derivatives are almost exclusively 16:0, 18:0 and 18:144,17~ . The phospholipids of freshwater species tend to contain higher proportions of saturated fatty acids and Cls PUFA and lower levels of C20 and (222 PUFA, than the equivalent lipids from marine fish96. Ackman pointed out that, for fish from northerly latitudes, it is the high proportions of C1s PUFA, rather than the low C20 and C22 PUFA, that are typical of freshwater species 1. The lower (n-3) to (n-6)PUFA ratio in freshwater fish (1.6 to 2.0) in comparison to marine fish (7.8 to 18.5) was also noted 1,2.96. 3.2. Glycerophospholipid classes Studies on various tissues from several species have indicated that there is a pattern for the distribution of fatty acids among the glycerophospholipids which generally holds true despite tissue differences and extreme dietary effects. PtdCho is characterized by having high 16:0 (higher than any other class) and lower PUFA (i.e. lower than PtdEtn and PtdSer). PtdEtn is characterized by having intermediate levels of saturated and monounsaturated fatty acids and high levels of PUFA which

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are relatively evenly split between C20 and C22 PUFA. PtdSer is characterized by high 18:0 and high PUFA, predominantly C22 PUFA. PtdIns also has high 18:0, but relatively lower PUFA which is predominantly C20, particularly 20:4n-6, and a low (n-3) to (n-6) ratio. The above pattern has been most frequently observed in various neural tissues including trout and cod brains and retinas 22a, turbot (Scophthalmus maximus) brain 148, herring brain 146, carp and pike brain and spinal cord 153, hake and sea bass brain and spinal cord 12, and brains from Caribbean fish 123. However, the same pattern of distribution was observed in whole turbot 132, cod gills and dogfish rectal glands 29, marine fish eggs23~ trout spleen 241 and the electric organ of some elasmobranchs 19~ It is noteworthy that this pattern of distribution was clearly demonstrated by various long established cultured fish cell lines including rainbow trout gonad (RTG-2), turbot fin (TF), Atlantic salmon (Salmo salar) (AS), chinook salmon (Oncorhynchustshawytscha) embryo (CHSE-214), bluegill (Lepomis macrochirus) fry (BF-2) and fathead minnow (Pimephales promelas) (FHM) 239. Some of these cells lines have been in culture for over 25 years 239. As indicated earlier, this pattern of distribution holds despite some differences between species and tissues, such as lower PUFA, especially 22:6n-3, in spinal cords 12,153 and dogfish rectal gland 29 or higher PUFA in retinas, especially increased 22:6n-3 in PtdCho 228, or lower (n-3) to (n-6) ratios as in some Caribbean fish brains 123 and the cell lines which have been cultured in mammalian sera 239. Similarly, several studies on Atlantic salmon showed that dietary effects did not abolish these patterns 2~ nor did direct supplementation of PUFA to cultured fish ce11s225,227,229. Fish glycerophospholipids have some features worthy of additional comment with respect to fatty acid composition. The composition of Ptdlns in fish, so similar to that in mammals, showing selective retention of 20:4n-6, has suggested a potential role for this class in eicosanoid metabolism (see section V.2.1) 29,230. The high levels of 22:6n-3 in fish neural tissues (as indicated by high 22:6n-3 to 20:5n-3 ratios) parallels the situation in mammals 199. In particular, the high level of PtdEtn coupled with the very high levels of 22:6n-3 in PtdEtn in fish neural tissues may suggest a special role for this glycerophospholipid class in neural functions. Fish neural tissues are an ideal system in which to study this possibility. PtdCho in fish neural tissues is unusual in that it contains relatively large amounts of the very-long-chain monoene 24:112,25,29,153,228. This may be to compensate for the lower amounts of sphingomyelin, which is rich in long chain monoenes, found in fish neural tissues in comparison to mammalian neural tissues 199'228.

4. Molecularspecies It has been increasingly appreciated recently that it is not simply the fatty acyl composition of glycerophospholipids that is important, but also the particular combinations of fatty acids, i.e. the molecular species, that are present within the individual glycerophospholipid classes. In an early study the possible fatty acid molecular species of cod PtdCho and PtdEtn were estimated based on fatty

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acid compositions alone 169. However, the first analysis of the molecular species composition of fish PtdCho appeared in 1985221. In this study the experimenters used HPLC to analyze diacylglycerol acetate derivatives which had been prepared from diacylglycerol produced by the action of bacterial phospholipase C on PtdCho purified from the dorsal muscle of 12 species of fish. The principal molecular species in muscle PtdCho from most species, including marine and freshwater species, was 16:0-22:6 (i.e. 16:0 in the 1-position and 22:6 in the 2-position) 221. However, the two flatfish studied were different in that the main molecular species of their muscle PtdCho was 16:0-20:5. The di-PUFA molecular species 20:5-22:6 and 22:6-22:6 were significant components of muscle PtdCho, particularly in the marine fish, although they were not quantified in that study221. Some two dozen different molecular species of muscle PtdCho were identified and quantified in chum salmon 22~ In addition to the major di-PUFA species mentioned above, minor amounts of several other di-PUFA species including 20:5-20:5, 20:5-22:5, 22:522:6 and 20:4-22:5 were reported 22~ These workers also reported surprisingly high percentages of molecular species with PUFA in the 1-position and a saturated or monoenoic fatty acid in the 2-position, such as 22:6-16:0 and 22:6-18:122~ The molecular species of total glycerophospholipids was determined in bonito white muscle by selected-ion monitoring gas chromatography/mass spectrometry using electron impact ionization 171. The predominant molecular species were 16:022:6n-3 (--,36%), 18:1-22:6n-3 (~,20%) and 16:0-18:1 ('-,13%) with the di-PUFA species 22:6n-3-22:6n-3 and 20:5n-3-22:6n-3 accounting for almost 9 and 6%, respectively 171. About 17 different species were identified, but species with PUFA in the 1-position and a saturated or monoenoic fatty acid in the 2-position were not found. Ohshima et al. used the same method to analyze the molecular species composition of 1-O-alk-l'-enyl-2-acylglycerophospholipids (plasmalogens) from bonito white muscle 172. Less than a dozen species were found, with the predominant species being 16:0"-22:6n-3 (alkenyl"-acyl), which accounted for over 57% of the total, followed by 16:0"-18:1 (,~15%) and 18:0"-22:6n-3 (,-.13%) 172. Hazel and Zerba investigated the molecular species compositions of PtdCho and PtdEtn from trout mitochondrial and microsomal membranes using gas chromatography of trimethylsilyl ethers of the diacylglycerols92. The predominant species of PtdCho were 16:0-22:6, 16:0-18:1, 16:0-20:3 and 16:0-22:5, whereas the predominant species of PtdEtn were 18:1-20:4, 14:0-16:0, 18:0-22:6 and 18:1-22:692. Mitochondria contained higher proportions of long-chain, polyunsaturated molecular species of PtdEtn, but less of the corresponding species of PtdCho, than microsomes 92. Using 3,5-dinitrobenzoyl derivatives of glycerophospholipids and a combination of three different isocratic solvent systems, Bell and coworkers have identified approximately 70 different molecular species in a range of different fish tissues23-25.2s. In cod roe, four species 16:0-20:5n-3, 18:1-20:5n-3, 16:0-22:6n-3 and 18:1-22:6n-3 contributed 67 and 62% of the total species in PtdCho and PtdEtn, respectively23. The 16:0-containing species predominated in PtdCho, whereas the 18:l-containing species predominated in PtdEtn. These four species accounted for only 23% in cod roe PtdIns, where 18:0-20:4n-6 was the predominant species at 37% of the total

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molecular species23. Di-PUFA species totalled less than 3% in cod roe PtdCho and PtdEtn, and were not found in Ptdlns. In contrast, retina from rainbow trout and cod contained high levels of diPUFA molecular species25,28. Trout retina contained 14.6, 43.5 and 29.0% diPUFA species, almost all di-22:6n-3, in PtdCho, PtdEtn and PtdSer, respectively28. In cod retina, di-22:6n-3 accounted for 29, 72 and 60% of the total molecular species in PtdCho, PtdEtn and PtdSer, respectively25. Trout and cod brain tissue also contained between 16 and 26% di-PUFA species in PtdEtn and PtdSer 25,28. However, in brain, PtdCho contained only ~1% di-PUFA species, and the di-22:6n3 species accounted for less of the total di-PUFA species, compared to retina 25,28. Fish brain PtdCho was also characterized by containing high percentages (9 and 13% in trout and cod, respectively) of 18:1-24:1/24:1-18:1 species25,28. In cod muscle and liver, saturated fatty acid-PUFA and monounsaturated fatty acid-PUFA species predominated, particularly 16:0-20:5 and 16:0-22:6 in PtdCho, 16:0-22:6 and 18:1-22:6 in PtdEtn and 18:0-22:6 and 18:1-22:6 in PtdSer 25. Muscle had the widest range of di-PUFA species, at least 6, totalling 21 and 38% in PtdCho and PtdEtn, respectively. Liver contained the lowest amount of di-PUFA species of the four tissues studied in cod 25. Ptdlns in cod muscle and liver had very low levels of di-PUFA species24. In cod liver Ptdlns, 49% was the 18:0-20:4n-6 species and in retina the major species of Ptdlns were 16:0-20:4n-6 (26%) and 18:0-20:4n-6 (24%) 24. However, in cod brain Ptdlns, 18:0-20:5n-3 was the most abundant species (41%) and in muscle 40% was 18:0-22:6n-324. The finding that 18:0-20:4n-6 was not the predominant species in Ptdlns in all fish tissues was particularly noteworthy. In all the above studies, di-saturated fatty acid species were notably uncommon.

5. Dietary effects The main effects of diet on glycerophospholipids are related to fatty acid composition and these are briefly summarized here. Algae commonly feature in the diet of the early life stages of some freshwater fish. Freshwater algae contain higher levels of C18 PUFA than C20 or Czz PUFA, although 20:5n-3 can be present in significant amounts in some diatoms 96,198. However, 22:6n-3 is rarely found. Aquatic insects are a major food source for freshwater fish, particularly salmonids, and although C18 PUFA predominate in these insects, 20:4n-6 and 20:5n-3 can account for up to 7 and 25%, respectively, of the total fatty acids96. Therefore, the lipids at the different trophic levels of the freshwater food chain are characterized by 18:2n-6, 18:3n-3, 20:5n-3 and 20:4n-6. In contrast, the marine food chain contains lipids in which 18:3n-3, 20:5n-3 and 22:6n-3 predominate and which have low levels of (n-6)PUFA 195. Therefore, these differences in the dietary input between the freshwater and marine environments are primarily responsible for the differences in glycerophospholipid fatty acid composition observed in freshwater and marine fish described above 195,198. As eluded to above, the PUFA composition of glycerophospholipids in fish tissues is highly dependent upon dietary PUFA composition 254.255 . The absence of PUFA in the diet, leads to the deposition of 20:3n-9, derived from the desaturation and

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elongation of 18:1n-9, in tissue phospholipids in salmonids 255,256. Feeding increased levels of 18:3n-3 to trout resulted in deposition of 18:3n-3 in phospholipids and particularly neutral lipids, whereas the level of its conversion products, 20:5n-3 and 22:6n-3, increased in phospholipids 254. The level of 22:6n-3 in phospholipids is also directly dependent on its level in the diet 257. Similarly, when increased levels of 18:2n-6 are included in the diet of salmonids, it is incorporated into neutral lipids and to a lesser extent phospholipids, whereas its conversion products 20:4n-6 and 22:5n-6 are particularly incorporated into phospholipids 255-257. This may not be the case in fish that lack the full spectrum of desaturases (A6, A5 and A4, see ref. 95), such as the turbot which appears to lack specifically the A5 desaturase 132,197. Low or absent A5 desaturase activity may be a common characteristic of marine fish or fish that are extreme carnivores, although there are few fish species in which the desaturase complement has been sufficiently characterized. In addition, dietary studies are lacking in turbot and other species that may lack A5 desaturase activity. However, in cultured turbot cells, 22:6n-3 and 22:5n-6 could not be produced by supplementing 18:3n-3 and 18:2n-6, respectively, to the cultures 229. 6. Adaptation to environmental factors

Due to the implications for homeoviscous adaptation of biological membranes of poikilothermic animals, this is a subject which has been the focus of considerable attention in fish over the years. This section is a brief summary of the main findings on lipid adaptations to environment in fish. For a detailed account of the experimental data and a very comprehensive review of the literature, the reader is directed to recent articles by Hazel and Williams91, Hazel 89 and Volume 5 of this series 98. There are several other reviews specifically on the effects of temperature on membrane lipids in fish51,53,87. 6.1. Temperature Changes in membrane composition and phospholipid structure in response to cold acclimation include changes in head group composition, acyl chain composition, phospholipid structure and molecular species composition 87. Cold acclimation has been associated with increased proportions of PtdEtn and decreased proportions of PtdCho 42,63'144'242. The proportion of plasmalogens, primarily alkenylacylglycerophosphoethanolamine, also decreased, particularly in neural membranes, in response to cold ac Climation 63 . Cold adaptation resulted in reduced proportions of saturated fatty acids and increased proportions of PUFA and to a lesser extent monounsaturated fatty acids 42,5~ Increased proportions of (n-3) and/or (n-6) PUFA (often at the expense of (n-9) acids) and increased average acyl chain length have also been reported 242. Changes in glycerophospholipid and fatty acid metabolism directly at the cellular level in response to temperature have also been demonstrated in fish cells in culture 233.234. Structural changes induced by cold acclimation include increased proportions of PUFA at the 2-position, increased unsaturation at the 1-position and generally increased proportions of highly unsaturated molecular species 144. These changes

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are perhaps linked to gross compositional changes. However, a further adaptation to environmental changes is the redistribution of fatty acyl chains into different glycerophospholipid molecular species, which can have significant effects on the properties of membranes without gross compositional changes. For instance, the phase behavior of 18:0-18:1 PtdCho (Tin = +3"C) is totally different to that of a binary (1:1) mixture of 18:0-18:0 PtdCho and 18:1-18:1 PtdCho (two transitions at -22"C and 30-53"C) despite having an identical fatty acyl chain composition la4. Hazel and Z e r b a 92 showed that PtdCho in mitochondrial membranes from trout acclimated to 5"C contained proportions of 16:0-22:6n-3 and 16:0-20:5n-3 that were 2- and 3-fold greater, respectively, than those from 20*C-acclimated fish. The effects of increasing the proportion of 16:0-22:6n-3 PtdCho was investigated in model membrane systems 94. Increased 16:0-22:6n-3 PtdCho tended to fluidize the membranes and reduced the temperature sensitivity of electrolyte permeation 94. Whereas the compositional changes, briefly summarized above, are well documented, the enzymic mechanisms behind these changes are yet to be fully elucidated. Fatty acid desaturase activities, including A9, A6 and A5, are generally increased in response to lowered environmental temperature 59,85,161.2~176 and this alone could account for the increased degree of unsaturation in membrane glycerophospholipids. The deacylation/reacylation reactions of glycerophospholipid turnover have been studied to determine the effects of temperature 93,133. Studies on the microsomal acyl-CoA:lysoPtdCho acyltransferase from liver of thermally acclimated rainbow trout have suggested that, although the activity may not be affected by temperature, the substrate specificities may vary and so contribute to restructuring 133. A recent study on the pathways of de novo biosynthesis of glycerophospholipids showed that the activities of CDP-choline and CDP-ethanolamine phosphotransferase were reduced at 5"C, whereas PtdSer decarboxylase activity was increased 9~ The overall synthesis of PtdCho depended more on temperature than that of PtdEtn and so the ratio PtdCho: PtdEtn synthesis positively correlated with temperature 9~ This may be a mechanism underpinning the increased proportions of PtdEtn and decreased proportions of PtdCho in cold-acclimated fish. 6. 2. Salinity and hydrostatic pressure The major difference between freshwater and sea water is the high concentration of inorganic salts in the latter and so changes in the lipids of fish transferred from freshwater to sea water in the laboratory can be considered as adaptive responses to environmental salinity. In the wild, anadromous fish make the change from freshwater to sea water and vice versa but these changes are usually accompanied by great changes to the diet, including starvation. Therefore, changes in the lipids of wild anadromous fish will not be included here. The proportion of phospholipids to neutral lipids increased in all tissues of the guppy during sea water adaptation 56. Tissue specific changes occurred in the relative proportions of the phospholipid classes, with the proportion of PtdEtn increasing in gill, intestine, liver and eye and PtdCho decreasing in the kidney of the fish in sea water 56. However, there were no changes in.the relative proportions of phospholipid classes in brush-border membranes from the intestine of rainbow trout transferred

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from freshwater to sea water ns. Similarly, phospholipid class composition of the gills of Atlantic salmon was unaffected by keeping the fish in salinities from 0 to 2% over 48 h 222. In the guppy, the levels of 22:6n-3 and 20:4n-6 increased and decreased, respectively, in PtdCho and, especially, PtdEtn of the digestive tract during sea water adaptation 56. Similarly, the proportion of total (n-3)PUFA, almost entirely 22:6n-3, in PtdCho from trout intestinal membranes increased from 17.7 to 33.4% within one day of transfer from freshwater to sea water 128. There were concomitant decreases in the percentages of (n-6)PUFA and saturated fatty acids ns. In contrast, the level of (n-3)PUFA, particularly 22:6n-3, decreased in gill PtdCho in response to increased salinity in Atlantic salmon 222. Studies have suggested that there are similarities between the adaptive responses to low temperature and high pressures. Deep water and Antarctic fish both possessed elevated proportions of PUFA compared to tropical species 176. In a study comparing the compositions of liver mitochondrial PtdEtn from 13 species of teleost fish ranging in depth from 200 to 4000 m, the proportions of 16:0 and 18:0 decreased in abundance with depth, whereas PUFA levels remained relatively constant and monounsaturated fatty acid levels increased 52. Similar increases in the unsaturation index with depth/pressure were observed in PtdCho and PtdSer 52. Changes in the membrane lipid environment have been implicated in the pressure adaptation of Na +/K+-ATPase in fish gills77.

V Roles As the major lipid components in all biological membranes, glyeerophospholipids have a crucial role to play in both the structure and function of all animal cells. This is as true for fish as it is for mammals. Indeed, as fish are poikilothermie, it could be argued that the lipid components of the biological membranes have an additional important role in homeoviseous adaptation in relation to temperature. The following is a brief review of some specific roles of glyeerophospholipids that have, or may have, relevance to fish. 1. Structural roles

Glyeerophospholipids have the same central role in the structure of cell membranes in fish as they do in mammals. The dynamic changes in the composition and metabolism of the glycerophospholipids in biomembranes in response to environmental factors91, discussed above, is testament to their importance. Fish membranes, however, are characterized by high levels of (n-3)PUFA, including 22:6n-3. This is particularly the case in fish neural membranes where, like virtually all vertebrate brains and retinas, 22:6n-3 is the predominant fatty acid 1~. It is now established that 22:6n-3 is essential for the proper development and function of mammalian neural membranes 19'159. This is probably equally true in fish as we have recently established that 22:6n-3 is rapidly taken up by turbot brain when the

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post-larvae were weaned from a 22:6-poor diet to a 22:6-rich diet 147,148. Biochemical studies showed that the incorporation of 22:6 was particularly avid in PtdEtn 238. Recent biophysical studies have begun to elucidate some of the physical and structural properties that this acid, when esterified in glycerophospholipids, may impart to membranes. Increasing the number of double bonds in long chain fatty acids of a given chain length constrains the structures of the molecules such that they form increasingly compact conformations 9. This effect is maximal in 22:6n-3 whose minimum energy conformation is a relatively compact helix or 'angle iron' structure with an overall length shorter even than a saturated fatty acid, such as 18:0 9. In fish neural tissues, the 22:6n-3 is mainly esterified to PtdEtn and PtdSer, and a considerable portion of this is in the form of di-22:6n-3 species (see above). Given the marked helicity of 22:6n-3, it can be deduced that cell membranes, such as retinal rod outer segments, that are rich in di-22:6n-3PtdEtn, have a very specialized phospholipid bilayer, probably with novel liquid crystalline properties. Precisely how this specialization relates to the function of rhodopsin, and the retina in general, is not known in detail. It has been considered, on the basis of physicochemical studies of mammalian rod outer segment membranes, that the abundance of di-22:6n-3PtdEtn maintains the membrane bilayer with the required balance between fluidity and rigidity necessary to accommodate very rapid protein conformational changes initiated by the c/strans conversion undergone by the retinal chromophore of rhodopsin 62. In addition, it has recently been deduced on theoretical grounds that the minimum energy form of 22:6n-3 is conformationally stable over a wide range of temperature, so that membrane bilayers rich in 22:6n-3 may be essentially 'buffered' against environmental change, with obvious advantages to their associated physiological processes 185. The foregoing scenario can be extended in principle to encompass very fast conformational changes undergone by other signal transducers in cell membranes, e.g. those involved in ion current generation, thus accounting for the abundance of 22:6n-3 in neural tissue glycerophospholipids generally. It is well established in mammals, that phospholipids are asymmetrically distributed in cell membranes, with choline-containing phospholipids, PtdCho and sphingomyelin, being concentrated in the outer leaflet and PtdEtn, PtdSer and, to a lesser extent, PtdIns being concentrated in the inner leaflet of the membrane 6~ This aspect of membrane structure has not been well studied in fish. However, the localizations of the amine-containing glycerophospholipids, PtdEtn and PtdSer, were studied in the photoreceptor membranes of the retina from walleye pollock (Theragra chalcogramma) and an asymmetric distribution was apparent 113. 2. Metabolic roles 2.1. Eicosanoid metabolism Eicosanoids are a range of highly bioactive molecules so named because they are derived, via the action of dioxygenase enzymes, primarily from the C20 PUFA 20:3n-6, 20:4n-6 and 20:5n-3. Two main enzymes are involved: (1) cyclooxygenase, which produces cyclic oxygenated derivatives, collectively called prostanoids, in-

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eluding prostaglandins (PG), prostacyclins (PG I) and thromboxanes (TX); and (2) lipoxygenases, which produce linear oxygenated derivatives including hydroperoxy and hydroxy fatty acids, leukotrienes (LT) and lipoxins (LX). Eieosanoids are produced by a wide range of tissues in response to a variety of extracellular stimuli. Glyeerophospholipids are important as the source of the substrate fatty acids for the cyclooxygenase and lipoxygenase enzymes. The pathway from extraeellular stimulus to the production of eieosanoids forms a cascade termed the 'arachidonic acid cascade' as araehidonie acid (20:4n-6) is the primary precursor in mammals. Briefly, activation of cell surface receptors results in the production of free precursor acid either via phospholipase A2 activity or via the sequential action of phospholipase C and DAG lipase. The activation of phospholipase A2 may occur through elevation of intraceUular Ca 2+ (it is activated in vitro by high Ca2+levels) or it may be regulated by a G protein, as phospholipase C appears to be in the phosphoinositide cycle4~ It appears that the increased concentration of the free precursor acid is itself the key stimulus for the activity of the eyclooxygenase or lipoxygenase enzymes214. The pathways for the synthesis of individual eieosanoids are complex with many different steps2t4 and will not be discussed in detail here, although work is progressing in characterization of these pathways, particularly lipoxygenase pathways, in fish. In fish there is little mechanistic data on most of the steps involved in this cascade, particularly up to the production of free fatty acid precursor. However, due to the very obvious differences in the C20 PUFA composition of the glyeerophospholipids between mammals and fish, there has been considerable interest in the general production of eicosanoids in fish. Although the roles in the cascade of the glycerophospholipids themselves are not well understood, eieosanoid metabolism is nonetheless an important aspect of glycerophospholipid metabolism. The following sections offer a brief review, therefore, of the existing knowledge of eicosanoids in fish with particular emphasis on glycerophospholipids. Henderson and Tother ~ give a more complete review on eieosanoid metabolism in fish in relation to essential fatty acid metabolism.

2.1.1. Species and tbsue distribution of eicosanoids in fish Prostanoids have been found in a large range of freshwater fish, including c a r p 17'49'120'164~ , rainbow trout rig, brown trout 49, brook trout (Salvelinus fontinalb) 8t'82, tench 49, tilapia (Tilapia mossambica [Oreochromis mossambicus]) tT, Asian catfish (Heteropneustes fossili~ and Clarias batrachus) t7, pond loach (Mbgumus anguillacaudatua) 1~, eel (Anguilla anguilla) 2ta, sheat-fish (Parasilurus asotus) 164 and goldfishsl, and marine fish, such as plaice (Pleuronectes platessa) 7, turbot 97, dogfish 192, flounder (Par. alichthys olivaceus) 143, black (Acanthopagrus schlegeli) and red (Pagrus major) sea breams 143, black rockfish (Sebastes schlegeli) t43 and Atlantic salmon 2~ Virtually every fish tissue so far studied has shown eyclooxygenase activity, including gills, kidney, spleen, intestine, stomach, liver, heart and isolated eardiomyoeytes, skeletal muscle, brain, fin, skin, air sac, ovaries and ovarian fluid, testes and milt, blood thrombocytes and leukoeytes20,22,49,u9,120,164.16s,167,16s.In some of the above studies exogenous fatty acid precursors were added, but synthesis of prostanoids from

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endogenous fatty acids has been shown in ovarian tissues 168, gills49, leukocytes 22, isolated cardiac myocytes2~ and testes, liver, heart, intestine, brain, air sac and kidney 164. Gills were found to have the greatest capacity for the synthesis of prostanoids from exogenously added fatty acid precursors 49,167, whereas intestine, heart, air sac and skin synthesized more prostanoids from endogenous substrates than gill164. Lipoxygenase products have been identified in tissues from American eel (AnguiUa rostrata)183, rainbow trout 73'75'76'177'179-181'235, dogfish 178'192, plaice 232, salmon 21'22'191, and catfish, carp, rudd (Scardinius erythrophthalmus) and tilapia (Oreochromis niloticus) 191. Tissues studied include gills 21,22,73,75,183, skin 76, whole blood 21'22'178'179, blood total leukocytes 177,191,192, peripheral blood neutrophils 232, tissue macrophages 18~ and brain cells 23s. A third group of eicosanoids, epoxides formed by the action of cytochrome P-450 enzymes, have been demonstrated in mammalian systems214. To date, there has been no report of these derivatives being found in fish tissues. However, this is probably simply because they have not been looked for. By analogy with how the characterization of lipoxygenase products in the fish field lagged slightly behind the mammalian field and how similar the fields appear today, it is likely that epoxide derivatives will be discovered and characterized in fish tissues in the future.

2.1.2. Range of eicosanoids in fish PGE 17'49'120'143'164'167'192,PGD 17'120'143'192,and PGF 17'49'120'143'167'169'192have most commonly been reported for many fish tissues96. TXB, the stable metabolite of TXA, was the major prostanoid producedby rainbow trout thrombocytes 119'12~and Atlantic salmon blood 22 and was also produced by salmon cardiomyocytes2~ and isolated gill ceUs 22, and dogfish leukocytes 192. No metabolites of PGI were produced by dogfish leukocytes, but 6-keto-PGFl~, the stable metabolite of PGI2, was detected in Atlantic salmon blood, gill cells and cardiomyocytes2~ The 12-1ipoxygenase product, 12-hydroxy fatty acid, was the major lipoxygenase product from trout gill75, skin 76, head kidney macrophages 179,1sl and brain cells23s. It was also reported in studies with plaice neutrophils 232. The 12-1ipoxygenase activity has now been found in the gills and skin of 14 species of fish and well characterized in rainbow trout gill 1~ The presence of 15-1ipoxygenase in fish tissue has also been indicated by the presence of 15-hydroxy products in trout brain cells235 and dogfish blood cells 178. The 15-1ipoxygenase activity has been identified and more thoroughly characterized in various fish gills72. The 5-hydroxy products of the 5-1ipoxygenase were found in studies with plaice neutrophils 232 and rainbow trout head kidney macrophages lsl and brain cells235. LTB has been the most widely reported lipoxygenase product, being produced by various tissues including plaice neutrophils 232, dogfish leukocytes 192, trout macrophages ls~ leukocytes 177, whole blood 179 and brain cells23s. The peptido-leukotrienes, LTC, D and E, were produced by American eel gill tissue 183. Various other di- and tri-hydroxy-products have also been noted in fish tissues71,73,74,17a. Lipoxins, LXA and LXB, were major lipoxygenase products in rainbow trout head kidney macrophages 180.181. Head kidney cells, mainly macrophages, from Atlantic salmon and carp also produced

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lipoxins, but the same preparations from catfish, rudd and tilapia did not produce lipoxins 191. Overall, it appears that fish produce the same wide range of eicosanoids as in mammals. Also, as in mammals, the precise eieosanoid profile varies with tissue and, to some extent, species. Although relatively few species have been investigated, there appears to be no major differences between elasmobranehs and teleosts, or between freshwater and marine fish. However, as more species are studied, differences or patterns may become apparent.

2.1.3. Stimuli for production of eicosanoids In early studies, production of eicosanoids in experimental systems in fish was most often stimulated simply by the addition of exogenous fatty acid substrate %. More recently, eieosanoid production has been stimulated by incubation with the calcium ionophore A2318720''22,177-180'183'191'192'232'235.Therefore, a rise in intracellular Ca2+ion coneentration was effective in stimulating the production of a variety of eicosanoids in a wide range of fish tissues. However, there have been no specific eicosanoid studies using more physiological stimuli in fish. 2.1.4. Fatty acid precursors of eicosanoids Production of PGE2 and PGF2a from endogenous 20:4n-6 in carp, tench and trout tissues 49,167 and PGE3 from endogenous 20:5n-3 in tissues from carp and sheat-fish 164 have been reported. The amounts of PGE3 produced were generally lower than those for endogenous PGE2 production 49'1u. Production of 1-series prostaglandins from endogenous 20:3n-6 was not detected 49. Approximately 5-fold more 20:4n-6 was converted to eicosanoids by plaice skin microsomes compared to 20:5n-37. Only prostanoids of the 2-series (20:4n-6 products) and LTB4 were produced by dogfish leukocytes exposed to calcium ionophore A23187192. Bell and colleagues 21,22 measured the production of TXB2, 6-ketoPGFla, 12-hydroxyeicosatetraenoate (HETE) and LTB4 from endogenous 20:4n-6 and 12-hydroxyeicosapentaenoate (HEPE) and LTB5 from endogenous 20:5n-3 in Atlantic salmon tissues. In salmon fed a normal diet the production of 12-HETE exceeded that of 12-HEPE in blood leukocytes, whereas the levels of 12-HEPE in gills and LTB5 in blood leukocytes exceeded the levels of the respective 20:4n-6 products 21'22. TXB2 and 6-ketoPGFta were produced in isolated eardiomyocytes from Atlantic salmon 2~ Rainbow trout blood cells stimulated with A23187 produced both LTB4 and LTB5 from endogenous precursors, with LTB4 production exceeding that of LTB5179'180.Rainbow trout macrophages stimulated with A23187 produced LTB4 and LXA4, and also LTB5 and LXA5 from endogenous 20:4n-6 and 20:5n-3, respectively 18~ with the 20:4n-6 products predominating 181. Similarly, the levels of lipoxygenase products from 20:4n-6 exceeded those from 20:5n-3 in Atlantic salmon, carp and tilapia 191. More exogenously added [1-14C]20:4n-6 compared to [1-t4C]20:5n-3 was incorporated into prostaglandins by turbot tissues 97 . Incubation of thromboeytes from several fish species with A23187 in the presence of [1-14C]20:4n-6 resulted in the formation of labeled PGF2~, PGE2 and PGD2 in all cases 143. In contrast, when

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the thrombocytes were stimulated with A23187 in the presence of [1-14C]20:5n-3, production of PGE3 and PGD3 were only produced by one species 143. In plaice neutrophils labeled with [1-14C]20:4 n-6 or [1-14C]20:5n-3231, the production of labeled LTB4 and 12/15-HETE exceeded that of LTB5 and 12/15-HEPE, respectively 232. Overall, the results of these studies indicate that both 20:4n-6 and 20:5n-3 serve as eicosanoid precursors in fish tissues. However, in almost all cases the evidence suggests that 20:4n-6 is the preferred substrate despite the preponderance of 20:5n-3 in the tissue glycerophospholipids. Dihomo-y-linolenic acid (20:3n-6) can serve as a substrate for fish cyclooxygenase enzymes when added exogenously 49,167. However, there was no production of labeled prostanoids when thrombocytes from several fish species were incubated with [1-14C]22:6n-3 in the presence of A23187147. Lipoxygenase-derived derivatives of endogenous 22:6n-3 have been reported in fish tissue 74. There have been several studies characterizing different lipoxygenase-derived metabolites of exogenously added 22:6n-3 in various fish tissues 73'75'76. It remains to be seen how many of these are produced in vivo. The 12-1ipoxygenase derivative of 22:6n-3, 14-hydroxydocosahexaenoate (14-HDHE) was produced from endogenous 22:6n-3 in Atlantic salmon gill cells stimulated with A23187 22. 2.1.5. Glycerophospholipid sources of precursor fatty acids The precise glycerophospholipid class that serves as a source of precursor fatty acids for eicosanoid synthesis is still a controversial area in mammalian metabolism. The wide range of tissues studied using a variety of stimuli has probably confused this area. Overall, it appears that one glycerophospholipid class may not be the sole supplier of eicosanoid precursor in all systems. In contrast, this area is virtually unstudied in fish. The evidence, summarized above, indicating, in all systems studied so far, that 20:4n-6 is the predominant eicosanoid precursor in fish invites a very obvious question. How do fish selectively utilize 20:4n-6 for eicosanoid synthesis when the tissues are rich in 20:5n-3? One possible answer to this would be to selectively concentrate the eicosanoid precursor in one glycerophospholipid species. The characteristic fatty acid composition of Ptdlns in fish tissues, with high 20:4n-6, led Sargent and coworkers to speculate that Ptdlns may be an ideal source of eicosanoid precursor in fish 29'230. However, in a time course study on the loss of radioactivity from labeled glycerophospholipids in plaice neutrophils in response to A23187, the labeling appeared to decrease firstly in PtdCho and to a lesser extent PtdEtn prior to loss of label from Ptdlns 226. A recent study examining 3H/14C ratios in trout brain cells dual-labeled with [3H]20:4n-6 and [14C]20:5n-3, suggested that the glycerophospholipid source of eicosanoid precursors was unlikely to be Ptdlns and that PtdCho was the most likely source 235. If PtdCho is the preferred source of eicosanoid precursor in fish, then the specificity for 20:4n-6 may result from a 20:4n-6-specific phospholipase A2 or from the specificity of the cyclooxygenase and lipoxygenase enzymes. These aspects are virtually unstudied in fish. However, the 3H/14C ratio of released fatty acids obtained in the study above suggested that there was no specificity for 20:4n-6 at the level of the phospholipase 235. There is

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conflicting evidence in fish regarding the possibility that the specificity may reside mainly in the dioxygenase enzymes. In one study on sardine skin lipoxygenase, the enzyme activity towards 20:4n-6 was twice that towards 20:5n-3145. Interestingly, the enzyme was as active towards 22:6n-3 as it was towards 20:4n-6145. However, the 12-1ipoxygenase activity from rainbow trout gill was equally active with 20:4n-6, 20:5n-3 and 22:6n-31~ 2.1.6. Functions ofeicosanoids Considering the relatively large number of studies characterizing eicosanoid production in fish, there are surprisingly few studies on the functions of eicosanoids in fish. An exception to this is fish reproduction, where the roles of prostanoids are well known 96. Prostaglandins are involved in inducing ovulation, and may be involved in eliciting behavioral changes, such as spawning activity96. This subject has been comprehensively reviewed previouslya1,219. The production of thromboxane by rainbow trout thrombocytes 119,n~ Atlantic salmon blood 21,22 and dogfish leukocytes 192, and the presence of prostacyclin (PGI) in Atlantic salmon plasma 22 suggests that the control of thrombocyte aggregation and blood clotting in fish may involve a "IXA/PGI balance as in mammals. Series-2 prostanoids have been shown to have various cardiovascular effects when injected into fish 182'184'246. Similarly, series-1 prostanoids were also shown to have vasoactive properties in snakehead 253. It is not known if these effects have any physiological significance in fish. The peptido-leukotrienes from eel gills had the same biological effects on mammalian lung tissue as mammalian LTC4, and so they may play a role in modulating gill function at a local level 183. An LTB extract from plaice neutrophils stimulated with A23187 had chemotactic activity for plaice leukocytes 96. Similarly, it was found that LTB4 enhanced the migration of fish leukocytes in vitro 1~ Therefore LTB may play similar roles in inflammatory and immune processes as it does in mammals. 2.2. Thephosphoinositidecycle The phosphoinositide cycle, in which phosphorylated derivatives of PtdIns, such as PtdIns4,5P2, are converted by the action of phospholipase C into two intracellular second messengers, DAG and inositol phosphates (e.g. InsP3), in response to various hormones and effectors is well characterized in mammals 33. Considerably less is known about the phosphoinositide cycle in fish. However, studies in various fish tissues have suggested that the basic components of the cycle do operate in fish 10'39'78'196'207. In an early study in seawater eel gills, it was shown that depression of salt secretion by 10-5 M adrenalin was accompanied by enhanced turnover of inositol lipids78. Dogfish rectal gland is rich in inositol phospholipids (reflecting the extensive plasma membranes in chloride cells) with PtdIns, PtdIns4P and PtdIns4,SP2 comprising 9.1, 1.0 and 0.9% of total cellular phospholipid, respectively 2~ Studies with 32p-labeled orthophosphate established that inositol phospholipids are metabolically active in non-stimulated rectal glands, i.e. an active inositol lipid cycle exists in the resting gland, and that elevation of intracellular cAMP depresses inositol lipid turnover 207. Interestingly, the situation in fish

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is opposite to that in the avian salt gland where activation of salt secretion by aeetyleholine is accompanied by enhanced turnover of inositol lipids99. Cilia isolated from the olfactory epithelium of channel catfish were shown to contain an active phospholipase C 39. Thg enzyme was shown to hydrolyze exogenously added radioactive Ptdlns4,5P2, with IP3 the major inositol phosphate product 39. The optimum pH was 6.7 compared to the more acidic optima of about pH 5 for mammalian Ptdlns-speeifie phospholipase C 39'186. Similar to the mammalian phosholipase C activity, the activity in catfish cilia had multiple molecular forms with Mr >100,000, 82,000 and 60,000 which compare reasonably well with the molecular weights found in a range of mammalian tissues 39,186. The metabolism of inositol phospholipids was studied in metabolically active electrocytes from the electric ray (Discopyge tschudii) after labeling with myo[3H]Insl~ Ptdlns displayed the highest level of labeling which was inhibited by lithium l~ Incubation of the labeled electrocytes over 24 h showed that labeled inositol phosphates were produced in the rank order InsP > InsP3 > InsP2, and that the presence of lithium ions enhanced the accumulation 1~ These results were interpreted as indicating the participation of phospholipase C and of Li-sensitive phosphatases in the modulation of phosphoinositide metabolism in the eleetrocytes of the electric ray. 2.3. Other metabolism 2.3.1. Protein kinase C One consequence of an active phosphoinositide cycle is the production of increased intracellular Ca 2+ and DAG, both activators of protein kinase C 124. PtdSer also has an important metabolic function in mammals as a specific activator of protein kinase C, along with DAG and Ca2+ions 124. Protein kinase C has been found in trout spleen and dogfish rectal gland, and the activities were stimulated by the addition of PtdSer 26. There was no difference between PtdSer from bovine brain and PtdSer from trout liver (7.5-times greater 22:6n-3 and very low (n-6)PUFA) in their ability to stimulate fish protein kinase C26. In contrast, trout liver PtdSer was not as effective as bovine brain PtdSer in the activation of rat spleen protein kinase C27. Studies using the calcium ionophore A23187 and phorbol esters (analogues of DAG) in goldfish ovarian follicles and testes have implicated a role for protein kinase C in the stimulation of steroidogenesis in these tissues 243'245. 2.3.2. Platelet-activatingfactor Platelet-activating factor (PAF), 1-O-alkyl-2-acetylsn-glycero-3-phosphocholine, is a biologically active phospholipid synthesized by inflammatory cells 215. In mammals, PAF is implicated in the activation and/or aggregation of platelets and leukocytes and may be a mediator of hypotensive activities as well as causing increased vascular permeability, vasoconstriction and contraction of smooth muscle 21s. PAF synthesis has been demonstrated in gill, kidney, liver and spleen of rainbow trout and its production was stimulated by calcium ionophore A2318724~ The enzyme responsible for deactivation of PAF, PAF acetylhydrolase, has been shown in fish serum 41. In mammals, the reacylation of lyso-PAF is highly specific for arachidonic acid, and it is the 1-alky-2-arachidonylglycerophosphocholine species that is the substrate for the synthesis of PAF via

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an arachidonate-specific phospholipase A2 that also produces arachidonate for eicosanoid synthesis 2~s. The lipid substrates, most of the enzymes involved and their specificities, and the roles of PAF, are all unknown in fish.

3. Nutritional roles 3.1. Embryonic development Upon hatching, the larvae of many fish species are unable to feed as the intestinal tract and mouthparts are not fully developed 37. Therefore, throughout embryogenesis in the egg and then early larval development up to first feed, the larvae gain nutrition from the endogenous energy reserves of the yolk. In some fish, particularly marine fish, phospholipids account for the great majority of lipid stored in the egg23~ Phospholipids are not normally regarded as an energy source, and in most situations this generalization is true. However, there is evidence that, during embryonic and early larval development of some fish, phospholipids are preferentially utilized not only for cell division and organogenesis but also for energy. During development of rainbow trout, phospholipid was continuously metabolized, whereas triacylglycerol was not utilized until after hatching 213. In the Atlantic salmon, PtdCho, which originally accounted for over 94% of the total phospholipids of the egg, was continuously metabolized so that in the fry a PtdCho:PtdEtn ratio approaching that of salmon muscle was obtained s4. In the whitefish (Coregonus sp.), total phospholipid decreased slightly and PtdCho decreased from 76 to 61% of the total phospholipids by the time of hatching ~s7. During development of the grass carp (Ctenopharyngodon sp.) phospholipids were utilized continuously during development ~2e. The data suggested that a significant portion of the phospholipid was utilized for energy ~22. PtdCho declined in absolute terms during embryonic development of the Atlantic herring and cod, whereas catabolism of neutral lipid, primarily triacylglycerol was initiated after hatching 6s,237. Actual oxidation of the phospholipid fatty acids was not measured in any of these studies. However, the data clearly show a decrease in the absolute amount of phospholipid, usually PtdCho, during embryogenesis in the egg, particularly in marine fish species. Therefore, more than simple redistribution of PtdCho from lipid stores into larval tissue is occurring. One consequence of complete catabolism of phospholipid for energy would be the loss of important PUFA. In herring, however, there was selective retention of PUFA during embryonic development, as PUFA produced by the hydrolysis of PtdCho were selectively retained in the neutral lipid pool at the expense of monoenes 236. A similar process occurred in cod eggs during embryonic development, where it was calculated that approximately one third of the 22:6n-3 produced by PtdCho catabolism was incorporated into triacylglycerol and sterol ester 65. 3.2. Larval diet From the data obtained on lipid metabolism during embryonic development in cod eggs, it was postulated that PtdCho, replete in (n-3)PUFA, should represent a major portion of the lipid in artificial diets for fish larvae 6s. This was the same conclusion

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as obtained from earlier work 114, although the experimental route to the conclusions was rather different. Several studies suggested that glycerophospholipids were superior to triacylglycerols in promoting growth in larval ayu (Plecoglossus altivelus) 115-117, red sea bream (Chrysophrys major) 114, white sturgeon (Acipenser transmontanus) 1~ and cod 173. The addition of glycerophospholipids to the diets of larval ayu also reduced the incidence of malformations 115. Kanazawa concluded that glycerophospholipids containing a PUFA at position-2 and with an inositol or choline head group were indispensable for normal growth and survival of larval ayu 114. This requirement was in addition to the essential fatty acid requirement of the fish. The mechanism behind the growth-promoting ability of Ptdlns and PtdCho in larval fish has never been conclusively established. Although glycerophospholipids will present a higher percentage of PUFA in their total fatty acids compared to triacylglyeerols, this itself cannot account for the effects as one of the growth-promoting glycerophospholipids, soybean lecithin 117, is not a rich source of the (n-3)PUFA required in large amounts by larval fish 224. Furthermore, the inherent differences in fatty acid compositions between PtdCho and Ptdlns suggest that supply of particular fatty acids is not a factor. It may be that the glyeerophospholipids are superior to triacylglycerols because, being more polar, they are more easily emulsified and, therefore, more rapidly hydrolyzed and assimilated. Triacylglycerol assimilation may be more susceptible to bile salt limitation than glycerophospholipid assimilation, especially in larval fish fed diets rich in triacylglycerols whose digestive capacity may not be fully developed. Definition of the development of larval hepatic, intestinal and pancreatic functions in larval fish is required to elucidate this area. Glycerophospholipids have a number of other effects - when added to formulated pelleted diets - related to their polarity, and physical properties in general. Glycerophospholipids may improve the mixing, binding and texture of pelleted diets. The improved binding effects may help to reduce the leaching of water-soluble vitamins or components of the diet, such as choline and inositol. With specific reference to choline and inositol, supplying these essential components of the diet in the form of glycerophospholipid may, therefore, be more efficacious. Finally, as well as being more easily emulsified themselves, glycerophospholipids may improve the emulsification of the neutral lipid in the diet and so aid the digestion and absorption of all dietary lipid.

VI. Conclusions and perspectives The class and fatty acid compositions of glycerophospholipids in fish tissues are now well characterized. However, the enzymic systems underpinning the compositions, including those responsible for the adaptation of the compositions in response to dietary and environmental influences, are poorly characterized. More work is required to elucidate the general pathways of glycerophospholipid biosynthesis, catabolism (tissue and subcellular sites) and interconversion in fish. Fundamental questions related to the digestion and absorption of glycerophospolipids remain

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unanswered. For instance, what are the intestinal enzymes that act on glycerophospholipids, what are the absorbed products and how exactly are they metabolized in the mucosal ceils? There is little information on the appearance of intestinal phospholipases in larval fish, especially marine fish, which is a crucial question with important consequences for aquaculture. Ontogenetic studies of the development of hepatic, intestinal and pancreatic functions in larval fish are required in this area. The specific uptake of glycerophospholipids from serum lipoproteins, including enzyme involvements, mechanisms and tissue specificities are all virtually unknown in fish. The significance and roles of individual molecular species of glycerophospholipids needs to be studied, including the effects of diet and environmental factors on specific molecular species. The precise mechanisms that allow 20:4n-6 to be the main eicosanoid precursor in (n-3)PUFA-rich fish are still unclear. What are the specificities of phospholipase A, cyclooxygenase, lipoxygenases and other enzymes in the eicosanoid pathways? The operation of the Ptdlns cycle, including the tissues and stimuli linked to the cycle, and the glycerophospholipid class specificities of phospholipase C are unstudied in fish. The above list of areas requiring study has a common link. The enzymes involved in glycerophospholipid metabolism, such as phospholipases and acyltransferases require isolation and characterization in fish tissues. Only then will it be possible to answer most of the questions raised above.

VII. References 1. Ackman, R.G. Characteristics of the fatty acid composition and biochemistry of some fresh water fish oils and lipids in comparison with marine oils and lipids. Comp. Biochem. PhysioL 22: 907-922, 1967. 2. Ackman, R.G. Fish lipids, Part 1. In: Advances in Fish Science and Technology, edited by J.J. ConneU, Farnham, U.K., Fishing News Books, pp. 87-103, 1980. 3. Ackman, R.G. Fatty acid composition of fish oil. In: Nutritional Evaluation of Long Chain Fatty Acids in Fish Oil, edited by S.M. Barlow and M.E. Stansby, London, U.K., Academic Press, pp. 25-88, 1982. 4. Ackman, R.G. (Editor). Marine Biogenic Lipids, Fats and Oils, Boca Raton, FL, CRC Press, 1989. 5. Ackman, R.G. and C. McLeod. Total lipids and nutritionally important fatty acids of some Nova Scotia fish and shellfish food products. Can. Inst. Food ScL Technol. J. 21: 390-398, 1988. 6. Amthauer, R., J. Villanueva, M.I. Vera, M. Concha and M. Krauskopf. Characterization of the major plasma apolipoproteins of the high density lipoprotein in the carp (Cyprinus carpio). Comp. Biochem. PhysioL 92B: 787-793, 1989. 7. Anderson, A.A., T.C. Fletcher and G.M. Smith. Prostaglandin biosynthesis in the skin of the plaice Pleuronectes platessa L. Comp. Biochem. PhysioL 70C: 195-199, 1981. 8. Ando, S. and M. Hatano. Isolation of apolipoproteins from carotenoid-carrying lipoprotein in the serum of chum salmon, Oncorhynchus keta. J. Lipid Res. 29: 1264-1271, 1988. 9. Applegate, ILR. and J.A. Glomset. Computer-based modelling of the conformation and packing properties of docosahexaenoic acid. J. L/pid Res. 27: 658-680, 1986. 10. Arias, H.R. and EJ. Barrantes. Phosphoinositides and inositol phosphates in Discopyge tschudii electrocyte membranes. Int. J. Biochen~ 22: 1387-1392, 1990. 11. Audley, M.A., K.J. Shetty and J.E. KinseUa. Isolation and properties of phospholipase A from pollock muscle. J. Food. ScL 43: 1771-1775, 1978. 12. Ayala, S., C.E. Castuma and R.R. Brenner. Fatty acid composition and dynamics of phospholipids from hake (Merluccius hubbsi) spinal cord and brain and sea bass (Acanthustius brasilianus) brain. Biochem. Int. 23: 163-174, 1991. 13. Ayrault-Jarrier, M., J. Burdin, L. Fremont and M.-T. Gozzelino. Immunological evidence for

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14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

27. 28. 29. 30. 31. 32. 33. 34. 35. 36.

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common antigenic sites in high-density lipoproteins from rainbow trout and man. Biochem. J. 254: 927-930, 1988. Babin, P.J. Plasma lipoprotein and apolipoprotein distribution as a function of density in the rainbow trout (Salmo gairdneri). Biochem. J. 246: 425-429, 1987. Babin, P.J. Apolipoproteins and the association of egg yolk proteins with plasma high density lipoproteins after ovulation and follicular atresia in the rainbow trout (Salmo gairdneri). J. Biol. Chem. 262: 4290-4296, 1987. Babin, P.J. and J.-M. Vernier. Plasma lipoproteins in fish. J. Lipid Res. 30: 467-489, 1989. Bandyopadhyay, G.K., J. Dutta and S. Ghosh. Synthesis of diene prostaglandins in freshwater fish. Lipids 17: 755-758, 1982. Bauermeister, A.E.M., B.J.S. Pirie and J.R. Sargent. An electron microscopic study of lipid absorption in the pyloric caeca of rainbow trout (Salmo gairdneri) fed wax ester-rich zooplankton. Cell. Tissue Res. 200: 475-486, 1979. Bazan, N.G. Supply of n-3 polyunsaturated fatty acids and their significance in the central nervous system. In: Nutrition and the Brain, Vol. 8, edited by R.J. Wurtman and J.J. Wurtman, New York, NY, Raven Press, pp. 1-24, 1990. Bell, J.G., J.R. Dick, J.R. Sargent and A.H. McVicar. Dietary linoleic acid affects phospholipid fatty acid composition in heart and eicosanoid production by cardiomyocytes from Atlantic salmon (Salmo salar). Comp. Biochem. Physiol., 103A: 337-342, 1992. Bell, J.G., R.S. Raynard and J.R. Sargent. The effect of dietary linoleic acid on the fatty acid composition of individual phospholipids and lipoxygenase products from gills and leucocytes of Atlantic salmon (Salmo salar). Lipids 26: 445-450, 1991. Bell, J.G., J.R. Sargent and R.S. Raynard. Effects of increasing dietary linoleic acid on phospholipid fatty acid composition and eicosanoid production in leucocytes and gill cells of Atlantic salmon (Salmo salar). Prostaglandins Leukotrienes Essent. Fatty Acids 45: 197-206, 1992. Bell, M.V. Molecular species analysis of phosphoglycerides from the ripe roes of cod (Gadus morhua). Lipids 24: 585-588, 1989. Bell, M.V. and J.R. Dick. Molecular species composition of phosphatidylinositoi from the brain, retina, liver and muscle of cod (Gadus morhua). Lipids 25: 691-694, 1990. Bell, M.V. and J.R. Dick. Molecular species composition of the major glycerophospholipids from the muscle, liver, retina and brain of cod (Gadus morhua). Lipids 26: 565-573, 1991. Bell, M.V. and J.R. Sargent. Protein kinase C activity in the spleen of trout (Salmo gairdneri) and the rectal gland of dogfish (Scyliorhinus canicula), and the effects of phosphatidylserine and diacylglycerol containing (n-3) polyunsaturated fatty acids. Comp. Biochem. Physiol. 87B: 875-880, 1987. Bell, M.V. and J.R. Sargent. Effects of the fatty acid composition of phosphatidylserine and diacylglycerol on the in vitro activity of protein kinase C activity from rat spleen: influences of (n-3) and (n-6) polyunsaturated fatty acids. Comp. Biochem. Physiol. 86B: 227-232, 1987. Bell, M.V. and D.R. Tocher. Molecular species composition of the major phosphoglycerides in brain and retina from trout: Occurrence of high levels of di-(n-3) polyunsaturated fatty acid species. Biochem. J. 264: 909-914, 1989. Bell, M.V., C.M.E Simpson and J.R. Sargent. (n-3) and (n-6) polyunsaturated fatty acids in the phosphoglycerides of salt-secreting epithelia from two marine fish species. Lipids 18: 720-726, 1983. Bell, M.V., C.M.E Simpson and J.R. Sargent. Fatty acid analyses of polyphosphoinositides from the gills of cod (Gadus morhua). Biochem. Soc. Trans. 13: 182-183, 1985. Bergot, P. Fat absorption. In: Nutrition des Poissons, Acres du Colloque CNERNA, Paris, 1979, edited by M. Fontaine, Paris, France, Centre National de la Recherche Scientifique, pp. 123-129, 1981. Bergot, P. and J.-E. Flechon. Forme et voie d'absorption intestinale des acides gras a chaine longue chez la truite arc-en-ciel (Salmo gairdneri rich.) I. Lipides en particules. Ann. Biol. Anim. Biochem. Biophys. 10: 459-472, 1970. Berridge, M.J. Inositol trisphosphate and diacylglyceroh two interacting second messengers. Ann. Rev. Biochem. 56: 159-194, 1987. Black, D., S.A. Kirkpatrick and E.R. Skinner. Lipoprotein lipase and salt-resistant lipase activities in the livers of the rainbow trout and cod. Biochem. Soc. Trans. 11: 708, 1983. Black, D., S.G. Mackie and E.R. Skinner. A lecithin :cholesterol acyltransferase-like activity in the plasma of rainbow trout. Biochem. Soc. Trans. 13: 143-144, 1985. Black, D., A.M. Youssef and E.R. Skinner. The mechanism of lipid uptake by tissues in the

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gairdneri. Lipids 19: 258-263, 1984. 155. Neas, N.P. and J.R. Hazel. Partial purification and kinetic characterization of the microsomal phospholipase A2 from thermally acclimated rainbow trout (Salmo gairdneri). J. Comp. Physiol. 155: 461-469, 1985. 156. Neas, N.P. and J.R. Hazel. Phospholipase A2 from liver microsomal membrane of thermally acclimated rainbow trout. J. Exp. Zool. 233: 51-60, 1985. 157. Nefoda, Z.A., E.I. Lizenko, V.V. Kosheleva, V.P. Sorokin and V.S. Siderov. Lipid composition of whitefish during embryogenesis. Sravn. Biokhim. Vodn. Zhivotn. 43-52, 1983. 158. Nelson, G.J. and V.G. Shore. Characterization of the serum high density lipoproteins and apolipoproteins of pink salmon. J. Biol. Chem. 249: 536-542, 1974. 159. Neuringer, M., G.J. Anderson and W.E. Connor. The essentiality of omega 3 fatty acids for the development and function of the retina and brain. Ann. Rev. Nutr. 8: 517-541, 1988. 160. Nevenzel, J.C., A. Gibbs and A.A. Benson. Plasmalogens in the gill lipids of aquatic animals. Comp. Biochem. Physiol. 82B: 293-297, 1985. 161. Ninno, R.E., M.A.A.P. De Torrengo, J.C. Castuma and R.R. Brenner: Specificity of 5- and 6-fatty acid desaturases in rat and fish. Biochim. Biophys. Acta 360: 124-133, 1974. 162. Noaillac-Depeyre, J. and N. Gas. Fat absorption by the enterocytes of the carp (Cyprinus carpio L.). Cell Tissue Res. 155: 353-365, 1974. 163. Noaillac-Depeyre, J. and N. Gas. Electron microscopic study on gut epithelium of the tench (Tinca tinca L.) with respect to its absorptive functions. Tissue Cell. 8:511-530, 1976. 164. Nomura, T and H. Ogata. Distribution of prostaglandins in the animal kingdom. Biochim. Biophys. Acta 431: 127-131, 1976. 165. Nomura, T., H. Ogata and M. Ito. Occurrence of prostaglandins in fish testis. Tohoku J. Agric. Res. 21: 138-144, 1973. 166. Norberg, B. and C. Haux. Induction, isolation and a characterization of the lipid content of plama vitellogenin from two Salmo species: rainbow trout (Salmo gairdneri) and sea trout (Salmo trutta). Comp. Biochem. Physiol. 81B: 869-876, 1985. 167. Ogata, H., T. Nomura and M. Hata. Prostaglandin biosynthesis in the tissue homogenates of marine animals. Bull. Jpn. Soc. Sci. Fish. 44: 1367-1370, 1978. 168. Ogata, H., T. Nomura and M. Hata. Prostaglandin PGF2a changes induced by ovulatory stimuli in the pond loach, Misgurnus anguillacaudatus. Bull. Jpn. Soc. Sci. Fish. 45" 929-931, 1979. 169. Ohshima, T., S. Wada and C. Koizumi. Estimation of possible fatty acid combinations of phospatidylcholine and phosphatidylethanolamine of cod. Bull. Jpn. Soc. Sci. Fish. 49: 123-130, 1983. 170. Ohshima, T., S. Wada and C. Koizumi. 1-O-alk-l'-enyl-2-acyl and 1-O-alkyl-2-acyl glycerophospholipids in white muscle of bonito Euthynnus pelamis (Linnaeus). Lipids 24: 363-370, 1989. 171. Ohshima, T, S. Wada and C. Koizumi. Application of selected-ion monitoring gas chromatography/ mass spectrometry to the analysis of molecular species of 1,2-diacylglycerophospholipids of bonito white muscle. Nippon Suisan Gakkaishi 55: 875-883, 1989. 172. Ohshima, T, S. Wada and C. Koizumi. Molecular species of 1-O-alk-l'-enyl-2-acylglycerophospholipids of bonito white muscle. Nippon Suisan Gakkaishi 55: 885-890, 1989. 173. OIsen, R., R.J. Henderson and T Pedersen. The influence of dietary lipid classes on the fatty acid composition of small cod (Gadus morhua L.) juveniles reared in an enclosure in northern Norway. J. Exp. Mar. Biol. Ecol. 148: 59-76, 1991. 174. Ota, T and T Takagi. Fatty acids in lipids of mature chum salmon, Oncorhynchus keta, with special reference to phytanic acid. Bull. Fac. Fish. Hokkaido Univ. 40: 313-332, 1989. 175. Pagliarani, A., M. Pirini, G. Trigari and V. Ventrella. Effects of diets containing different oils on brain fatty acid composition in sea bass (Dicentrarchus labrax L.) Comp. Biochem. Physiol. 83B: 277-282, 1986. 176. Patton, J.S. The effect of pressure and temperature on phospholipid and triglyceride fatty acids of fish white muscle: a comparison of deep water and surface marine species. Comp. Biochem. Physiol. 52B: 105-110, 1975. 177. Pettitt, TR. and A.E Rowley. Uptake, incorporation and calcium-ionophore-stimulated mobilization of arachidonic, eicosapentaenoic and docosahexaenoic acids by leucocytes of the rainbow trout, Salmo gairdneri. Biochim. Biophys. Acta 1042: 62-69, 1990. 178. Pettitt, T.R. and A.E Rowley. Fatty acid composition and lipoxygenase metabolism in blood cells of the lesser spotted dogfish, Scyliorhinus canicula. Comp. Biochem. Physiol. 99B: 647-652, 1991. 179. Pettitt, TR., A.E Rowley and S.E. Barrow. Synthesis of leukotriene B and other conjugated triene lipoxygenase products by blood cells of the rainbow trout, Salmo gairdneri. Biochim. Biophys. Acta

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Hochachka and Mommsen (eds.), Biochemistry and molecular biology of fishes, vol. 4 9 1995 Elsevier Science B.V. All rights reserved. CHAPTER 7

Amino acid metabolism in fish KARL JORSS AND RALF BASTROP

Universitdt Rostock, Fachbereich Biologic, Zoologisches lnstitut, Universiti~tsplatz 2, D-02500 Rostock 1, Germany

I. II. III. IV. V. VI. VII. VIII. IX.

Introduction Pools of free amino acids Blood Intestine (gut) Liver Kidney Muscle Interorgan amino acid fluxes Acknowledgement References

I. Introduction From the nutritionist's standpoint, the animal organism has one common pool of free amino acids (FAA or AA) into which all amino acids freed by the decomposition of food and body protein and all non-essential amino acids (NEAA) produced by biosynthesis flow. In turn, this pool also feeds all reactions in which AA are consumed (Fig. 1). However, this standpoint is too simplistic for a mechanistic analysis of amino acid metabolism. The following facts must be taken into account: (1) the total FAA pool is broken down by the cell membrane into an intracellular and an extracellular pool; (2) the intraceUular pool is organ specific and probably also cell specific. The intracellular pools of different organs are linked by the extraceUular pool (blood circulation). The various organs can therefore interact with each other as the nutritional status of the organism changes; (3) transport mechanisms are needed for movement of amino acids from the extracellular to the intracellular pool. They are also needed for the flow of amino acids from the 'milieu ext~rieur'; in this case both the intestinal epithelium (apical and basolateral cell membrane) must be crossed instead of a single cell membrane; and (4) the composition of the intracellular AA pool is strongly influenced by the enzymes present in the cell and their transport across the cell membrane. The same factors also govern the amino acid distribution among cellular compartments such as the cytosol and mitochondda. In the following we shall discuss mainly the roles and interactions of the organs in the fish AA metabolism on the basis of our knowledge of the mammalian organism,

K. Jfrrss and R. Bastrop

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,.,,,,,.

DIETARY PROTEINS

[

BODY PROTEINS

1

AMMONIA

<x-KETO ACIDS

GLUCOSE

CO2, H20

LIPID

Fig. 1. Pool of free amino adds.

and draw attention to corresponding control points, since exhaustive descriptions of the sequences of biochemical reactions in which AA are involved in fish have already been published 27,136.145,149.To simplify the discussion, we shall consider the amino acids as being metabolized mainly in just four organs: the intestine, liver, kidney and skeletal muscle.

II. Pools of free amino acids The only comparative analysis published so far about the AA pools in different organs deals with the channel catfish (Ictaluruspunctatus), a freshwater species 155. Presenting FAA concentrations in the serum, liver, muscle, gill, gut tract, kidney, heart, brain and spleen after 48 h of fasting, this analysis permits the following conclusions to be drawn. 1. Each organ has a specific FAA concentration. 2. In general, the liver and kidney contained the highest concentration, expressed as #mol/g tissue, of most of the amino acids. 3. The total serum level of proteinogenic FAA of about 2.0/tmol/g is strikingly low compared to that, say, of the liver (130/~mol/g) and muscle (16.5 #mol/g). This indicates the importance of ATP dependent transport mechanisms for the uptake of the various amino acids into the fish cell at least after the postabsorptive phase. 4. If the total amount of each AA is considered as split up among the different organs, it can be seen that in the channel catfish the muscle contains the largest fraction of them all except aspartic acid.

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Less comprehensive analyses dealing with other teleosts (e.g. rainbow trout 27) lead to the same conclusions.

IlL B l o o d The intracellular AA pools of the different organs are linked by the blood circulation system. It is striking that the blood of the channel catfish contains less than 1% of the total of each proteinogenic amino acid after 48 h of starvation ~ss. This fraction naturally increases more or less temporarily after feeding 1~ The blood itself has an intracellular AA pool (mainly in the red blood cells, RBC) and an extracellular pool (plasma). An interaction between erythrocytes and the plasma may play a role in convectional AA transport. The AA concentrations in the plasma relative to those in the cells of the various organs and the variations in these ratios after feeding by the fish are also of interest. In carp (Cyprinus carpio), coho salmon (Oncorhynchus kisutch), rainbow trout (Oncorhynchus mykiss) and channel catfish (Ictalurus punctatus), plasma contains 47.5, 58.2, 52.6 and 9.0% of the sum of the essential amino acids (EAA), respectively and 32.8, 38.3, 33.4 and 22.3%, respectively, of the sum of the NEAA. The remainder resides in the intracellular pool. Ogata and Arai 111 calculated the FAA content of erythrocytes from the levels found in plasma and whole blood. Their studies also revealed that intraceUular concentrations of taurine (2-aminoethane sulfonic acid), aspartic acid and glutamic acid were the highest (> 10-fold) relative to those found in the plasma. The AA must be actively transported across the membrane to achieve the concentrations found in the RBC. An Na+-dependent mechanism which transports L-serine has already been identified in the erythrocytes of brown trout, Salmo trutta 44. The species-specific nature of the intracellular and extracellular AA distribution is shown by the fact that the lowest intracellular FAA concentrations were found as a rule in the coho salmon. Moreover, the concentrations of all EAA in erythrocytes of the channel catfish is distinctly higher (>3-fold) than in the plasma. It is conceivable that erythrocytes perform a transport function particularly for amino acids whose concentrations in the RBC change rapidly after feeding. The faster an amino acid can enter the erythrocytes, the more probable it seems that the RBC are involved in its transport, thereby increasing the transport capacity of the blood for that AA. The blood amino acid pattern reflects the net results of digestion, absorption and subsequent utilization. The free amino acid pattern in the plasma is influenced particularly by the composition of the protein in the food 48. This applies especially to the EAA. Plasma FAA levels generally undergo a marked increase after feeding to reach a peak level or a plateau, then return to prefeeding values. The time at which the peak post-feeding concentrations are reached may vary considerably among fish species and experimental conditions. A corresponding correlation has been found between dietary and systemic free essential amino acids at least in the rainbow trout 1~ the carp 115 and the channel catfish 153. Wilson and coworkers 153 interpret this as a form of metabolic conservation of EAA to ensure adequate circulation

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g. Jarss and R. Bastrop

levels for protein synthesis, whereas the NEAA in the diet are metabolized and altered to a great extent. Postprandial changes in plasma and erythrocyte FAA levels have been studied in parallel only for the carp. According to Ogata 1~ the RBC appear to be implicated in convectional transport only for tyrosine, phenylalanine, tryptophan, histidine, isoleucine and leucine. These EAA may be particularly easily transferred from the plasma to erythrocytes and back. They probably enter the cell mainly by diffusion because, except for tyrosine, their concentrations in the plasma and in erythrocytes scarcely differ 111. More species should be studied to define more precisely the function of erythrocytes in the transport of FAA. Knowledge of these mechanisms involved in the exchange of FAA between the plasma and erythrocytes will probably be of decisive importance in this question, although attention should focus on its capacity and speed. Attention should also be given to AA consumption by erythrocytes for metabolism in addition to the question of their transport function. Moderate activities of glutamate dehydrogenase (GDH, EC 1.4.1.2), aspartate aminotransferase (AspAT, EC 2.6.1.1) and alanine aminotransferase (AIaAT, EC 2.6.1.2), the most important enzymes implicated in AA catabolism, have been found in the erythrocytes of the rainbow trout 36. However, alanine oxidation rates are quite low in trout erythrocytes 144. In carp erythrocytes, glutamine is more important than glucose as an oxidative substrate. Its importance in this respect is exceeded only by lactate and pyruvate 13~ Glutamate, aspartate and isoleucine are oxidized to a much smaller extent. The oxidation of glutamate was only one tenth that of glutamine in the presence of other substrates. Tiihonen and Nikinmaa ~3~ attribute this to the properties of the carp erythrocyte membrane. The report that catfish hepatocytes produce five times more ammonia when using glutamine as a substrate than when using glutamate substantiates these findings 16.

IV. Intestine (guO The intestine plays a special role in maintaining AA homeostasis. The passage of the AA from the intestinal milieu across the intestinal wall into the circulation involves transceUular transport. When considering the absorption process, it is necessary to distinguish between uptake of AA by the brush-border membrane and its basolateral release into the circulation. In addition, depending on the physiological status of the fish, basolateral transport mechanisms selectively remove FAA from the circulation for organ-specific AA metabolism. When considering the AA requirements of the intestine, the rapid turnover of enterocytes and their proteins must be taken into account besides the AA needed for intermediary metabolism. The general anatomical structure of the fish intestinal wall is similar to that of the higher vertebrate intestine. An absorptive epithelium faces the luminal contents and is responsible for the transport of nutrients (e.g. amino acids), ions and water. However, in contrast to mammals, the intestinal epithelium of fish is not divided into villus and crypt regions 37. According to Vernier 141, a regional differentiation of the intestine in teleost fish gives rise to an anterior intestine, in which amino acids

Amino acid metabolism in fish

163

and peptides are absorbed, and a posterior intestine where proteins are absorbed in macromolecular form. Epithelial cells of the posterior intestine are characterized by a heterophagous process for dietary proteins. Vernier ~4~ also attributes nutritional as well as immunological functions to the posterior intestine. Basically, amino acids can cross the apical and basolateral cell membrane with the aid of Na+-dependent carriers, Na+-independent carriers or simple diffusion. Studies of amino acid uptake into intact epithelia and brush-border membrane vesicles (BBMV) have shown that the transapical step consists of 'active' accumulation. Much less is known about the mechanisms of AA transport across the basolateral membrane ~2~ In the eel (Anguilla anguilla), the intestinal brush-border transport of all AA tested (a-aminoisobutyric acid, a-(methylamino)isobutyric acid, N-methylglycine, alanine, glycine, glutamic acid, lysine, phenylalanirie and proline) was performed by Na +dependent secondary transport systems. Na+-independent systems were detected for alanine, glycine and lysine. An additional significant diffusional transfer across the brush border was also observed for all of the above amino acids. At least four distinct Na+-dependent carrier mechanisms exist in the brush-border of the ee118,127,128,142:

1. A cationic system transporting exclusively lysine and arginine. 2. A second specific system for the anionic amino acids glutamic acid and aspartic acid. 3. A third mechanism responsible for the transport of proline and N-methylated amino acids; this system is inhibited completely by alanine and partly by phenylalanine. 4. A neutral amino acid system accepting most neutral AA such as alanine, glycine, serine and cysteine. Comparable systems also exist in the brush border of the mammalian small intestine s8. Like the mammals, fish also have multiple transporters with overlapping specificities. The Na+-dependent nutrient transport mechanisms are energized in the final analysis by Na+/K+-ATPase. The intracellular Na + level is maintained at a low level by this enzyme, which pumps Na+across the basolateral membrane. The energy produced by ATP hydrolysis and invested in the Na + pump is thus stored in the inwardly directed Na + gradient. A group of carrier proteins in the mucosal membrane provide the means by which the Na + influx is coupled to the 'uphill' movement of nutrients (AA) into the cell. This mechanism can only function if K +channels are present for recycling the K + ions, and these were recently found by Loretz and Fourtner as in the basolateral membrane of intestinal epithelial cells of a goby (Gillichthys mirabilis). Thus, the intestinal intake of amino acids is possibly controlled at least at three points (Fig. 2): (1) carrier protein; (2) Na+/K+-ATPase; and (3) K + channel. In this context it is interesting to note that Reshkin and coworkers 119 induced parallel changes in the apical glucose transporters and the basolateral Na+/K+-ATPase of the upper intestine (anterior intestine) of tilapias (Oreochromis mossambicus) by means of hormone treatment. The apical enterocyte membrane of fish transports dipeptides besides FAA 12'118. A comparative study into the uptake of phenylalanine

164

K. Jarss and R. Bastrop

LUMEN ii

i

iiiiii

i

9~|

a

.

i

I

BLOOD Fig. 2. Intestinal intake of amino acids.

and glycyl-L-phenylalanine into BBMV of the anterior intestinal epithelium of Ore-

ochromis mossambicus acclimated to sea water showed that glycyl-L-phenylalanine is transported intact by a cationic-independent facilitated diffusion mechanism during which the dipeptide is rapidly hydrolyzed in the vesicle. The dipeptide transport mechanism is independent of the systems carrying L-phenylalanine ns. Such systems might perform an important function during the early stages of digestion when peptide concentrations in the intestinal lumen are high. The transepithelial amino acid absorption system requires exit pathways across the basolateral membrane. It must be assumed that basolateral uptake mechanisms also exist. Their function would be to nourish the epithelial cells from the blood when amino acid intake from the intestinal lumen is limited. Less is known about the mechanisms serving the transport across the basolateral membrane of the vertebrate intestine than about the transport mechanisms of the apical membrane because the former are less accessible for experimentation ~9'Ss'n~ The fact that FAA are transported into the blood on the one hand while, as we have seen, transport mechanisms must simultaneously exist for the uptake of amino acids out of the circulation on the other is an additional complication. Experiments with basolateral membrane vesicles (BLMV) from the intestinal epithelium of seawater-adapted European eels (Anguilla anguilla) have shown that the mechanisms vary considerably. Among the six free L-AA that were tested, only proline and glutamic acid appeared to be transported through the basolateral membrane by Na+-dependent carriers. The simultaneous presence of an inward Na +gradient and an outward K + gradient was necessary for glycine, while alanine, lysine and phenylalanine were transported by Na+-independent facilitated diffusion and, apparently, simple diffusion n~ Owing to the ionic gradients existing in vivo,

Amino acid metabolism in ftsh

165

ion-dependent transport mechanisms will not serve the translocation of AA into the circulation. They must in some way be linked to the specific metabolic requirements of the enterocytes and serve the active uptake of glutamic acid and glycine from the circulation into the intestinal epithelial cell. The decisive processes taking place in the basolateral membrane and serving as the last step in the transcellular translocation of amino acids from the intestinal lumen into the circulation are probably facilitated diffusion and free diffusion. Facilitated diffusion by means of carriers and free diffusion are possible due to the accumulation of FAA in the intestinal epithelial cell and may, in the final analysis, therefore also be dependent upon the step energized by Na+/K+-ATPase, i.e. the Na+-dependent uptake of free amino acids across the apical membrane. This raises the question of the degree to which the various A.A, transport mechanisms of the intestine are involved in the control of AA homeostasis, because the luminal milieu varies according to diet composition and time after feeding. In addition, the regulation mechanism must affect both the brush border and the basolateral transport mechanisms. Experimental results have been published only for brush-border transport in fish. Buddington and coworkers 14 compared proline uptake across the brush border membrane in nine fish species of differing natural diets which all received the same diet. The proline transport rates varied much less among species than glucose uptake, since species with different natural diets still have similar protein requirements. In adaptation experiments with Oreochromis mossambicus, a proteinrich and a protein-deficient diet revealed no differences in the maximum rate of proline transport 131. In other words, regulation apparently serves to match transporter activity to a running average of diet composition over many days. However, fish might also possess a basic set of AA transporter proteins, and their activity would be capable of instantaneous modulation. This kind of regulation mechanism would match the different AA transport systems to the current gut content. Allosteric actions of amino acids on AA carriers are conceivable as triggers. The most important functions of the intestinal epithelial cells in quantitative terms are probably proliferation, protein synthesis and the transport of solutes and ions. All three of these are linked in two ways with amino acid metabolism or transport: protein synthesis, the basic condition for the synthesis of enzymes and transporter proteins, form the basis for cell proliferation. It requires a balanced spectrum of FAA. Protein synthesis and transport functions, like regulation processes involving phosphorylation, require ATP, which is probably obtained to a great extent by the oxidation of amino acids. Of the total protein synthesis taking place in a rainbow trout weighing 80 g one hour after the last feeding, the proportion found in the whole digestive tract is 31% at 10~ and 39% at 18~ However, only 10% of this is deposited 33,34. Thus, in absolute terms, the digestive tract has a higher protein turnover than any other fish organ. The presence of key enzymes in the gut implies that the oxidation of FAA accounts for a substantial fraction of the ATP production. Wilson 152 studied the tissue distribution of the three most important enzymes of amino acid catabolism in the channel catfish. The activities of alanine aminotransferase and aspartate aminotransferase per gram organ fresh weight and

166

K. J~irss and R. Bastrop

per mg protein were distinctly lower in the intestine than in the kidney and liver. However, consideration of the total organ activities per 100 g body weight revealed that gut glutamate dehydrogenase was quantitatively the most important enzyme (see below).

AspAT AIaAT GDH

Liver (U/IO0 g fish weight) 40.7 28.0 3.4

Gut tract (U/100 g fish weight) 18.2 10.3 4.9

Since this applies only to GDH, it does not necessarily imply that transdeamination plays an important role. As stated earlier, active basolateral uptake of glutamic acid from the blood has been observed in the eel (Anguilla anguilla). It is also interesting that, of 22 AA tested in the sea water adapted Japanese eel (Anguilla japonica), only L-glutamine, L-glutamic acid, L-alanine and v-alanine stimulated ion and water transport in the intestine 2. GDH serves to introduce glutamine and glutamate carbons into the citrate cycle in the form of a-ketoglutarate. Neither of these AA appears to undergo transamination in this case because, unlike in the case of ~.alanine, the transport effects were not inhibited by aminooxyacetate. Interestingly, the rate of glutamine and glutamate uptake into the intestinal epithelial cell probably limits the transport effect of these AA in the Japanese eel and not their intraceUular enzymatic turnover 3. The effect of v-alanine is particularly interesting and requires further study. D-Alanine is probably deaminated oxidatively to pyruvate by a D-amino-oxidase. Such an enzyme has been found in the pyloric caeca of the rainbow trout 3s. Another important fact was reported by Mural et al.l~ a considerable temporary (3-6 h) increase in the ammonia concentration was observed in the hepatic portal vein of force-fed rainbow trout receiving a casein diet relative to that in starved fish. This transient effect had almost vanished within 12 h, whereas the aspartate and glutamate levels did not increase. In summary, the intestine (gut tract) of fish has a high AA requirement relative to that of the whole organism due to its high protein turnover. Moreover, FAA must be oxidized on a considerable scale in the gut, so that during the postabsorptive phase certain amino acids must be extracted from the circulation. This probably applies to glutamine and glutamic acid. This implies that the gut is also involved in the ammoniogenesis of the fish to an extent that has yet to be defined. Glutamine is an important energy source for the mammalian intestine, which extracts it from the blood. However, in mammals the intestine releases alanine into the circulation22,35. Gluconeogenesis from FAA and the channeling of the carbon skeletons of FAA into fat synthesis probably fail to play a role in the actual mucosal epithelium.

V. Liver The function of the liver is very closely intertwined with that of the intestine in that it filters the products of digestion and releases them into the circulation

Amino acid metabolism in fish

167

for use by other organs. The amino acids transported through the basolateral membranes of the enterocytes pass along the hepatic portal system to the liver of the fish. The postprandial changes in the FAA have been studied in the plasma and hepatopancreas of the carp (Cyprinus carpio11~ and in the plasma and liver of the rainbow trout is~ In the case of the carp, a close correlation was found particularly between EAA in the diet, in the blood plasma and in the hepatopancreas four hours after the last feeding. In the rainbow trout, however, the FAA in the liver remained fairly stable throughout the postprandial period. Taking the postprandial changes in the AA concentrations into account, it was found that in the trout the liver concentrations of taurine, aspartie acid, glutamic acid, glutamine and alanine were several times higher (5- to 33-fold) than the plasma concentrations. Van der Boon and colleagues 134 reported similar differences between blood and liver concentrations of taurine, aspartic acid, glutamic acid, glutamine, asparagine and glycine in the goldfish (Carassius auratus). It can safely be assumed that ATP-dependent mechanisms are involved in AA uptake from the circulation. Such mechanisms can possibly regulate the metabolism of various amino acids in the fish liver in the same way, for instance, as alanine metabolism is regulated in the mammalian liver 24,32. However, Haschemeyer 5~ showed that the uptake of a mixture of 15 radioactively marked amino acids supplied through the hepatic portal vein is very rapid in the oyster toadfish, Opsanus tau. AA uptake rates into the intracellular space peak within one minute at a body temperature of 24"C and within about two minutes at ll*C. It is therefore improbable that AA transport limits protein synthesis in the liver. This need not apply to other reactions, however. The amino acyl tRNA synthetases are known to have much higher substrate affinities than amino acid catabolizing enzymes 145. According to Campbell and coworkers 16, the higher rates of ammoniogenesis from glutamate, glutamine and aspartate by isolated mitochondria than by the whole cell suggest that the transport of these three amino acids may be limiting for their metabolism by intact catfish hepatocytes. Since the liver is the central site of intermediary metabolism, it is not surprising that it is also the most important organ governing AA homeostasis in the fish. Hepatic parenchymal cells are the main site of transdeamination 91,146 and gluconeogenesis 82 ,93 ,96 ,98,100 Moreover, the liver is an organ with a high protein turnover 33,34. Blood AA levels rise considerably in hepatectomized eels (Anguilla anguilla and Anguilla japonica) 67,77, while sham operated and hepatectomized eels do not differ in their levels of ammonia excretion. If L-alanine is injected into starved fish, ammonia excretion increases perceptibly only in the sham-operated specimens 77. In fed fish, ammonia is produced mainly in the liver, whereas in the postabsorptive state, kidney, musculature and intestine also contribute significantly to total amino acid deamination. As a rule, activities of AA catabolizing enzymes are high in the liver 136,145,152. Apart from direct deamination of a few AA by specific enzymes 27, the main pathway for ammoniogenesis in the liver is transdeamination (Fig. 3). The purine nucleotide cycle88, which is an important route for release of ammonia in fish muscle, is of little quantitative significance for ammoniogenesis in the fish liver 17,135. The transfer of the ammonia to c~-ketoglutarate (2-oxoglutarate) is a basic

K. 1arss and R. Bastrop

168 NADH

ot-KETOGLUTARATE

AMINO ACID

AMINOTRANSFERASES

GLUTAMATE DEHYDROGENASE

m,,.

~-KETO ACID

,

GLUTAMATE

,

.,

NAD

+

+ H_O z

Fig. 3. "l~ansdeamination.

prerequisite for transdeamination. In the liver of the goldfish (Carassius auratus) glutamate is formed rapidly from alanine, tyrosine, aspartate and branched-chain AA. Ammonia from histidine is also transferred quickly to a-ketoglutarate 135 in either of two ways. Ammonia may be liberated by histidase and transferred to a-ketoglutarate by glutamate dehydrogenase as suspected by van Waarde 135. Alternatively, the ammonia may be transferred to pyruvate by a histidine aminotransferase. However, reports on the presence of the latter enzyme in the fish liver are contradictory 27. Glutamate dehydrogenase (GDH), the key enzyme in transdeamination, is located in the mitochondrial matrix. Ammoniogenesis by transdeamination is therefore a mitochondrial process. Being an allosteric enzyme, GDH is the decisive point of control for catabolism in the AA metabolism. It is inhibited by ATP and GTP and stimulated by ADP, AMP and leucine 39,53.1~. In hepatocytes, AlaAT and AspAT are present both inside and outside of the mitochondria 1~,147. If ammonia production from a particular AA is stimulated by ADP, then transdeamination is probably involved. Similarly, inhibition of ammonia production from a particular AA by the transamination inhibitor aminooxyacetate can be regarded as a sign of the deamination of the AA through transdeamination. Experiments of this kind showed that aspartate and alanine are deaminated by transdeamination in the mitochondria of catfish hepatocytes, whereas serine is deaminated only to a small extent by this pathway. In the catfish hepatocyte, serine is probably deaminated mainly by a cytosolic serine dehydratase (EC 4.2.1.13) 16. In the rainbow trout hepatocyte, serine is reported to be mainly transaminated in the gluconeogenesis process 147. However, the main route of serine catabolism in fish liver is probably through the action of serine hydroxymethyltransferase (EC 2.1.2.1). In this reaction, the serine reacts with tetrahydrofolate to form glycine and N ~N 10 -methylenetetrahydrofolate 27 ' 107 . In vitro, ammonia production from various AA by hepatocytes of Ictalun~ punctatus decreases in the order asparagine, glutamine > alanine, serine > aspartate, glutamate. The order for isolated mitoehondria is

Amino acid metabolismin fish

169

glutaminc > glutamate > alaninc, serine, aspartatc and asparaginc 16. The authors drew two conclusions from this result: (1) the higher rates of ammoniogencsis from glutamate, glutamine and aspartate by isolated mitochondria suggest that the metabolism of these AA in the intact channel catfish hepatocyte is limited by their transport through the hepatocyte membrane; and (2) the higher or equal rates of ammoniogenesis from glutaminc, glutamate, aspartate and alanine by mitochondria suggest that the mitochondrial compartment is the main site of their catabolism. Conversely, the markedly higher rate of ammoniogenesis from asparagine by intact hepatocytes than by isolated mitochondria implies that asparagine catabolism takes place mainly in the cytosol, although only 24-34% of total asparaginase (EC 3.5.1.1) activity is found in the cytosolic fraction of channel catfish liver 17. The teleost liver can produce ammonia from glutamine (Gin) and asparaginc (Asn) at high rates 17,126,139. Both glutaminase I (EC 3.5.1.2) and glutaminase II (EC 2.6.1.5) are present in the teleost liver 17,2~ The phosphate dependent glutaminase I predominates quantitatively and is located in the hepatocyte mitochondria. The intracellular localization of asparaginase is apparently still not known exactly 16. At any rate, both enzymes catalyze reactions yielding substrates that can flow directly into transdeamination: Asparaginase A s n + H 2 0 ~ Asp + NH3

(1)

Glutaminase I Gin + H 2 0 ~ Glu + NH3

(2)

Finally, all or some carbon atoms of many AA end up in pyruvate or certain intermediates of the citric acid cycle. In carnivorous fish, where the natural diet is high in protein and low in carbohydrate, amino acids constitute a major source of energy 136. Liver cells utilize AA as an energy source either directly by oxidation of the carbon skeleton or indirectly by conversion of the carbon skeleton to glucose. AA can also be used in fat synthesis 149. Glutamic acid, both from the body protein of the fish 45'154 and from food protein 1~ is probably the AA present in the largest quantities. In whole body tissue of fish, this is followed by aspartic acid, lysine, leucine and alanine (g/100 g amino acid). It should be remembered that glutamate and aspartate can be produced from glutamine and asparaginc respectively in a single, hydrolytic step. In view of the quantitative predominance of glutamic acid in protein and its close link to the citric acid cycle, one might think that these AA would be used mainly for oxidation. This also applies to a lesser degree to aspartic acid and alanine. On the other hand, all three of these AA are glucogenic. Moreover, oxidation is the only possibility for the essential ketogenic AA lysine and leucine. All these AA or their carbon skeletons can naturally be used in the synthesis of protein and fat respectively. The turbot (Scophthalmus maximus) oxidizes about twice as much glutamic acid and alaninc as it does leucine and phenylalanine, regardless of the protein content of its diet 26. High glutamic acid and alanine oxidation rates have also been reported in the hepatopancreas of the carp (Cyprinus carpio L.; refs. 105 and 106). In an experiment with alanine, histidine, proline, lysine, leucine, serine, asparagine, glycine and valine it was found that hepatocytes of rainbow trout

170

K. Jarss and R. Bastrop

oxidized mainly asparagine, serine and alanine 42. The main AA oxidized by little skate (Raja erinacea) hepatocytes was glutamate followed at some distance by glutamine, aspartate, alanine, proline and serine 1~ Summing up, it can be stated that, despite all species-specific differences, glutamic acid, aspartic acid, alanine, serine, asparagine and glutamine are the most important AA in fish liver with respect to oxidation. Gluconeogenesis in the liver or in isolated hepatocytes of the fish is among the main topics of current research 93.9s,1~176 The most important AA for gluconeogenesis in the fish liver appear to be alanine, glutamic acid, serine and aspartic acid 56,1~176 whereby alanine and glutamic acid appear to be channeled preferably into this pathway. In the fish liver, gluconeogenesis from serine 147 and aspartic acid 117 is less important in quantitative terms than that from alanine. Alanine obtained by proteolysis in the musculature forms the starting point for de novo glucose synthesis in the liver of migrating salmon (Oncorhynchus sp.; refs. 41 and 95). In the teleost Paralabrax clathratus, gluconeogenesis from alanine and glutamate increases 3.9- and 2.2-fold respectively in the course of starvation for 14 days, whereas gluconeogenesis from aspartate decreases 16.7-fold 7. The question remains how the AA are distributed between direct oxidation via the citric acid cycle and gluconeogenesis in the liver? In terms of the stoichiometry of the carbon, glucose synthesis from alanine dominates over oxidation to CO2 in hepatocytes from sockeye salmon (Oncorhynchus nerka) 41. The same also applies to alanine in hepatocytes of the American eel (Anguilla rostrata); but in these cells aspartate is converted to CO2 rather than used for glucose synthesis4~ After a major analytic effort, Jungas and coworkers 69 came to the following conclusion regarding the liver of omnivorous mammals: the amount of ATP produced by AA oxidation is roughly the same as the amount needed to convert amino acid carbon to glucose. According to these authors, the daily supply of amino acids in the diet cannot be totally oxidized to CO2 in the liver, mainly because ATP production would then exceed demand in the liver. At present, no such budget can be formulated for fish owing to the lack of experimental data. In fish, the liver is also the main site of fat synthesis m. Although protein would seem to the major natural source of carbon for fatty acid synthesis in fish, little is known concerning the flow of amino acid carbon into fat 55.m. After intraperitoneal injection of U-14C-labeled glutamate into juvenile carp (Cyprinus carpio), much more radioactivity was found in hepatopancreatic lipid than in glycogen. [U-14C]Glucose was hardly incorporated into hepatopancreatic lipid l~ Correspondingly, more [14C]alanine than [14C]glucose was converted into fatty acids by slices of rainbow trout liver in vitro54. However, radioactivity from [14C]aspartate was not incorporated into fatty acids in hepatocytes from the eelAnguilla rostrata ~7. The distribution of AA in the fish liver between protein synthesis on the one hand and oxidation, gluconeogenesis and lipogenesis on the other is probably influenced strongly by nutritional status and the consequent hormone balance. The most important exogenous factors influencing AA homeostasis in the liver are food quantity and composition. Intracellular AA concentrations are also strongly influenced by salinity in euryhaline teleosts64.

Amino acid metabolism in fish

171

It has often been shown that the liver enzymes serving AA metabolism in omnivorous mammals possess a certain degree of adaptability to food composition 47. After analyzing the corresponding experimental findings for fish, Cowey and Walton 27 came to the conclusion that 'there is little effect on the activities of amino acidcatabolizing enzymes'. At this point, we consider this conclusion a little premature for the following reasons: (1) too few species have been studied so far; (2) in the studies published to date, the units chosen to express enzyme activities were often unsuitable for such generalized conclusions; and (3) the experimental conditions (e.g. diets in which protein was replaced by poorly metabolizable carbohydrate) led in some cases to inappropriate conclusions. To assess the importance of changes in liver enzyme activity for the whole fish organism it is necessary to consider the total liver activities relative to body weight (U/g liver fresh weight x liver-somatic index (%) -- U/100 g body weight). Conclusions relating to the liver cell itself can be obtained by expressing enzyme activities relative to DNA levels. The decisive enzymes for transdeamination, i.e. glutamate dehydrogenase, aspartate aminotransferase and alanine aminotransferase, should be considered on the basis of our own results in this respect. It should be noted that aminotransferase activities were all expressed as total activities (cytosolic + mitochondrial isoenzymes). It is therefore possible that any adaptive effect would affect only one isoenzyme, and the activity of this isoenzyme would in turn change more relative to the total activity. GDH activity is usually measured as glutamate formation in the presence of the allosteric effector ADE An adaptive change in GDH activity, should it reflects a change in the amount of enzyme, must be considered specially because the activity of the existing molecules of this aUosteric enzyme can already be greatly modulated by ligands 53. In a two factor (feeding x salinity) experiment 73 with tilapia, Oreochromis mossambicus, starvation reduced the activities (U/100 g body weight) of AspAT and AIaAT in the liver to less than half of the original levels. GDH activity was reduced almost to half the original level. Salinity, which strongly affects the free amino acid concentration in the liver of O. mossambicus 4, had no effect on the activities of these enzymes 73. A series of experiments with rainbow t r o u t 72,74-76 led to quantitatively similar results as far as salinity effects are concerned. Food deprivation also significantly reduced GDH activity (U/mg DNA) per liver cell in carp (Cyprinus carpio) 5. The activity of GDH in the rainbow trout liver cell appears to play a different role of the enzyme is regulated by a route differing from that observed in other teleostean fishes. After starvation or a reduced ration, the activity of the trout liver enzyme stabilizes at the level of a well nourished fish, whereas AspAT activity is slightly, but significantly,

reduced71,75, 76. Generally reduced activities of important AA catabolism enzymes were found in the livers of tilapia and carp in accordance with the lower NH3 excretion by starved fish reported by Fromm 43 and Infante 66. This suggests that the activity of this metabolic pathway is reduced as a result of starvation. The fact that there is no loss of GDH activity (U/100 g body weight) in the liver of starved rainbow trout can only mean that the liver plays a greater role in gluconeogenesis in this species than in others. After intraperitoneal injection of [U- 14C]glutamate into

K. Jiirss and R. Bastrop

172

rainbow trout after a 30 day starvation, De la Higuera and Cardenas 56 found more radioactivity in blood glucose and liver glycogen than in fish receiving a high protein diet. Gluconeogenesis from [14C]alanine increases by similar amounts in rainbow trout fed a high protein diet or if the animals are starved 2s. In ammoniotelic fish, gluconeogenesis is linked to ammoniogenesis by a route that appears to be functionally similar to the way gluconeogenesis is linked to urea cycle in ureotelic mammals 69. The function of gluconeogenesis is supported by the fact that gluconeogenesis from pyruvate and alanine is activated by leucine in the same way as GDH in Anguilla japonica 52,53. Although the role of transdeamination must be sought primarily in connection with gluconeogenesis during starvation, in fish receiving a high protein diet transdeamination is probably mainly responsible for channelling AA to the citric acid cycle and fatty acid synthesis despite the increased level of gluconeogenesis under these conditions. The oxidation of AA increases considerably when fish are fed a high protein diet 26,146. In rainbow trout, this is accompanied by increased conversion of food protein into body fat 74'83 if the fish are given an isoenergetic protein rich, but low fat diet, the liver activities (expressed in units per 100 g body weight) of AspAT and AIaAT increase substantially7~ This also applies to GDH and the lipogenic enzymes which, under these conditions, are also adjusted to a higher activity level (Table 1). This effect is most obvious when the activities obtained for the diets A and C are expressed as ratios. Similar adaptive response of enzymes involved in transdeamination will be scarcely discernible, if at all, when the protein in the diet is replaced by carbohydrate (see ref. 27 for a review of various findings). In other words, the enzymes implicated in transdeamination in the teleost liver respond adaptively in several fish species, although not to the same degree as has been recorded for mammals 47. Some enzymes metabolizing amino acids are also involved in biosynthetic pathways. As the case of ornithine decarboxylase (ODC; EC 4.1.1.17) demonstrates, such enzymes may also be subject to marked adaptive changes in fish. ODC is the first and rate-limiting enzyme in polyamine biosynthesis. After a 12 clay starvation period, hepatic ODC activity in TABLE 1 Effect of diet on glutamate dehydrogenase and NADPH-generating enzymes in rainbow trout (Oncorhynchus mykiss) liver Diet A

Diet B

Diet C

Raw protein (% of dry weight) Raw fat (% of dry weight)

24.0 25.0

46.2 12.1

57.8 6.7

Enzyme Glutamate dehydrogenase Glucose 6-phosphate dehydrogenase 6-Phosphogluconate dehydrogenase

Enzyme activity in units/mg DNA 11.7 4- 1.0 12.2 4- 1.1 18.0 4- 0.8 3.1 4- 0.1 3.9 4- 0.4 9.8 4- 1.2 1.7 4- 0.1 2.2 4- 0.3 5.6 4- 0.7

Ratio C/A 1.5 3.2 3.3

Glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) and 6-phosphogluconate dehydrogenase (E.C. 1.1.1.43) are considered lipogenic enzymes, since they produce cytosolic NADPH equivalents required for fatty acid synthesis 46. Data from Bastrop and Jiirss (unpublished results).

A m i n o acid metabolism in fish

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brook charr (trout), Salvelinus fontinalis, increased from almost zero to about 60 pmol CO2/h/mg protein within 24 h after refeeding 6.

VI. Kidney Formally, amino acid homeostasis in the kidney (excretory part) is similar to that in the intestine. Amino acids not only enter the tubular cells from the lumen, but also from the peritubular blood. This flux opposes the cellular exit of AA of luminal origin. Amino acids taken from both sides of the tubular cell enter the same pool. Owing to the specific enzymes in the renal cell, certain AA may be removed from the pool or the concentrations of certain NEAA may increase is a result of synthesis. In the channel catfish (I. punctatus), the concentrations of most FAA are quantitatively about the same in both the kidney and the liver. Only the methionine concentration is about 2.6 times as high in the kidney as in the liver in this species ls5. In the rainbow trout, in contrast, the methionine concentration in the liver is higher by a factor of 5 than in the kidney 149. The differences between FAA concentrations liver and kidney are altogether more pronounced in the rainbow trout than in the channel catfish. Renal taurine and alanine concentrations are very high in the latter species (> 20 /zmol/g) 155. Relatively high glutamate concentrations are feature common to the renal FAA pools of the catfish (14/zmol/g), rainbow trout (6.9/tmol/g) and goldfish (2.3 /.tmol/g) 79'149'155. The concentrations of most FAA are higher in the kidney than in the plasma of all three of these teleost species. ATP-dependent transport mechanisms are probably mainly responsible for maintaining the concentration difference. This applies equally to FAA extracted from the ultrafiltrate and to any taken up from the peritubular blood vessels. The process of AA absorption from the ultrafiltrate appears to be more efficient in fish than in mammals because the carp and channel catfish excrete fewer AA in their urine than humans 112. In vertebrates, the amino acids filtered out at the glomeruli are re-absorbed in the early proximal tubule 12s. The FAA concentration differences between the ultrafiltrate, tubulus cell and blood plasma suggest that an uphill gradient has to be overcome during amino acid resorption across the brush-border membrane, whereas the peritubular exit step is a downhill process. Eveloff and colleagues 31 published a comparative study of the BBMV in the kidney and intestine of winter flounder, Pseudopleuronectes americanus. The uptake of L-alanine was stimulated by sodium and inhibited by the addition of 10 mmol L-phenylalanine in BBMV from both organs. Much of what we know about FAA uptake into the intestinal epithelial cells probably also applies in principle to the less studied cells of the proximal tubulus of the fish kidney. The peritubular exit of amino acids is not well understood even in mammals 125. Active transport has been shown to be involved in the peritubular uptake of glutamine, glycine, taurine and acidic AA for protein synthesis, gluconeogenesis and ammoniogenesis at least in the case of mammals 125. In the case of P. americanus it was found that taurine is actively secreted in

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the tubulus. This also applies to species of fishes with aglomerular kidneyss. The movement of taurine across the peritubular membrane was identified as the Na + and Cl- dependent concentrating step in taudne secretion 124. Unlike fish, mammals do not actively secrete taurine in the kidney 125. In most species, cellular, organ and whole body taurine concentrations are regulated by transport; biosynthesis and metabolism are of only minor importance in this respect 6s. This invites one to conclude that, since taudne is of great importance to fish as an osmotic effector, the ability to secrete it actively through the kidney represents a specially developed control point for regulating the amount of taurine present in fish. Taurine enters the fish organism with food and is probably also synthesized in the fish liver by a pathway resembling that found in mammals 157. If expressed in units per g fresh weight, the activities of the three most important transdeamination enzymes (glutamate dehydrogenase, aspartate aminotransferase and alanine aminotransferasr in the kidney of Ictalur~ punctatus 152, Cyprinus carpio and Oncorhynchus mykiss 122 are close to those in the liver. In the teleost kidney, AIaAT and AspAT are distributed extra and intramitochondrially in the same ways as in the liver79. In an experiment in which the organ-somatic index was used to express activities relative to 100 g fish weight, Jfirss and colleagues7s showed that GDH capacities were slightly higher in the kidney of rainbow trout than in the liver, while renal AspAT activity was twice as high as the liver AspAT level. In Salmo salar, however, renal GDH and AspAT activities expressed in U/g were only about one tenth as high as in the liver~. At any rate, as in the liver, transdeamination appears to be quantitatively the main process by which ammonia is formed in the fish kidney. The purine nucleotide cycle does not appear to contribute to ammonia production in the kidney of the fish79'146. GDH is regulated by an aUosteric process and, therefore, is also important controlling AA metabolism in the kidney. Bittorf 9 compared the hepatic and renal GDH of the rainbow trout after nearly 150-fold purification. Both renal and hepatic forms of the enzyme were inhibited by ATP (1 mmol/l), GTP (1 retool/l), malate (5 retool/l) and isocitrate (5 mmol/l). Among 15 AA (administered at a final concentration of 5 mmol/1) tested, only leueine activated hepatic and renal GDH. The renal activity was activated to similar degrees in both directions: in the presence of leucine, the rate of oxidative deamination was enhanced to 197% of control. Conversely, reductive amination was increased to 212%, respectively. Liver GDH was activated much more strongly towards reduetive amination: oxidative deamination increased to 139% of control, while reduetive amination reached 313%. Hughes et al. 62 found that, among five Salvelinus namaycush tissues tested (anterior kidney, posterior kidney, gill, liver and muscle), specific activity was highest for branched-chain amino acid aminotransferase (EC 2.6.1.42; BCAAT) in the posterior kidney. Similar results relating to this extra and intramitoehondrial enzyme have been obtained for other teleost specieszT. BCAAT catalyzes the first step in the catabolism of leueine, isoleucine and valine, in which the amino group is transferred either to a-ketoglutarate or to the corresponding branched-chain a-ketoaeid. By synthesizing glutamate, this enzyme leads to the oxidative deamination of branched-

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chain AA (BCAA) by GDH. The ketoacids remaining after transamination serve as substrates for irreversible oxidative decarboxylation, which is the second step in the BCAA degradation pathway and is catalyzed by branched-chain a-ketoacid dehydrogenase (EC 1.2.4.4; BCKAD) a multienzyme complex located on the inner surface of the inner mitochondrial membrane 49. Unlike BCAAT, BCKAD is a highly regulated enzyme at least in mammals. Among the AA-degrading enzymes only BCKAD and phenylalanine hydroxylase are regulated by phosphorylationdephosphorylation mechanisms 49. BCKAD has so far been studied only in the liver and red muscle of the A. rostrata 97 and in the liver of the rainbow trout 3~ The reaction catalyzed by BCKAD yields the corresponding CoA compounds: BCAAT

BCKAD

leucine - , a-ketosiocaproate --, isovaleryl-CoA

(3)

isoleucine - , a-keto-~l-methylvalerate - , r

(4)

valine - , a-ketoisovalerate - , isobutyryl-CoA

(5)

The subsequent reactions are comparable to those involved in fatty acid oxidation 49. Naturally, these reaction sequences do not necessarily all take place completely in the kidney of fish. Part of the ketoacids from transamination of the branched-chain AA can leave the renal cells and be translocated to other tissues by the circulation. The activities of serine catabolizing enzymes in the kidney and liver respectively of the rainbow trout reported by Walton and Cowey 148 yield the following activity quotients between these organs (Table 2). The direct deamination of serine is catalyzed by serine dehydratase in the cytosol: Serine dehydratase serine --, pyruvate + NH + + H 2 0

(6)

In other words, the available tissue distribution of the serine catabolizing enzymes indicate that the kidney is an important site of serine catabolism in fish. The mammalian kidney releases serine into the circulation 1. A major comparative study of ammoniotelic teleost species 28 showed that specific arginase (EC 3.5.3.1) activity in the kidney was exceeded only by that in the liver. However, if the arginase activities in the various organs are compared with the total arginase activity in the whole fish, it is found that renal arginase activity in fed rainbow trout is only about one third lower than in the liver 21,76. Van Waarde and Kesbeke ~39 report that in the TABLE 2 Serine catabolizing enzymes in rainbow trout liver and kidney Kidney/liver activity ratio Serine dehydratase (E.C 4.2.1.13) Serine pyruvate transaminase (E.C. 2.6.1.51) Serine-hydroxy methyltransferase (E.C. 2.1.2.1) Data from Walton and Cowey14s.

2.1 0.5 0.3

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goldfish (Carassius aumtus) renal asparaginc synthetase and glutamine synthetase activities are 0.6- and 2.1-fold as high as in the liver. They could not detect asparaginase (EC 3.5.1.1) in the kidney of this species. Mitochondrial glutaminase I (EC 3.5.1.1) is found with relatively high activities in the kidneys of various fish species 79'146. Altogether, it is evident that the kidney possesses the enzymes needed for the degradation of substantial amounts of AA. The catabolized FAA are probably only oxidized there and used for gluconeogenesis, whereby changes in the proportions between the AA will naturally also occur due to the synthesis of N A A and the release of degradation products into the circulation. The first step towards channeling the AA to oxidation or gluconeogenesis is direct deamination (e.g. of histidine) and/or indirect transdeamination. As in other vertebrates 8~ ammoniogenesis in the fish kidney could serve a special function in acid/base regulation. Glutamine serves as a source of ammonia for regulation of the acid/base balance in mammalian kidneys~ Pequin 113 and Pequin and Serfaty 114 showed that the blood ammonia level increases from 0.68 to 4.60/zg/ml during passage through the kidney. In view of the previously mentioned results achieved by Kenyon 77 with the hepatectomized eel it can be assumed that the kidney contributes appreciably to total ammoniogenesis in the postabsorptive fish. In obligate air-breathers such as Arapaima gigas, the renal contribution is probably highest of all regardless of nutritional status. In this species, the enzymes found in the unusually large kidney which is not divided into head and trunk regions 57 suggest that transdeamination is the main form of ammoniogenesis. Kidney and liver homogenates from Stizostedion vitreum deaminated more glutamine and asparagine than any of the other 18 AA tested by Stieber and Cvancara 126. However, according to King and Goldstein 79, intact renal cells (teased renal tubules) from the goldfish (C. auratus) produced most ammonia from aspartate. These authors report that AA were utilized in the following order for ammoniogenesis: aspartate > > alanine > glutamine > glycine > glutamate. After incubating kidney slices from the elasmobranch Squalus acanthias with AA, King and Goldstein 7s found that the quantitative sequence for ammonia formation was glutamine > > aspartate, glutamate, alanine, glycine. However, when analyzing the results of experiments with intact renal cells, it is necessary to take possible variations in the transport capacity of the renal cell into account. According to King and Goldstein 78'79 acidosis leads to an ammoniogenic response in the kidney of the goldfish and dogfish. The authors suspect that ammonia production in the kidney of Squalus acanthias is regulated by the relative activities of glutaminase and glutamine synthetase working in a substrate cycle between glutamine and glutamate + NH +. Such a cycle is scarcely conceivable for the teleost kidney owing to the low glutamine synthetase activity79. Ammonia is produced in the teleost kidney and is released mainly into the circulation (for subsequent excretion via the gills), but is also excreted to a lesser degree with the urine 116. The remaining carbon skeletons are oxidized directly in the citric acid cycle or used for gluconeogenesis. In view of the enzymes it possesses, the kidney must be considered second only to the liver in importance as a site of gluconeogenesis s2'96. Slices of posterior kidney from Salmo salar, like liver slices from the same species,

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channel much more alanine derived carbon into oxidation (to CO2) than into gluconeogenesis. The gluconeogenic flux in the kidney, on a gram wet weight basis, is somewhat lower than in the liver 96. However, since it has been shown in the case of the liver that the distribution of AA between oxidation and gluconeogenesis varies according to the specific AA and is also species specific, the above findings can not be generalized. The experiments performed by Jiirss and colleagues 75,76 with the rainbow trout show that renal amino acid catabolism decreases relative to that of the liver after several weeks of starvation. Total renal activities of GDH, AspAT and arginase were markedly suppressed at the end of the starvation period, while the corresponding hepatic activities remained almost unchanged. After experiments with plaice, Pleuronectes platessa, Moon and Johnston 99 came to the conclusion that the role of the liver in amino acid metabolism is maintained and slightly enhanced by food deprivation, whereas that of the kidney is reduced. In the rainbow trout, renal GDH and AspAT activities scarcely respond to diets with different protein:lipid ratios, whereas activities of these enzymes in the liver increase significantly as the protein content of the diet increases 74. Lupianez et al. 89 obtained corresponding results for the different responses of GDH and AIaAT activities in the rainbow trout kidney and liver to increasing diet protein content also when protein was replaced by carbohydrate. However, the kidney seems to be important for the metabolism of branched-chain AA. Hughes et al. 63 showed that the addition of leucine or valine to the diet induced a significant increase in the activity of branched-chain amino acid transferase only in the posterior kidney of Salvelinus namaycush, and not in the muscle or liver. Leucine therefore appears also to be an important substrate in the teleost kidney.

VII. M u s c l e

The somatic muscle system accounts for over 50% of the body weight of most fish 151 and thus possesses the largest FAA pool in absolute terms lss. Many fish species possess both red and white musculature, but in most species red muscle accounts only for about 6% of the total muscle weight 87. Therefore, only the white muscle represents the largest FAA pool in fish. It is evident from the enzyme profile that red muscle is aerobic, while the white muscle is anaerobic 13,29. The morphological and biochemical differences between the two muscle types form the basis for their different functions. Electrophysiological studies show that during slow swimming the propulsive force is brought about entirely by red muscle and that, as the swimming velocity increases, white muscle is progressively recruited 61. With respect to protein turnover, the muscle differs fundamentally from other organs. According to in vivo experiments undertaken by Fauconneau and Arna133,34 only about 33% of total protein synthesis in fed rainbow trout takes place in the muscle. However, 52-76% of the synthesized protein is deposited in the muscle. This is considerably more than in such metabolically active organs as the liver or intestinal tract, which deposit only about 5 and 11% of the synthesized protein, respectively 33,34. The red and white musculature differ in this respect, too. The red muscle of rainbow

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trout retains 30.4% of the synthesized muscle, whereas white muscle retains no less than 79.7% ~~ It can therefore be concluded that fewer amino acids flow into the FAA pool from protein degradation in the muscle of non-starved fish than in other organs. Since neither lipogenesis nl nor gluconeogenesis from amino acidss2,1~ play a quantitatively important role in fish muscle, the main physiological purposes of AA catabolism in the muscle must be two-fold. First, to support ATP production for locomotion, and second, to supply other organs with substrates during periods of depletion. The different functions of the two muscle types are also reflected in qualitative and quantitative distinctions between their AA pools. Van der Boon and coworkers z33 report the following quantitative AA sequences in the goldfish after 6 days of starvation: in white muscle histidine is most prevalent, followed in decreasing abundance by glutamine, glutamate, asparagine, glycine and alanine. In red muscle, the sequence is: His > Gin > A s n > Gly > Ala > Glu. Individual ratios for white over red muscle are: His = 1.7, Asn = 1.4, Gln = 1.1, Gly = 2.8, Glu = 0.28, Ala = 2.8. The concentrations of these and a few other AA in the circulation system of the goldfish are low, and the high intracellular concentrations in the muscle must therefore be achieved by means of active transport mechanisms or, in a few cases, synthesis. Since, in addition, taurine reaches its highest concentration (>20/~mol/g wet weight) in the muscle of the goldfish - as it does in many other fish species - the FAA pool of the goldfish muscle can be considered typical of cyprinids 134. It is interesting to note that the taurine concentration is high in the liver of rainbow trout, whereas its concentration in the muscle is relatively low~Ss. AIaAT, AspAT, GDH and branched-chain amino acid transferase (BCAAT) activities are many times higher in the red, oxidative muscle of teleosts than in the white, glycolytic muscle99'135. The highest AspAT activities are found in the red muscle and heart of fishs3'135. The intra and extramitochondrial AspAT in this case, forming a component of the malate-aspartate shuttle, very probably serves the transport of electrons. Of the three enzymes catabolizing serine, low activities of serine-hydroxy methyltransferase have been detected in the red muscle of the rainbow trout only1~. Van Waarde 135 reports that aspartate is the only actively transaminated amino acid in the epaxial white muscle of goldfish, whereas significant transamination of aspartate and the branched chain AA leucine, isoleucine and valine takes place in the lateral red muscle. Asparagine and glutamine synthetase activities are higher in the red muscle of the goldfish than in the white muscle, while asparaginase and glutaminase activities were too low to be measured (activity <0.05 units) 139. Phosphate-dependent glutaminase (EC 3.5.1.2) has been detected in mitochondria isolated from the mudskipper (Periophthalmus chrysospilos68). While red muscle shows a much greater ability to catabolize AA than white muscle 135, the anaerobic white muscle needs glycogen to perform work. One of the functions of the AA pool in the white muscle of fish is probably to provide a reserve AA supply for other organs and tissues. In salmonids, the bulk of the energy used for sustained swimming (with the red muscle) is obtained by oxidizing amino acids. Van den Thillart 132 injected 14 C-labeled glucose, palmitate or glutamate, alanine and leucine individually into rainbow trout (O. mykiss) and measured the rates of

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oxygen consumption and CO2 production at rest and while swimming at 80% of the maximum speed. Glucose oxidation was extremely low in both resting and actively swimming fish. Protein and lipid oxidation were estimated to provide 80 and 20% respectively of the total substrates oxidized in the whole fish at rest and 90 and 10% respectively during sustained swimming. The rate of oxidation of leucine increased much more during the transition from the resting to the swimming state than that of alanine and glutamate. Amino acids must be deaminated before their carbon skeletons can enter the oxidative metabolism. Two major pathways have been described for the liberation of ammonia from amino acids: the mitochondrial glutamate dehydrogenase route using glutamate and the cytosolic purine nueleotide cycle using aspartate as the ultimate substrate. Intact aerobic red muscle mitochondria from goldfish (Carassius auratus) produce large amounts of aspartate and small quantifies of ammonia when incubated with glutamate, ADP and orthophosphate. Aspartate is the only nitrogenous end product during anaerobiosis 136. Malate, IMP, AMP and NH3 levels increased in the white muscle after direct electrical stimulation of the goldfish myotome 14~ whereas aspartate decreased. The concentration of lactate in the red muscle increased 2.6-fold, while the corresponding increase in the white muscle was 13.2-fold. However, blood ammonia levels did not change. The results cited here suggest that the enzymes of the purine nucleotide cycle play some role in NH3 production, but do not prove that the purine nucleotide cycle supplies the NH3 from the muscle for excretion through the gills. Mommsen and Hoehachka 94 obtained data sets which gave additional insight into the role of white muscle purine nucleotide cycle in the rainbow trout by measuring white muscle metabolites after exhaustion and during the time of their recovery. The changes in the metabolite concentrations in the exhausted muscle were similar to those reported by van Waarde and Kesbeke 14~ for electrically stimulated goldfish muscle. The specific dynamics of ATP, IMP, NH3, malate and aspartate during the 25 h recovery period led Mommsen and Hochachka to the conclusion that the purine nucleotide cycle as such cannot operate as a functional unit. It seems more probable that two temporally and functionally separate phases exist. Phase 1. Exhaustive exercise: A M P 2- + H 2 0 ---, IMP 2- + NH3. T h e NH3 can

accept protons released during the cleavage of ATP and thus serve as an H+buffer with the concomitant formation of NH +" NH3 + H + ---, NH +. Phase 2. The bulk of the NH + is retained in the white muscle and transferred to a-ketoglutarate by GDH during recovery. Transamination yields aspartate, and the proton picked up from the medium by NH3 is released backinto the medium when aspartate is added to IMP. Only further experiments will show whether this reinterpretation for the function of the components of purine nucleotide cycle is correct. However, from the results presented by Mommsen and Hochachka 94 it seems certain that the purine nucleotide cycle of the muscle does not contribute appreciably to ammoniogenesis for excretion. Experiments performed by Ip and coworkers 20.68 with mitochondria prepared from the lateral muscle of different species of mudskippers (Perioph. thalmus chrysospilos, Boleophthalmus boddaerti, Periophthalmodon schlosseri) also

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K. :l~rss and R. Bastrop

suggest that the purine nucleotide cycle does not play a leading role in the net ammoniogenesis. Muscle mitochondria preparations from all three species produced most ammonia after the addition of glutamine, but only very small amounts after the addition of alanine or aspartate. No ammonia at all was produced when serine was added. The quotients of the ammonia produced with the two most important substrates (glutamine/glutamate) were 34.0 for P. chrysospilos 35.0 for B. boddaerti and 36.0 for P. schlosseri, respectively. Since glutamine can supply only twice as much ammonia as glutamate, it must be concluded that glutamine enters the mitochondria more quickly and/or to some extent produces ammonia more quickly and without transdeamination. Aminooxyacetate and bromofuorate (an inhibitor of glutamate dehydrogenase) completely inhibit ammonia production from glutamate and aspartate in all three species. In 1?.chrysospilos, ammonia production from glutamine was curtailed to 21% of control by bromofuorate and reduced to 32% of control by aminooxyacetate 6s. Apart from illustrating the importance of transdeamination in ammoniogenesis from glutamine, these findings suggest that the permeability of the internal mitochondrial membrane may not be the same for glutamine and glutamate (transport mechanisms). Moreover, in view of the inhibitory effect of aminooxyacetate, an as yet undetected aminotransferase in the fish muscle must be involved in ammoniogenesis from glutamate. Finally, we must remark, without commenting on their quantitative significance, that the musculature may contribute to ammonia excretion by fish in one of the two following ways: (1) direct release into the circulation system of ammonia from transdeamination (in view of the enzyme profile, predominantly in the red musculature) and breakdown of the glutamine (white muscle; ref. 133); or (2) transport of the amino group to the liver in the form of alanine or glutamine (cf. section VIII). Clearly, the enzymatic machinery responsible for the production of ammonia from glutamine requires more detailed investigation.

VII. Interorgan flux of amino acids The physiological necessity of an amino acid flux between organs stems from the biochemical specialization of the various tissues and organs and the need to guarantee supplies of their substrates during the postabsorptive status and long periods of food deprivation. Hormones are doubtless important for regulating the parts played by the organs in AA metabolism, but their role will not be discussed here. Any discussion on interorgan amino acid nutrition presupposes knowledge of the blood circulation, transport mechanisms of the cell membrane, and AA metabolism in the various tissues and organs. In the case of fish, our knowledge in these areas is far from complete and based on only a few species. Therefore, this section will discuss problems rather than present known facts. Unlike that of fish, the flow of amino acids between organs in mammals has been a topic of metabolic research for many years 5'22'23'35'69'92. It therefore seems appropriate to take the mammalian interorgan AA flux as a starting point for considering its counterpart in fish. Theoretically, of course, the fate of every single AA should be analyzed, but this

Amino acid metabolism in .fish

181

has not yet been done even for mammals. However, the quantitatively important fluxes of alanine, glutamine and branched-chain amino acids (BCAA) have been analyzed quite extensively. In the mammalian system, AA interconversion takes place after the amino acids from a protein rich diet have been taken up into the mucosal cells. In other words, the liver receives a mixture of AA processed by the intestine, whereas the peripheral organs receive a different AA mixture from the liver. In the mammalian mucosal cells, glutamine, glutamate and aspartate are converted into alanine, citrulline, ornithine, proline and ammonia 92. Except for the synthesis of citrulline and ornithine, a similar transformation appears to take place in fish. Citrulline and ornithine are synthesized in the mammalian intestine and likely fuel the hepatic urea cycle. This route of carbon/nitrogen flux could therefore be useful only for ureotelic fish such as a species of tilapia living in an alkaline lake (Oreochromis alcalicus grahami156)and the Gulf toadfish, Opsanus beta 143. However, nothing is known in these two species about the role of the intestine in this respect. Murai et al. 1~ report that levels of alanine, proline and ammonia in the hepatic portal vein increase rapidly regardless of diet composition in force-fed rainbow trout receiving a defined casein or amino acid diet. Surprisingly, considerably less glutamate and aspartate appear in the plasma than might be expected from the AA content of the diet. This finding suggests that glutamate and aspartate are oxidized (intestinal ammoniogenesis) or converted into proline and alanine. Glutamate is the precursor for the biosynthesis of proline 129. At present, however, no enzymes are known to be involved in proline biosynthesis in fish 27. Alanine might be produced from glutamate and pyruvate by transamination, and ammonia is probably liberated from glutamate by oxidative deamination and from aspartate by transdeamination. As noted in section IV, intestinal GDH, AIaAT and AspAT activities are sufficiently high for these reactions. It is therefore evident that the AA mixture in the hepatic portal vein of fish has been modified by the intestinal metabolism. Murai and colleagues 1~ also measured the differences between the AA concentrations in the hepatic portal vein and the hepatic vein. AA uptake by the liver was very intense for up to 12 h after feeding. The intensity of hepatic uptake accounted for almost half of the amounts absorbed in the portal blood for almost all EAA, including the BCAA. In mammals, however, the liver extracts only small amounts of BCAA from the portal blood 49. Perhaps the fish liver can utilize BCAA better than the mammalian liver. This is suggested not only by the findings of Murai and coworkers 1~ but also by the enzymes present in the fish liver. Upon being taken up into the intracellular hepatic AA pool, BCAA are used for protein synthesis or undergo transamination to yield BCKA and glutamate. In the rat, BCAAT activity in the liver amounts to only 11 or 12% of the activity found in skeletal muscle49. Hepatic BCAAT activity in Salvelinus namaycush is about 57% of the activity found in the muscle 62, and Moon 97 reports that BCAAT activity in the liver of Anguilla rostrata is even higher than in red muscle. However, activities of the second enzyme in BCAA catabolism, BCKAD, are almost equal in the liver and red muscle of A. rostrata, whereas in mammals its activity in the muscle is only 5-8% of that found in the liver97. Of the NEAA, the liver extracts quantitatively appreciable amounts of alanine

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and proline from the portal blood in the rainbow trout ~~ Although a considerable amount of information is available concerning the catabolism of alanine in the fish liver (section V), proline has not been studied in this respect zT. Its carbon skeleton could be used for glutamate synthesis 129 and thus ultimately flow into the large liver pools of glutamate and glutaminr The interaction between liver and musculature in AA homeostasis is seen most distinctly in the postabsorptivr fish and during food deprivation. It is important to note that reversals in direction while fasting are not characteristic of the major AA fluxes2z. Proteolysis in the muscle is the main source of AA for other organs after starvation. Houlihan and colleagues s9 reported that white muscle contributes 66% to the whole body protein loss during starvation. This means that white muscle is mainly responsible for the maintenance of plasma AA concentrations during food deprivation. In view of the concentration gradient, the transport of the AA from the muscle into the circulation probably does not consume ATE Blasco r al. 11 published a detailed study into the variation in time of plasma AA levels in starved carp (C. carpw) without, unfortunately, analytically distinguishing glutamine from glutamate. According to Ogata and Arai 111, the glutamine concentration in the plasma of the carp is 28 times higher than the glutamate concentration. We shall therefore assume that the changes recorded by Blasco et al. 11 for glutamine plus glutamate refer only to glutamine. If this assumption is correct, it would mean that the glutamine concentration in the carp plasma was elevated from the 8th to the 50th day of starvation. The alaninr concentration was abnormally high only from the 5th to the 19th day. BCAA levels in the plasma of the starved carp were significantly depressed during the first week of starvation. The plasma leucine and isoleucine concentrations in these fish were significantly higher than normal from the 19th to the 50th day of starvation. Muscle is the most important tissue for the oxidation of BCAA in mammals 2z,49 and fish 13z. This applies particularly to the red muscle owing to its special role in sustained swimming, its blood flow and its enzyme profile. From the results published by Blasco et al. 11, we can conclude that the demand for BCAA exceeds proteolytic BCAA liberation in the carp during the first week of starvation. According to Loughna and Goldspink s6, protein degradation in the white muscle of starved rainbow trout does not increase until after about one week. The increased amounts of glutamine and alanine very probably entering the blood stream from the muscle during starvation might sometimes be destined for organs. In mammals, for instance, glutamine may be supplied to the intestine and kidneys 1'9~ Under starvation conditions, alanine must be the main substrate for de novo glucose synthesis in the liver. Hayashi and Ooshiro 51 observed that 14C-glucose synthesis from 14C-alanine increased by 100% in A. japonica during starvation for periods ranging from 1 to 2 months. Moreover, alanine is known to be the single important source for de novo synthesis of glucose in the liver of migrating sockeye salmon (Oncorhynchus nerka) 41,95. There are signs that alanine is synthesized at the expense of other AA in the fish muscle. Knapp and Wieser sl showed that the alanine concentration in the muscle of starved rudd (Scardinus erythrophthalmus) increased 2.5-fold, whereas the concentrations of other AA decreased. Leech and colleagues 84 reported that the tail muscle of the spiny dogfish (Squalus acanthias) releases alanine after fasting

Amino acid metabolism in fish

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for one day. Moreover, in vitro experiments with isolated pelvic fin muscles revealed that alanine biosynthesis was dependent on a source of added isoleucine or leucine, but not glucose 84. The following sequences of reactions seem conceivable for the synthesis of alanine and glutamine in the light of what we know about mammalian metabolism 49. Because a-ketoglutarate is an efficient amino-acceptor for BCAAT, BCAA amino groups could be used to form glutamate which in the presence of pyruvate and alanine aminotransferase can readily pass BCAA amino groups on to form alanine. The amino group of the BCAA would then be carded to the liver by alanine for transdeamination. In addition, in the presence of oxaloacetate and aspartate aminotransferase, glutamate can, via aspartate, liberate the amino groups of BCAA as ammonia via the purine nucleotide cycle, and ammonia can be transformed into glutamine by glutamine synthetase. Carbon skeletons (pyruvate and a-ketoglutarate) are needed for the de novo synthesis of alanine and glutamine. Pyruvate could be produced by glycolysis and ketoglutarate could be taken from the citric acid cycle, into which the carbon skeletons of the BCAA might also flow. The enzyme profile (BCKAD) of the fish liver suggests that some of the branched-chain a-ketoacids derived from the BCAA in the muscle are used in the fish liver in the same way as Harper et al. 49 describe their use in mammals. The kidney has scarcely been mentioned in this brief discussion of the interorgan amino acid flux owing to lack of data. Its position in the AA homeostasis of the fish urgently needs studying. Acknowledgement. The authors wish to express their thanks to Brian Patchett, Intertext Rostock, for his help in translating the manuscript.

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117. Renaud, J.M. and T.W. Moon. Starvation and the metabolism of hepatocytes isolated from the American eel, AnguiUa rostrata LeSueur. I. Comp. Physiol. 135: 127-137, 1980. 118. Reshkin, S.J. and Ahearn, G.A. Intestinal glycyi-L-phenylalanine and L-phenylalanine transport in a euryhaline teleost. Am. I. Physiol. 260: R563-R569, 1991. 119. Reshkin, S.J., M.L. Grover, R.D. Howerton, E.G. Grau and G.A. Ahearn. Dietary hormonal modification of growth, intestinal ATPase, and glucose transport in tilapia. Am. I. Physiol. 256: E610-E618, 1989. 120. Reshkin, S.J., S. Vilella, G. Cassano, G.A. Ahearn and C. Storelli. Basolateral amino acid and glucose transport by the intestine of the teleost, Anguilla anguilla. Comp. Biochem. Physiol. 91A: 779-788, 1988. 121. Sargent, J., R.J. Henderson and D.R. Tocher. The lipids. In: Fish Nutrition, 2nd edn., edited by J.E. Halver, San Diego, Academic Press, pp. 153-218, 1989. 122. Scheinert, P. and R. Hoffmann. Qualitative und quantitative Verteilung yon sieben Enzymen in Organen der Regenbogenforelle (Salmo gairdneri R.) und des Karpfens (Cyprinus carpio). J. Vet. Med. A, 34: 339-343, 1987. 123. Schlisio, W. and B. Nicolai. Kinetic investigations in the behaviour of free amino acids in the plasma and of two aminotransferases in the liver of rainbow trout (Salmo gairdnerii Richardson) after feeding on a synthetic composition containing pure amino acids. Cornp. Biochem Physiol. 59B: 373-379, 1978. 124. Schr6ck, H., R.P. Forster and L. Goldstein. Renal handling of taurine in marine fish. Am. I. Physiol. 242: R64-R69, 1982. 125. Silbernagl, S. The renal handling of amino acids and oligopeptides. PhysioL Rev. 68: 911-1007, 1988. 126. Stieber, S.E and V.A. Cvancara. Tissue deamination of L-amino acids in the teleost Stizostedion vitreum (Mitchill). Comp. Biochem. Physiol. 56B: 285-287, 1977. 127. StoreUi, C., S. Vilella and G. Cassano. Na-dependent D-glucose and L-alanine transport in eel intestinal brush border membrane vesicles. Am. I. Physiol. 251: R463-R469, 1986. 128. Storelli, C., VileUa, M. Paola Romano, M. Maffia and G. Cassano. Brush-border amino acid transport mechanisms in carnivorous eel intestine. Am. J. Physiol. 257: R506-R510, 1989. 129. Stryer, L. Biochemistry, 3rd edn., New York, Freeman and Company, 1988. 130. Tiihonen, K. and M. Nikinmaa. Substrate utilization by carp (Cyprinus carpio) erythrocytes. Jr. Exp. Biol. 161: 509-514, 1991. 131. Titus, E., W.H. Karasov and G.A. Ahearn. Dietary modulation of intestinal nutrient transport in the teleost fish tilapia. Am. J. Physiol. 261: R1568-R1574, 1991. 132. Van den Thillart, G. Energy metabolism of swimming trout (Salrno gairdneri). Oxidation rates of palmitate, glucose, lactate, alanine, leucine and glutamate. J. Comp. Physiol. 156B: 511-520, 1986. 133. Van der Boon, J., EA. Eelkema, G.E.E.J.M. Van den ThiUart and A.D.E Addink. Influence of anoxia on free amino acid levels in blood, liver and skeletal muscles of the goldfish, Carassius auratus L. Comp. Biochem Physiol. 101B: 193-198, 1992. 134. Van der Boon, J., G.E.E.J.M. Van den Thillart and A.D.E Addink. Free amino acid profiles of aerobic (red) and anaerobic (white) skeletal muscle of the cyprinid fish, Carassius auratus L. (goldfish). Comp. Biochem. PhysioL 94A: 809-812, 1989. 135. Van Waarde, A~ Nitrogen metabolism in goldfish, Carassius auratus (L.). Activities of transamination reactions, purine nucleotide cycle and glutamate dehydrogenase in goldfish tissues. Cornp. Biochem. Physiol. 68B: 407-413, 1981. 136. Van Waarde, A. Aerobic and anaerobic ammonia production by fish. Comp. Biochem. Physiol. 7413: 675-684, 1983. 137. Van Waarde, A. and M. de Wilde.Van Berge Henegouwen. Nitrogen metabolism in goldfish, Carassius auratus (L.) Pathway of aerobic and anaerobic glutamate oxidation in red muscle and liver mitochondria. Comp. Biochem. Physiol. 72B: 133-136, 1982. 138. Van Waarde, A. and E Kesbeke. Nitrogen metabolism in goldfish, Carassius auratus (L.). Influence of added substrates and enzyme inhibitors on ammonia production of isolated hepatocytes. Cornp. Biochem. Physiol. 70B: 499-507, 1981. 139. Van Waarde, A. and E Kesbeke. Nitrogen metabolism in goldfish, Carassius auratus (L.). Activities of amidases and amide synthetases in goldfish tissues. Comp. Biochem. Physiol. 71B: 599--603, 1982. 140. Van Waarde, A. and E Kesbeke. Goldfish muscle energy metabolism during electrical stimulation. Comp. Biochem. Physiol. 75B: 635-639, 1983.

Amino acid metabolism in fish

189

141. Vernier, J.M. Intestine ultrastructure in relation to lipid and protein absorption in teleost fish. In: Animal Nutrition and Transport Processes. 1. Nutrition in Wild and Domestic Animals, Vol. 5, edited by J. Mellinger, Basel, Karger, pp. 166-175, 1990. 142. Vilella, S., G.A. Ahearn, G. Cassano, M. Maffia and C. Storelli. Lysine transport by brush-border membrane vesicles of eel intestine: interaction with neutral amino acids. Am. J. Physiol. Am. J. Physiol. 259: Rl181-Rl188, 1990. 143. Walsh, P.J., E. Danulat and T.P. Mommsen. Variation in urea excretion in the gulf toadfish Opsanus beta. Mar. Biol. 106: 323-328, 1990. 144. Walsh, P.J., C.M. Wood, S. Thomas and S.E Perry. Characterization of red blood cell metabolism in rainbow trout. J. Exp. Biol. 154: 375-489, 1990. 145. Walton, M.J. Aspects of amino acid metabolism in teleost fish. In: Nutrition and Feeding in Fish, edited by C.B. Cowey, A.M. Mackie and J.G. Bell, London, Academic Press, pp. 47-67, 1985. 146. Walton, M.J. and C.B. Cowey. Aspects of ammoniogenesis in rainbow trout, Salmo gairdneri. Comp. Biochem. Physiol. 57B: 143-149, 1977. 147. Walton, M.J. and C.B. Cowey. Gluconeogenesis from serine in rainbow trout Salmo gairdneri liver. Comp. Biochem. Physiol. 62B. 497-499, 1979. 148. Walton, M.J. and C.B. Cowey. Distribution and some kinetic properties of serine catabolizing enzymes in rainbow trout Salmo gairdneri. Comp. Biochem. Physiol. 68B: 147-150, 1981. 149. Walton, M.J. and C.B. Cowey. Aspects of intermediary metabolism in salmonid fish. Comp. Biochem. Physiol. 73B: 59-79, 1982. 150. Walton, M.J. and R.P. Wilson. Postprandial changes in plasma and liver free amino acids of rainbow trout fed complete diets containing casein. Aquaculture 51: 105-115, 1986. 151. Weatherley, A.H. and H.S. Gill. The Biology of Fish Growth, London, Academic Press, 1987. 152. Wilson, R.P. Nitrogen metabolism in channel catfish, Ictalurus punctatus - I. Tissue distribution of aspartate and alanine aminotransferases and glutamic dehydrogenase. Comp. Biochem. Physiol. 46B: 617-624, 1973. 153. Wilson, R.P., D.M. Oatlin Ill and W.E. Poe. Postprandial changes in serum amino acids of channel catfish fed diets containing different levels of protein and energy.Aquaculture 49: 101-110, 1985. 154. Wilson, R.P. and C.B. Cowey. Amino acid composition of whole body tissue of rainbow trout and atlantic salmon. Aquaculture 48: 373-376, 1985. 155. Wilson, R.P. and W.E. Poe. Nitrogen metabolism in channel catfish, Ictalurus punctatus - I I l . Relative pool sizes of free amino acids and related compounds in various tissues of the catfish. Comp. Biochem. Physiol. 48B: 545-556, 1974. 156. Wood, C.M., S.E Perry, P.A. Wright, H.L. Bergman and D.J. Randall. Ammonia and urea dynamics in the Lake Magadi tilapia, a ureotelic teleost fish adapted to an extremely alkaline environment. Resp. Physiol. 77: 1-20, 1989. 157. Yokoyama, M. and J.-l. Nakazoe. Induction of cysteine dioxygenase activity in rainbow trout liver by dietary sulfur amino acids. Proc. Third Int. Syrup. on Feeding and Nutr. in Fish, Toba, Japan, pp. 367-372, 1989. 158. Yokoyama, M. and J.-l. Nakazoe. Effects of dietary protein levels on free amino acid and glutathione contents in the tissues of rainbow trout. Comp. Biochem. Physiol. 99A: 203-206, 1991.

Hochachka and Mommsen (eds.), Biochemistryand molecularbiologyoffishes, vol. 4 9 1995 Elsevier Science B.V. All rights reserved. CHAPTER 8

Protein synthesis in fish D.E HOULIHAN,C.G. CARTER*AND I.D. MCCARTHY Department of Zoology, University of Aberdeen, TUlydroneAvenue, Aberdeen AB9 2TN, Scotland, U.K. and * Department of Aquaculture, University of Tasmania, P.O. Box 1214, Launceston, Tasmania 7250, Australia

I. II. III. IV. V. VI. VII. VIII. IX. X. XI.

Introduction Measurement of rates of protein turnover General model of protein turnover in fish Individual variation in protein turnover Protein synthesis and free pool amino acid concentrations Energy cost of protein synthesis Species comparisons Life history protein turnover Protein synthesis in tissues and cells Conclusions Acknowledgements References

I. Introduction This chapter will discuss rates of protein synthesis in fish. It will principally explore nutrition and protein synthesis in the whole animal and discusses the impact that protein synthesis has upon rates of oxygen consumption. We believe that the emphasis for future studies of protein metabolism should be on the individual animal as it is becoming increasingly clear that the rate of food consumption of the individual is a major determinant of individual rates of protein synthesis. Before discussing protein consumption, growth and synthesis of individual fish it is necessary to critically examine current methods for their measurement. Possible relationships between rates of protein synthesis and oxygen consumption have been the source of much discussion in mammals and are also an area of some uncertainty in fish. However, an understanding of protein synthesis rates can act as a mechanistic explanation for the linkage between energy consumption and growth rate most commonly expressed in the energy budget formulation. It provides a resolution of the incorrect assumption contained in models of fish (and other heterotrophs) growth, such as the Putter-von Bertalanffy model, that growth and metabolism are independent processes competing for the same energy source 1~ Higher metabolism does not necessarily imply that less energy is available for the synthesis of proteins. Estimation of the energy cost of protein synthesis therefore forms a major part of this chapter.

192

D.F. Houlihan, C.G. Carterand LD. McCarthy

Protein synthesis can be viewed at a number of levels. Whole-animal values can be integrated into descriptions of assimilation/growth or assimilation/metabolism patterns in different fish species and will be the focus of this chapter. The measurement of protein synthesis rates in body organs and tissues can provide information on the extent to which differences exist between various tissues and offer a challenge in understanding the integration of organ metabolism into whole animal physiology. For example, we still do not understand the significance of the ditferenees in rate of mitochondrial protein synthesis between different tissues in fish53. However, it is probably extremely hazardous to move between these levels to draw conclusions on the effects of protein synthesis on oxygen consumption. For example, it is unlikely that the stimulation in synthesis of individual proteins, except perhaps actin and myosin, will impinge upon whole animal energetics. It has been estimated that metallothionein synthesis in metal-challenged daphnids represents a very small proportion of oxygen consumption ~2.

II. Measurement o f rates o f protein turnover In all animals there is a continual cycle of synthesis and degradation of protein, in conditions where the rate of protein synthesis exceeds the rate of degradation there will be a net gain of protein and a net loss occurs when degradation rates exceed synthesis rates 114. The majority of methods for estimating protein synthesis measure the flux of an amino acid or nitrogen 1~ This involves the use of tracer substances, i.e. amino acids labelled with an isotope, that are given in a single dose or by continuous infusion (reviewed in refs. 5, 104, 122, 129). The measurements, parameters and formulae that are commonly employed in studies of protein growth, synthesis and degradation and which will be used in this chapter are described in Table 1. Most often protein synthesis rates are expressed in fractional terms, i.e. the proportion of the protein mass synthesised per day (ks, Table 1). The term protein turnover was used by Waterlow and coworkers 122 to describe both protein synthesis and degradation depending on conditions. The definition of protein turnover is dependent on nutritional status; under maintenance conditions where there is no net loss or gain of body protein, fractional rates of protein synthesis (ks) and degradation (kd) will be equal and equivalent to the rate of turnover (kt), i.e. ks kd -- kt. Under conditions of protein loss the rate of degradation exceeds synthesis and the rate of turnover will be equal to the synthesis rate, i.e. ks - kt; during weight gain, de novo synthesis and resynthesis of degraded proteins contribute to synthesis so turnover will be equal to degradation, kd -- kt (refs. 56, 127). Almost all estimates of protein synthesis in fish have been made by measuring the in vivo incorporation of a radiolabelled essential amino acid into body protein using either constant infusion (e.g. refs. 31, 49, 112) or a single flooding dose (e.g. refs. 37, 40, 54, 59, 103) to administer the radiolabel. The available data for in vivo whole animal estimates of protein synthesis, measured by a variety of methods, in rainbow trout, Oncorhynchus mykiss (Walbaum), are collected in Table 2. Despite the range

193

Protein synthesis in fish

TABLE 1 Measurements, parameters and formulae used to describe protein turnover Measurement

Parameter

Formula

Protein consumed (g)

Protein consumption rate kr: % day -1

[g prot. consumed/g fish prot./day] x 100

Protein-nitrogen absorption efficiency (AEpN: %)

Protein-nitrogen absorption rate ka: % day -1

kr x A EpN

Protein content (g)

Protein growth rate

[(In Pf - In Po)/time] x 100

kg: % day -1

Specific radioactivity of bound (Sb) and free pool (Sa) phenylalanine DPM nmo1-1

Protein synthesis rate

[(Sb/Sa) x (1440/incorporation time)] x 100

ks: day -1

Protein degradation rate kd: day -1

kd = k , - kg

(k~) RNA content (rag) Protein content (g)

Capacity for prot. synthesis Cs: mg RNA g protein -1

(mg RNA/g protein)

Capacity for protein synthesis (Cs) Protein synthesis

RNA Activity kRNA: g protein synthesised g-1 RNA day -l

(ks x 10)/Cs

Protein consumption Protein growth

Protein retention efficiency kg/kr : %

(kg/kr ) x 1O0

Protein synthesis Protein consumption

Anabolic stimulation efficiency ks/kr : %

(kslkr)x 100

Protein growth Protein synthesis

Synthesis retention efficiency

(kg/ks)x 100

Protein synthesis

ks~k,:

Abbreviation used: Prot. = protein.

in methods and experimental variables, the maximum values for whole animal fractional rates of protein synthesis are around 4-5% for rainbow trout of less than 200 g. In each of these studies, there was a range of measured synthesis rates which is likely to be explained by differences in the nutritional status of individual fish (see below). Although in vivo incorporation is the most commonly used method to measure rates of protein synthesis, several studies have used isolated polyribosomes from fish epaxial muscle to make in vitro measurements of rates of protein synthesis (e.g. refs. 65, 71, 72). In order to avoid the problems of variable free pool specific radioactivity and amino acid recycling, the majority of studies in fish have used the flooding dose method of Garlick and coworkers 43 to introduce the radiolabel in a single high dose injection. The most commonly used radiolabel is 3H-phenylalanine and although previous work has shown that a flooding dose of this amino acid does not affect rates of synthesis 73,85, more work is needed in order to confirm this assumption.

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Comparison of whole animal fractional rates of protein synthesis in fed rainbow trout, Oncorhynchus rnykiss, measured by different methods Method

Synthesis (% day -1)

Weight (g)

Temperature (*C)

Meals (day -1 )

Reference

Caudal vein infusion Flooding dose injection a Flooding dose injection b Flooding dose injection b Flooding dose injection b Fed 15N-protein

2.3-5.1 2.1 (4- 0.2) 4.4 (4- 0.5) 1.2-4.5 0.6-1.5 1.5-4.5

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a Injection of L-[U-14C]-arginine. b Injection of L-[2,6-3H]-phenylalanine. c McCarthy et al., in preparation.

The measurements that are needed for the successful calculation of protein synthesis rates using the flooding dose technique are shown in Fig. 1. The data are taken from the white muscle of 80 g rainbow trout at 10*C (McCarthy et aL, in preparation). The concentration of phenylalanine in the tissues after the flooding injection can (and should) be measured in order to demonstrate flooding of the tissue free pool. Assuming a free phenylalanine concentration in the white muscle of rainbow trout of 74 nmol g-1 wet weight 2~ a 12-fold increase above normal levels is achieved following injection (Fig. la). Measurement of the protein-bound and free pool phenylalanine-specific radioactivities are required to calculate the fractional rate of protein synthesis (Table 1). It is important to construct time courses for the specific radioactivity of the free and the protein-bound phenylalanine. Following injection, the specific radioactivity of the free pool must be elevated and either remain stable or show a slow linear decline over the incorporation period (Fig. lb) in order to calculate synthesis rates 43,~. The labelling of the body protein must be linear over the incorporation time (Fig. l c) and from the intercept of the regression line it is possible to estimate how soon after injection the radiolabel began to be incorporated into body protein. Several studies have validated the use of the flooding dose method in fish at a range of water temperatures and using an incorporation time of between 40 min and 6 h 16'41'54'59'73'123. The flooding dose technique has been used to measure rates of protein synthesis in fish from 1 g up to 500 g in weight by either intraperitoneal or intravenous injection 16,54,59,75,123. Recently this technique has been used to measure synthesis rates in fish of less than 1 g either by microinjection into file peritoneal cavity78,86

Fig. 1. (a) The mean white muscle free pool phenylalanine concentration (nmol Phe g wet wt-1), (b) the mean white muscle free pool phenylalanine-specific radioactivity (Sa, DPM nmol Phe -1) and (c) the mean white muscle protein-bound phenylalanine-specific radioactivity (S b, DPM nmol Phe -1) for rainbow trout (80 g, 10oc)_ at various time intervals following a single caudal vein injection of 135 mM phenylalanine and L-[2,6- 3H]phenylalanine (McCarthy et al. in preparation). The data are presented as mean4-SEM for each time interval.

196

D.E Houlihan, C.G. Carter and I.D. McCarthy

or by adapting the method of Fauconneau 3~ and bathing juvenile fish in a flooding dose of 3H-phenylalanine53'ss. The flooding dose method has also been applied to measure in vivo rates of protein synthesis in isolated organs and in vitro in cells54,59,s3,s4,1~176 As a result of its ease of application and adaptability, coupled with a sensitive and selective method of extraction and measurement of phenylalanine nS, the flooding dose technique has become a valuable tool in the study of protein synthesis in fish. Recently our attention has turned to measurements of protein synthesis using stable isotopes, principally 15N, which have been used extensively in the study of protein synthesis in mammals (e.g. refs. 109, 122, 129) as well as for one ectotherm, the mussel, Mytilus edulis L.5~ Initial results for rainbow trout indicate that protein synthesis rates obtained from feeding 15N enriched protein and collecting ammonia are similar to those obtained with radiolabcUed amino acids (Table 2). Feeding 15N enriched protein is advantageous because it allows non-invasive and nondestructive measurements of protein synthesis which can be repeated on the same animal. The use of stable isotopes should be used increasingly in the study of protein synthesis in fish. This would enable us to answer questions such as, how do changes in environmental conditions affect the protein turnover of the same fish? Furthermore, being able to 'track' the same fish provides the opportunity to measure ontogenetic changes in protein synthesis and construct synthesis/weight relationships for the same fish. In contrast to our detailed understanding of the mechanisms of protein synthesis (e.g. ref. 99), relatively little is known about the mechanisms of protein degradation. Several of the cellular pathways concerned with protein degradation have been identified 93,117, however, our understanding of the processes involved is far from complete and this has made quantification of the process difficult. In endotherms two main isotopic methods have been used to estimate in vivo protein degradation rates. The first involves measuring the loss of radiolabel from prelabelled proteins with time. However, this method can be complicated by amino acid reutilisation 122. An alternative isotopic method has involved constant infusion of radiolabel (e.g. 3H-leucinc) for a known period followed by the measurement of the amino acid flux and the increase during the infusion period of the 3H labelling of body water 42. Due to the complications of direct measurement, most commonly protein degradation has been estimated from the difference between synthesis and growth, i.e. kd = ks - k s (refs. 53, 91, 93). Since in fish protein synthesis is measured over a period of minutes or hours and protein growth over a period of days or weeks, this calculation can only be an estimate of the true value. However, recent work has shown little diurnal variation in the synthesis rates in the skeletal muscle of rats 1~ or sea bass, Dicentrarchus labrax L.7s measured over a 24 h period. Fuller and colleagues 42 found a highly significant correlation between protein degradation estimated from the difference between synthesis and growth and from the whole-animal flux of 3H-leucine. These results have given confidence in the use of this technique to estimate in vivo protein degradation rates in fish. The range of in vitro methods that are available to measure rates of protein degradation has recently been reviewed .

Protein synthesis in fish

197

Reliable determinations of the whole-body rates of protein growth, loss or accretion, are not particularly easy to calculate either since initial values for an individual fish cannot be measured directly (but see Ellis29). However, this measurement is a necessary prerequisite in understanding rates of protein turnover. In long term studies the best method seems to be to use frequent weighings of individual fish (fresh weights) combined with the regular analysis of the protein content of whole animals sacrificed during the course of the growth estimate.

III. General model of protein turnover in fish As a starting point for the more complete analysis of protein and amino acid metabolism, we have adapted the model of Millward and Rivers 9~ to describe the

Consumed protein nitrogen

6.75 mmol N

I

,

J

Faecal loss

I

1.01mmolN

Absorbed protein nitrogen

5.74 mmol N

Total nitrogen loss

I

Free amino acid pool

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l

,,7, I

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I I I

Protein degradation

4.97 mmol N

2.82 mmol N

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I

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Fig. 2. Daily protein-nitrogen flux for immature 80 g rainbow trout (Oncorhynchusmykiss) at 10~ based on the model of Millward and Rivers 9~ The data presented are for: (a) a growing trout fed a ration of 1.4% body weight per day of a commercial diet; or (see Fig. 2b).

198

D.F. Houlihan, C.G. Carter and I.D. McCarthy j

i

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Total nitrogen loss

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Fig. 2. (continued from Fig. 2a) (b) a starving trout (McCarthy et at. unpublished data). Protein-nitrogen = protein/5.85 (ref. 45). Apparent absorption etficiency is calculated assuming 85% absorption 6~ and the white muscle total free amino acid pool2o, in preparation) is used as an indication of the whole body free amino acid pool.

daily protein nitrogen flux in a growing immature rainbow trout following feeding (Fig. 2). The loss of nitrogen is shown as a single figure, although further subdivision of loss via ammonia from amino acid oxidation, urea and other nitrogenous molecules ultimately derived from the catabolism of non-protein substrates or via proteins lost in, for example, mucus, scales and digestive enzymes9~ would give a more complete picture. At the heart of the model is the relationship between dietary amino acid intake, the free amino acid pool and the protein pool linked via protein synthesis, protein degradation and amino acid metabolism (transamination/ deamination followed by oxidation and or synthesis of non-protein molecules). The model allows ratios between its components to be calculated. The nitrogen balance for a feeding trout indicates that 15% of the consumed nitrogen is lost v/a the faeces, 53% lost (the majority excreted as ammonia) and the protein nitrogen retention efficiency (kg/kr, Table 1) is 32%. (Fig. 2a). The anabolic stimulation efficiency (ks/kr) indicates that 74% of the consumed or 87% of the absorbed amino acids are used in protein synthesis. In an earlier report Houlihan and colleaguess5 estimated that for each gram of protein eaten by cod, 1 g of protein was synthesised. Thus the cod values are close to the model presented here for rainbow trout. This high proportion of the absorbed amino acids which are synthesised into proteins may be the first phase of the animal's response to the dietary amino acid influx, followed later by protein breakdown. Not all the synthesised protein is retained as growth, the synthesis retention efficiency (ks~ks, Table 1) is 43% and indicates an amount equivalent to 57% of the

Protein synthesisin fish

199

newly synthesised protein nitrogen is degraded and returns to the free amino acid pool, to be oxidised and excreted, or recycled in the synthesis of protein. It is likely that the processes of protein degradation follow at some time after the synthesis although experimental evidence for the time course of synthesis versus degradation remains to be elucidated. The frequency and size of meals clearly play a pivotal role in determining the daily protein synthesis and degradation rates. It may come as a surprise to realise that the dietary amino acids supplied by a normal sized meal are approximately double the size of the whole animal free pool. The evidence is that tissue free pools remain relatively stable after a meal and therefore the relative importance of protein synthesis in maintaining this homeostasis is evident. What we need to know now is what determines the relative rates of amino acid oxidation and protein synthesis and this is discussed further below. Compared with a growing animal, the rates of synthesis and degradation are substantially lower in a starving trout (Fig. 2b). The majority of amino acids from protein breakdown are used as energy substrates and the ratio between fractional rates of protein degradation and synthesis indicates that only 36% are recycled into protein. Reduced fractional rates of protein synthesis rates during starvation have been found in almost all animals, including fish (e.g. refs. 16, 34, 55) and means that turnover rates are lower in starved than in fed animals. In summary, this kind of model provides a useful framework for describing whole animal nitrogen flux and emphasises the importance of measuring dietary intake. However, it represents a simplification by the assumption that there is one free amino acid pool and one protein pool. Different tissues respond to a meal at different rates; protein synthesis may remain elevated for more than 24 h after a meal in the white muscle 83.

I V I n d i v i d u a l variation in protein turnover

We can move on from this static model of nitrogen flux to test relationships between rates of food consumption and protein turnovers6. The data set we have chosen comes from experiments where the rate of food consumption of individual rainbow trout (measured by radiography) a1,116 have been combined with whole animal protein synthesis measurements. Rates of protein growth increase linearly with increasing protein consumption rates (Fig. 3a) and in general about 70-80% of the observed variation in growth rate between individual fish can be explained by differences in food consumption 15,16,23,s~ There is also a linear relationship between protein consumption and protein synthesis which can be termed the anabolic stimulation of protein synthesis (Fig. 3b) and which is thought to be the result of the combination of dietary amino acid and hormonal stimulation of protein synthesis84,s9'114, mediated through an increase in the number of ribosomes and/or an alteration in ribosomal activitys3. The individual response to a given protein intake, in terms of the magnitude of synthesis is clearly variable (Fig. 3b). Whole-animal rates of protein degradation are independent of ration level (Fig. 3c) in contrast to previous results. Some studies have suggested that protein

200

D.E Houlihan, C.G. Cannerand LD. McCarthy

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Protein synthesis in fish

201

degradation is stimulated by increased consumption 55,89, whilst others have found rates of protein degradation to be independent of ration 16'52'82,95. It has been suggested that degradation rates are genetically determined, whilst synthesis rates are more responsive to nutritional status and environmental influences ~8. Not all the variation in growth is explained by variation in consumption (Fig. 3a) and individual fish can be eating similar amounts of food and yet show markedly different growth rates. Recently we have examined how individual differences in protein turnover can help to explain differences in protein-nitrogen retention efficiency 16,78,82. Seven pairs of rainbow trout consuming similar amounts of food, but growing at different rates, were selected from the group in Fig. 3a. The relationship between consumption and growth for these fish is shown in Fig. 4a. The mean protein-nitrogen retention efficiencies (Table 1) for the two groups of fish were 37.7 ~ 3.4% for the fish growing at a faster rate for a given food intake and 16.7 4- 2.2% for the fish growing at a slower rate for a given rate of food intake. There were no consistent differences in mean rates of protein synthesis but individuals with a higher protein-nitrogen retention efficiency were found to have reduced rates of protein degradation (Fig. 4b). Consequently, the trout with lower retention efiiciencies will have a higher cost of growth per gram of protein retained. This supports the finding that individual rates of protein degradation efficiency are negatively correlated with protein-nitrogen retention efficiency in grass carp, Ctenopharyngodon idella (Val.) 16 and with growth efficiency in chickens 118. It has been suggested by Hawkins 51 that individual differences in protein degradation are an indication of genotype-dependent differences in maintenance requirements and that this is a major influence on individual differences in performance between individuals. Superimposed on the influence of genotype on individual differences in protein turnover and growth performance is the modification of an individual's response as a result of social interaction. The social structure of juvenile salmonid fish is characterized by aggressive behaviour between individuals and the formation of dominance hierarchies 88. In Fig. 5 we attempt to model the relationship between social rank, protein turnover and growth performance of individual fish within a dominance hierarchy. This model is based upon experimental data obtained from groups of laboratory-reared rainbow trout and Atlantic salmon, Salmo salar L., hand-fed a variety of different ration levels. Disproportional food acquisition between individuals within the group is a common feature of social hierarchies with dominant individuals gaining preferential access to food 88, consuming a larger share of the group meal (Fig. 5a) 79 and growing at a faster rate compared to subordinates (Fig. 5b) 1. In Fig. 5a, the distribution of food between the individuals within the group would appear to indicate a linear Fig. 3. The relationship between the fractional rate of protein consumption (kr, % day-1) and: (a) the fractional rate of protein growth (ks, % day-l); (b) the fractional rate of protein synthesis (ks, % day- 1 ); and (c) the fractional rate of protein degradation (kd, % day-Z) for individual rainbow trout (80 g, 10~ s2. The regression equations are: (a) y - 0.366x - 0.347 (Rz ffi 0.736, n ffi 37, p <0.001); and (b) y = 0.445x + 1.686 (Re -- 0.736, n ffi 37, p <0.001).

202

D.E Houlihan, C.G. Carterand LD. McCarthy

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dominance hierarchy which have been reported for small groups of social fish of between 4 and 10 individuals 8s.97. However, the hierarchical structure may be more complex in larger groups of fish and non-linear hierarchies in large social groups of fish have been reported9'~. The low ranking of subordinate fish may impose a long term chronic stress on individuals (Fig. 5c) (reviewed by Jobling) 6a and influence both growth performance (Fig. 5d) and immunocompetence (Fig. 5f). Dominance has been associated with reduced metabolic costs and increased growth efficiency (Fig. 5d) 1,s7. It has been suggested that increased stress, mediated v/a cortisol, inhibits protein synthesis and stimulates protein degradation in fish (reviewed in Van der Boon et al.7). Decreased rates of degradation in dominant fish exhibiting

Protein synthesis in fish

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204

D.E Houlihan, C.G. Carter and LD. McCarthy

higher growth rates will therefore result in an increase in the percentage of the synthesized protein that is retained as growth (Fig 5e). The effect of social rank on the immunoeompetenee of individual fish within a group environment is still unclear. Much of the available data compares the responses of the dominant and subordinate individual within pairs of fish. These results have suggested that exposure to long term stress results in reduced immunocompetence in subordinate fish24,~~ However, in these studies the single subordinate fish is the sole focus of the aggressive behaviour of the dominant and this would serve to maximise the physiological stress imposed on that individual. In a large group the dominant fish may be more stressed by having to maintain their position. The immunological performance of individuals within a group environment is still little studied, especially in relation to individual feeding rates. Recent work has suggested that within a group the immunocompetence of dominant fish is reduced (Fig. 5f) (Thompson et al., in preparation). This may be due to the immunosuppressive effect of increased consumption by dominant fish or it may represent a trade-off whereby growth is maximised in the more active dominant fish at the expense of the immune system. The combined measurement of consumption, protein turnover and immunocompetence in individual fish of known social rank within a group will enable us to further examine the interplay between social rank and growth performance and to investigate whether these metabolic trade-offs do occur.

Thus in summary we find that the measurement of rates of protein synthesis in fish needs to be approached in an holisticfashion, considering the individual's ability to obtain food together with metabolism and growth efficiency. Using this approach, particularly with repeated, long-term measurements using stable isotopes, protein synthesis rates will give us a firm basis for understanding the role of protein turnover and in designing selection programmes. For example, we can already suggest that maximisation of protein synthesis rates is not the mechanism guaranteeing fast growth rate, rather it is reduced turnover which correlates with increased growth rate. We are only at the threshold of investigating the implications of these studies on for example, hormonal control, longevity and disease resistance.

V. Proteinsynthesisand freepool amino acid concentrations Despite the small size of the free amino acid pool in large fish, equivalent to approximately 2% of the protein pool and the large influx of dietary amino acids, approximately double the free pool size, there is comparatively little change in free amino acid concentrations in fish tissues following a mealz~176 This is true for both the total and the essential free amino acid pools (Fig. 6). It is interesting to note that although white muscle and liver have very different fractional rates of protein synthesis56, the free pool concentrations are very similar. Only the plasma free pool shows large changes in amino acid concentration lzs ; in rainbow trout the maximum concentration is twice the prefeeding leveP z~ The stability of the

Protein synthesis in fish

205

Fig. 6. Free amino acid concentrations (t~mol/g tissue wet weight) in the white muscle and liver of rainbow trout (200 g, 12"C) over a 24 h period after feeding2~ The data presented are the total essential amino acid (white bars) and total free amino acid (cross-hatched bars) concentrations in the liver and the total essential amino acid (hatched bars) and total free amino acid (black bars) concentrations in the white muscle respectively.

tissue free pools is strongly indicative of a homeostatic control mechanism. Indeed, Millward and Rivers 9~ proposed that the accumulation of essential amino acids may be harmful and are therefore maintained a low concentrations. It has been suggested that enzymes involved in amino acid metabolism have a relatively high Km (ref. 22) whereas aminoacyl-tRNA synthetases have relatively low Km values a n d t h e r e f o r e p r o t e i n synthesis will be ' p r e f e r r e d '26. H o w e v e r , q u a n t i t a t i v e e v i d e n c e

for this hypothesis is still lacking. In fish the major pathways of amino acid utilization are through protein synthesis or deamination and oxidation to ammonia. The model of protein-nitrogen flux indicated 87% of absorbed amino acids were partitioned into protein synthesis. In catfish, Ictalurus punctatus, approximately 21% of the total nitrogen in an essential amino acid infused was excreted as ammonia over the 24 h following infusion 1~ In unfed catfish the application of cycloheximide, to inhibit translation, led to an immediate increase in ammonia excretion which had doubled after four hours and suggested the importance of synthesis in regulating free amino acid concentrations 1~ It is very important to qualify these conclusions by investigating the flux of an individual amino acid. In rainbow trout 20-40% of the leucine metabolised in a day was oxidised 33,34 and it was demonstrated that the majority of this essential amino acid was utilised in protein synthesis. Oxygen consumption, ammonia excretion and protein synthesis all show postprandial increases 1~ The sequential pattern of all three variables has not been determined simultaneously in any fish but all the evidence suggests that the

206

D.E Houlilm~ C.G. Carter and I.D. McCarthy

postprandial stimulation of protein synthesis is the major contributor to the postprandial increase in oxygen consumption 1~ The few data available suggest that the oxygen consumption and ammonia excretion peaks are not synchronous. In catfish the ammonia peak was at 4 h and the oxygen consumption peak at 8 h after an infused meal 1~ In grass carp (Ctenopharyngodon idella) AQ values (mg ammonia/mg oxygen) peaked 5 h after feeding and indicated a relative increase in ammonia excretion which preceded the peak in oxygen consumption x3. Brown and Cameron ~~ also demonstrated that after cycloheximide administration, ammonia was excreted following a meal without an increase in oxygen consumption. Thus, overlapping peaks with maximum values of ammonia excretion preceding those of oxygen consumption and protein synthesis would be predicted. Reeds and Davis ~~ concluded that in endotherms it is rare to encounter circumstances in vivo in which the amino acid concentrations fall to levels that might inhibit translation. It has been suggested that amino acid imbalances may lead to increased amino acid oxidation and nitrogen excretion in fish3s,66,68 and examples of amino acid imbalances on protein synthesis rates are provided by Fauconneau 31 and Fauconneau et al.3s. Grass carp which were fed only lettuce and which had positive protein growth rates had a lower fractional rate of protein synthesis, lower synthesis retention efficiency, lower nitrogen retention efficiency and consequently a higher loss of nitrogen than grass carp fed a nutritionally balanced diet. Although rates of protein consumption were similar on the two diets it was not known whether these results were due to lettuce being deficient in methionine, the low energy content of lettuce or the low digestibility of lettuce protein 16.17. Such studies emphasise that much more work is needed to establish relationships between diet composition, protein turnover and amino acid metabolism. This question is of importance in the theoretical understanding for the use of lipids in commercial diets as a method of protein sparing6. In conclusion, analyses of amino acid fluxes in fish provide valuable support for separate estimates of rates of protein synthesis. The apparent relative constancy of the tissue free pools points to control mechanisms of protein synthesis and amino acid oxidation which are little understood. Perhaps the most intriguing feature is the similarity of the tissue free pools in different tissues with widely differing fractional rates of protein synthesis. This implies that the fluxes of amino acids, be they diffusional or active, into different tissues must be closely linked with the amino acid sinks of synthesis and oxidation.

VI. Energy cost of protein synthesis An alternative approach to the bioenergetic analysis of inputs and outputs in fish is to quantify the energy cost of the various life processes. There have been a number of attempts at quantifying the energy cost of protein synthesis but even in mammals it is an area of some uncertainty. One approach (termed the 'direct' by Waterlow and Millward nl) is to measure the rates of protein synthesis and multiply these by a protein synthesis cost factor, a theoretical cost of protein synthesis has frequently

Protein synthesis in fish

207

been employed. The minimum theoretical cost assumes that 4 ATP equivalents are required for each peptide bond synthesised which, with a molecular weight of peptides of 110, results in a cost of 36 mmol ATP g-I protein synthesised 1~ An additional ATP has been included for transport processe s giving a value of 8.3 mmol 02 g-~ protein synthesised (assuming 6 mmol ATP synthesised per mmol oxygen) 1~ It is important to note that the theoretical cost of peptide bond synthesis appears to be independent of temperature, body size, etc. Using theoretical values, protein synthesis has been estimated to account for a minimum of between 23 and 42% of the total oxygen consumption in cod, Gadus m o r h u a 59'75 and between 11 and 22% in the more active grass carp 16. In a variety of mammals, protein synthesis was calculated to account for 20-25% of the resting heat production 124. An alternative approach (termed the 'indirect' approach by Waterlow and Millward m ) is to compare protein synthesis rates and oxygen consumption in a variety of conditions and to calculate the oxygen consumption as a ratio of protein synthesis. A close relationship between oxygen consumption, growth and protein synthesis have been found for a variety of animals including fish 48'52'62'121. Comparisons between energy cost of protein synthesis in mammals estimated stoichiometrically and indirectly indicate that the latter give values approximately 5-fold higher than the former m, and generally aerobic costs of protein synthesis appear to vary widely (Table 3). Some of the data on energy costs of protein synthesis have been determined by measuring oxygen consumption and protein synthesis before and after the application of a protein synthesis inhibitor such as cycloheximide and have given values close to theoretical, e.g. juvenile tilapia, Oreochromis mossambicus 57. Cycloheximide-sensitive protein synthesis and oxygen consumption suggest that from 80 to 87% of oxygen consumption is used for protein synthesis in trout hepatocytes and juvenile tilapia. A possible explanation for these disconcertingly large variations in protein synthesis costs and the suggestion that different tissues have different energetic costs for protein synthesis 2,47,48,77 may lie in the observation that energy costs of protein synthesis appear to be related to the rate of protein synthesis, the higher the rate of synthesis the lower the aerobic cost (Table 3). An example of decreasing aerobic costs of protein synthesis with increasing rates of synthesis in isolated fish cells is given in Fig. 7a. These variable costs would arise if there were fixed costs at low rates of protein synthesis together with the peptide bond formation cost. In isolated fish hepatocytes 100 and other fish cells 113 plots of oxygen consumption and protein synthesis do reveal a large intercept (oxygen consumption) at zero protein synthesis. The slope of the line relating oxygen consumption may approach the theoretical peptide bond formation cost. Thus as the rate of protein synthesis increases the fixed costs make a decreasing contribution to the aerobic costs which at high protein synthesis rates will approach theoretical values (Fig. 7a). Variable protein synthesis costs would explain the apparent plateau of oxygen consumption with increasing protein synthesis rates found in a comparison of different fish cell types (Fig. 7b). Recent data from juvenile fish have also demonstrated that there is no further increase in oxygen consumption at growth rates above 8-10% per day 125,~26 and it has been suggested that variable protein synthesis

208

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209

Protein synthesis in fish

TABLE 3 Energetic costs for protein synthesis from different sources Species

ks (% day-1)

Calf-muscle Pig-muscle Trout hepatocytes Whole sheep Blue mussel Juvenile nase Sheep hepatocytes

0.7- 1.5 1 - 6 1 - 8.5 3 - 5 3 - 8 14 -25 30 -50

-

Trout hepatocytes 1 - 9 Trout scale cells 0.5 RTG-2 1.1 Larval herring 3 BF-2 9.1 Trout macrophages 2.9 Chickens 29 Juvenile tilapia 30

Oxygen consumption Method (mmol02 g-1 protein) Theoretical 8 Correlation 450 Correlation 158-575 Correlation 37-138 Correlation 49 Correlation 25 Correlation 25 Correlation 7 111-583 217 133 98 11 46 12 6

Inhibition with cycloheximide Inhibition with cycloheximide Inhibition with cycloheximide Inhibition with cycloheximide Inhibition with cycloheximide Inhibition with cycloheximide Inhibition with cycloheximide Inhibition with eycloheximide

Reference 62 47 2 100 48 52 58 77 100 113 113 a 113 113 4 57

a Houlihan et al., in preparation.

costs could contribute to this apparent cost-free growth 56. What these fixed cost components are remains speculative, but constant activation of t R N A as well as the production of r R N A which remains remarkably constant regardless of the rate of protein synthesis 98 most probably contribute to this fixed energy expenditure. It has been suggested by Cooper and Gibson 25 that accumulation of r R N A in the cytoplasm occurs only at half the rate at which it is synthesised in the nucleus. This would mean that the cost of r R N A synthesis would be underestimated by at least one half. The high proportion of the energy budget for protein synthesis in hepatocytes indicates that there is not much energy left for other purposes. Estimates of the cost of the N a + / K + ATPase pump vary from 26% of the total energy expenditure in rat diaphragm muscle to 40% and in calf muscle46,47 to 6% in perfused rat liver 39 and adipose tissue 21. In isolated trout hepatocytes, it was estimated that the Na+/K + ATPase pump contributes 2.8% of the oxygen consumption 1~176 In summary, the aerobic costs of protein synthesis, even using minimum theoretical values appears to make a large contribution to the oxygen consumption of inactive fish. Allocation of the costs to the individual tissues is complicated, as it will be the product of the mass of protein in each tissues, the tissue-specific rate of protein synthesis and the tissue-specific cost 56. However, in terms of the whole animal, it is interesting to relate the cost of protein synthesis to the cost of growth. Thus, in the example of the rainbow trout given above (Figs. 3 and 4) animals with similar rates of protein synthesis and hence similar protein synthesis costs (unless there are large differences in body composition) will have widely different costs of growth when the retention efficiency of synthesised proteins vary.

D.E Houlihan, C. G. Carter and I.D. McCarthy

210

VII. Species comparisons Comparisons of protein growth/synthesis/degradation between fish species are complicated because a single species-specific value for protein synthesis is not justifiable. Also differences in water temperature influence at least protein growth and synthesis rates. In addition, fish show indeterminate growth which means there is no maximum or 'adult' size as would be found in endotherms. What we can do is to compare the available data from different species in terms of the relationships between protein consumption, growth and synthesis and recognise that conclusions may be confused because of differences in body size, temperature or diet (Table 4). An interesting observation is that from the limited data there seems to be a positive correlation between the rate of protein synthesis at zero growth (maintenance synthesis, k, Cm)) and the maximum synthesis rate (k, Cmax)) calculated at the maximum ration or growth rate (Table 4: n = 9; r = 0.99; p <0.0001). This implies that fish need a high maintenance synthesis rate to allow a high protein synthetic scope. It is also noteworthy that although the anabolic stimulation efficiency and the synthesis retention efficiency vary widely between species, they are negatively correlated (n = 7; r = -0.93; p < 0.001). When consumption stimulates a high TABLE 4 Comparison of indices of protein turnover between different species of fish Species

kr(max) ks(m) ks(max) ks (% per day)

'ks/kr ks~ks 'ks/kr

69.8

7.3

1.4

3.8

2.0

52.0

53.0

22.4

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5.3

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44.7

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59.9 59.9

5.2 6.0

0.2 0.2

1.7 1.6

1.3 32.7 1.3 26.7

76.5 81.3

25.0 21.7

C C SP L Artemia

58.8 56.1 42.1 53.0 -

6.0 4.0 6.0 -

2.0 0.7 1.3 1.3 13.7

4.4 1.3 2.9 1.9 31.7

1.9 73.3 0.8 32.5 1.7 48.3 0.3 17.0 -

43.2 61.5 58.6 15.8 54.0

31.7 20.0 28.3 -

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Diets: C = commercial diet; SP = semi-purified experimental diet; FM = fishmeal based semi-purified diet; GM = Greaves meal based semi-purified diet; L = lettuce. PE/TE = protein energy to total energy of diets; krCmax) -- the maximum measured protein consumption rate; ks(m) = predicted fractional rate of protein synthesis at maintenance; ks(max) = predicted fractional rate of protein synthesis at maximum ration or growth; kg - protein growth rate; ks/kr -anabolic stimulation efficiency; kg/ks = synthesis retention efficiency; ks/kr = protein-nitrogen retention efficiency. Species: Atlantic cod (Gadus morhua); Atlantic salmon (Salmo salar); sea bass (Dicentrarchus/abrax); rainbow trout (Oncorhynchus myk/ss); grass carp (Ctenopharyngodon idella); nase (Chondrostoma nasus). a McCarthy et al., in preparation.

Protein synthesis in fish

211

rate of protein synthesis, less of the protein is retained as growth. Conversely, when there is a low anabolic stimulation a larger proportion of the synthesised protein is retained. This results in broadly similar nitrogen retention efticiencies between species. The data for the rainbow trout suggest the differences between high or low anabolic stimulation efficiency are not species-specific but due to the experimental conditions. The diet fed to cod has a higher protein to energy ratio than salmon diet and this may partly explain the lower anabolic stimulation of cod, proportionally more of the dietary amino acid is oxidised and less partitioned into synthesis. It is noteworthy that omnivorous and herbivorous fish are able to utilise a wider range of diets than carnivores 119. Compared with trout, higher synthesis, growth and nitrogen retention efficiency are expected for herbivorous grass carp feeding on a high carbohydrate diet. These species comparisons point the way for further studies. We need to measure synthesis at defined points in the ration-growth curve constructed under optimum conditions for each species. These points might be at maintenance (zero growth), maximum growth (nitrogen retention) efficiency (optimum growth) and at maximum growth. The problem is that the optimum conditions under which maximum growth can be achieved are not known for the majority of fish species. Indeed, we cannot yet answer the question as to whether maximum nitrogen retention efficiency varies between speciesa.

VIII. L i f e history p r o t e i n t u r n o v e r

Recently more information has become available on the protein synthesis rates of larval and juvenile fish and gives a more complete picture of protein turnover over the life history of fish and the influence of environmental variables. The rate of many physiological variables are proportional to body weight according to the relationship Y = aX b, where a is a constant and b is the weight exponent 54. For feeding fish the decline in weight-specific rates with increasing body size are similar for growth, whole animal fractional synthesis and growth rates and white muscle fractional rates of synthesis and degradation (Table 5). In its usual form the yon Bertalanffy growth function results from the integration: d W / d t = H W d - K W m, where W is body weight and H W d and K W m are synthesis (or anabolism) and the degradation (or catabolism) of body substances respectively; in both components body weight effects are recognised by the use of exponents. The exponents d and rn have been assumed to be 2/3 and 1 respectively94,1~ Reiss 1~ equates anabolism with assimilated energy and catabolism with average metabolic rate. As we have argued above, metabolic rate cannot be taken as an index of catabolism. The exponents in Table 5 for protein synthesis and degradation are more appropriate and suggest that both anabolism and catabolism scale in proportion to between, approximately, 0.7 and 0.6 of body weight. Very high fractional rates of protein synthesis have been reported for some juvenile fish with efliciencies of retention of synthesised proteins of between 6

D.E Houlihan, C.G. Carterand LD. McCarthy

212 TABLE 5

Effects of body weight W (g) on the ingestion rate, specific growth rate, endogenous nitrogen excretion, protein synthesis rates and protein degradation rate using the formula: log Y - log a + b log X for whole animals and white muscle. All parameters are in weight-specific units Species Various species t~ Various salmonids ~ Tilapia 57 Grass carp 14, a Rainbow trout 61 Rainbow trout 61 Trout white muscle 54 Trout white muscle 54

Parameter

Slope

(r)

(b)

Ingestion rate Growth rate Protein synthesis Endogenous nitrogen excretion Protein synthesis Protein growth Protein synthesis Protein degradation

-0.25 -0.3 to -0.45 -0.50 -0.37 -0.26 -0.25 -0.49 -0.42

a Calculated for unfed grass carp.

and 9% (refs. 35, 36). On the other hand using the techniques of either bathing the juvenile fish in tritiated phenylalanine or microinjection of the radiolabelled amino acids, much lower fractional rates of protein synthesis have been found and efficiencies of retention are around 40-50% (refs. 57, 58, 86). We need more work on fish larvae to resolve these differences. Environmental fluctuations effect rates of protein turnover in fish and the effects of temperature have recently been discussed 41,56. Koehn and Bayne~~ argued that the cost of increased protein turnover due to environmental stress would be reflected in less energy being available for growth. The effect of environmental stress on protein turnover in fish has been little studied. However, in a recent study with dab, Limanda limanda (L.), exposed to sewage sludge for three months, it was found that the sludge treated animals exhibited lower protein growth rates and increased protein turnover compared to control fish~. This is the first study in fish which demonstrates that an environmental stress may have a physiological cost in the form of increased protein turnover.

IX. Protein synthesis in tissues and cells This review has concentrated on protein synthesis rates in whole bodies of fish because we believe that it unlikely that we will make much sense out of protein synthesis rates of individual tissues or proteins by just pulling fish out of a tank with no regard to the details of their previous nutritional history. We also suspect that the individual tissues and proteins will follow, more or less, the pattern evident for the whole animal. There is good evidence for this in the white muscle (see below). It is also important to recognise that relatively few proteins make up a large part of the individual's total protein pool. Thus from estimates of the amounts of contractile and ribosomal proteins in the body of a rainbow trout it is found that they make up a third of the total bodyprotein (Table 6).

Protein synthesis in fish

213

TABLE 6 Contribution of specific proteins to total whole animal protein in an 80-g rainbow trout (Oncorhynchus mykiss) with a protein content of 9.2 g

Total muscle proteins Contractile Ribosomal Plasma Collagen Elastin

g 5.31 2.53 0.94 0.001 0.36 0.10

% of total protein 57.72 27.47 10.22 0.01 3.91 1.09

Muscle protein calculated from Houlihan et al.54. MyofibriUarprotein/muscleprotein ffi 0.476 (reference 28). Ribosomal protein/muscle protein ffi 0.177 (reference 27). Plasma proteins ffi 25.4 mg 100 m1-1 (reference 3). Blood volume -- 5.4 ml 100 g-I wet weight111.Connective tissue protein/total protein = 0.05; connective tissue = 78.5% collagen, 21.5% elastin74.

Recent information of protein synthesis rates in fish tissues has been reviewed by Houlihan 53 and Houlihan and coworkers 56. Fauconneau and colleagues 37 found a highly significant positive relationship between whole animal specific growth rates and white muscle synthesis for rainbow trout collected from a number of studies. With the addition of more data the white muscle fractional synthesis rates ( W M . ks, % day -l) can be predicted for rainbow trout from specific growth rate (SGR, % day-t), temperature (T, *C) and wet weight (W, g): W M . ks = 1.53 + 0.48 SGR - 0.10 T - 1.86 10 -4 W (n = 14; R2= 0.865; p <0.0001). With an increasing data base it should be possible to construct a similar relationship to describe whole animal rates of synthesis from white muscle protein synthesis rates.

X. Conclusions This review has emphasised 'supply side' economics in determining protein synthesis in fish. We believe that there is a good reason for doing this because if food consumption is not carefully monitored then the interpretation of any changes in protein turnover under different conditions may be severely compromised. In addition we have also emphasised the importance of the individual differences in rates of protein turnover and attempted to show that there are already indications of different strategies within species and possibly between species. It is justifiable to speculate that in rainbow trout some individuals are following a 'growth maximisation' strategy. This is achieved not by maximising protein synthesis rates but by reducing protein degradation rates. Other individuals may be following a 'turnover maximisation' strategy, not necessarily by reducing protein synthesis but by increasing rates of protein degradation. We are still not clear if there are 'energy minimisation' strategies, i.e. low maintenance costs and possibly low protein synthesis costs in individuals. The evidence points to the need for a high maintenance if growth rates are to be high and therefore high maintenance may be a feature of both growth and turnover

D.E Houlihan, CO. Carterand I.D. McCarthy

214

maximisation. There is evidence for some trade-offs in these strategies in that there may be reduced immunoeompetence in the 'growth maximisation' strategy. The evidence is that protein synthesis rates are highest after the arrival of dietary amino acids and that it is the timing and extent of the subsequent release of amino acids from protein degradation before the next meal which is a decisive factor controlling growth rates. It is still not dear whether the source of increased ammonia production after a meal is the amino acids from the meal or pre-existing proteins and amino acids. The likelihood of variable protein synthesis costs introduces some uncertainty in calculations of the contribution that protein synthesis makes to oxygen eonsurnption in fish. However, it is likely that the costs of protein synthesis are an important factor controlling the 'demand side' of energeties. For two fish synthesising proteins at similar rates, costs of protein synthesis will be the same but the fish which retains the highest proportion of the synthesised proteins will have the lowest costs of growth. In the context of estimating protein growth costs, two strands from this review are worth picking out. Flatfish living in a stressful environment synthesise proteins at a similar rates as control animals but because of increased protein breakdown exhibit reduced protein growth rates; their impaired immunocompetenee may or may not be related to this increased turnover (section VII) 6~176 In contrast, dominant salmonids in a group may exhibit reduced protein turnover, either as a consequence or a result of their dominant social position (section IV). These dominant fish may have reduced immunocompetenee. The costs of growth are higher in the environmentally stressed animals and lower in the dominant fish but in the face of a disease challenge increased protein turnover may prove vitally important despite the energy burden it imposes. If this review does nothing more than direct the focus of future research into integrating energy costs of protein turnover with the functional significance of the proteins that are being turned over it will have achieved the aims of the authors. Acknowledgements. We are grateful for financial support from the Agriculture and Food Research Council, the British Council, the Ministry of Agriculture Fisheries and Food, the Natural Environmental Research Council and the Science and Engineering Research Council.

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Hochachka and Mommsen (eds.), Biochemistryand molecularbiologyofftshes, vol. 4 9 1995 Elsevier Science B.V. All rights reserved. CHAPTER 9

Nutrient fluxes and regulation in fish intestine NATHAN L. COLLIE AND RONALDO P. FERRARIS *

Department of Biological Sciences and Institute for Biotechnology, Texas Tech University, Lubbock, TX 79409-3131, U.S.A. and *Department of Physiology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103-2714, U.S.A.

I. Introduction II. A primer on nutrient transport pathway characteristics III. Mechanisms of sugar absorption 1. Brush-border transport 2. Molecular biology of the Na+/D-glueose transporter 3. Site-density of Na+/D-glucose transporters in fish and other vertebrates 4. Basolateral transport 4.1. Basolateral transporter isoforms IV. Mechanisms of amino acid (AA) transport 1. Amino acid entry across the brush-border membrane 1.1. Na+-dependent uptake pathways 1.2. Na+-independent uptake pathways 1.3. Na + : AA coupling ratios in AA uptake pathways 2. Transport across the enterocyte basolateral membrane V. Regulation of nutrient transport 1. Adaptations to diet 1.1. Genetic adaptations 1.2. Phenotypic adaptations 2. Hormonal regulation 2.1. Steroids 2.1.1. Anabolic steroids 2.1.2. Interrenal steroids 2.2. Growth hormone (GI-I) 2.3. Thyroid hormones VI. Summary and perspectives Acknowledgements VII. References

I. Introduction Studies on intestinal nutrient absorption have increasingly turned to the question of how transport processes are regulated (for reviews, see refs. 8, 17-20, 26, 53 and 54). This shift in emphasis was possible, in part, because extensive work over the past three decades helped characterize nutrient transport mechanisms 11,16,45,79. Much of the early work on fish intestine that led to a basic outline of how nutrients move from the gut lumen to blood was summarized almost 10 years ago 24. This

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review focuses on research since then on mechanisms of glucose and amino acid (AA) transport and its regulation in fish intestine. We begin by examining the cellular mechanisms responsible for nutrient absorption, as a logical background for discussing adaptive changes in transport mechanisms. Recent advances in the molecular biology of glucose transport and its relevance to fish intestine are explored. However, work in this area for AA transport has only recently emerged 5~176 and no information is yet available in fish species. Despite these advances in mammalian species, we ask whether our knowledge of transport mechanisms is sufficient in fish to pinpoint which mechanisms underlie adaptive changes in nutrient absorption. In the final section we discuss the adaptive regulation of nutrient absorption, emphasizing two major aspects: adaptations to diet and hormonal regulation. We conclude by pointing out problems that remain unsolved and that future studies might profitably address. Related topics in intestinal nutrient absorption not covered explicitly here have been reviewed recently, including organismal aspects of digestion in fish 76, comparisons of techniques and nutrient transport models 67, intestinal development 8.2~ ecology and nutrient absorption 19,54, intestinal metabolism 11.32,67,and signal transduction in enterocytes 1~

II. A primer on nutrient transport pathway characteristics For readers whose primary interests lie outside epithelial transport, we first present a general, concise introduction to nutrient transport pathways, before turning to specific topics on glucose and AA absorptive mechanisms. As described below, these absorptive mechanisms can be distinguished by a minimum of four transport characteristics. First, nutrients may enter or exit absorptive cells by diffusion (non-mediated) or by a membrane transporter, which mediates solute movement. A plot of transport rate versus nutrient concentration is linear for diffusion and curvilinear (saturable) for mediated routes. Since some finite diffusion occurs for all solutes, the latter curvilinear relationship will always have a linear component superimposed on the nonlinear, mediated component. Second, carrier-mediated transport can be further resolved into facilitated (passive) or active mechanisms requiring energy input to drive nutrient entry against an electrochemical gradient. For sugars and AAs entering absorptive cells, the energy source for active uptake is provided secondarily by the Na + gradient, and maintained by Na + pumps (Na+/K+-ATPases) spanning the basolateral membrane. Hence, facilitated and active transport mechanisms are typically differentiated by the absence and presence of Na + dependency, respectively. However, other ionic requirements for active uptake have been reported, such as C1- in nutrient uptake by the marine teleost, Boops salpa, though the anion's mechanistic role is unclear 4's. Third, the specificity of carrier-mediated transport for related but discrete chemical classes of nutrients (e.g. acidic versus basic AAs) refines transport into additional uptake pathways. Measuring the uptake of a radiolabeled test solute

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in the presence of one or more potential competitors is an effective method of identifying which chemically related solutes share a given pathway. Fourth, particularly with the advent of membrane vesicle preparations, it has been possible to identify the coupling ratio (stoichiometry) between the number of Na + ions that enter the enterocyte per nutrient molecule transported. This is important for discriminating between different transporters as well as for estimating the gradients of nutrients that can be established across membrane barriers. Below we use these various characteristics to describe the absorptive pathways of nutrients entering the enterocyte from the gut lumen and exiting the cell across the basolateral membrane.

III. Mechanisms o f sugar absorption Fish possess the hydrolytic enzymes (amylase, maltase, sucrase and lactase) neeessary to digest dietary carbohydrates into their component monosaccharides Dglucose, D-galactose and D-fructose 3,63. These monosaccharides are then transported transcellularly by brush-border carriers from the intestinal lumen into the cell, and then by basolateral carriers from the cytoplasm into the blood. There is no significant absorption of monosaccharides through the paracellular pathway 19,3~

1. Brush-border transport In fish small intestine, early studies have shown that o-glucose and o-galactose absorption is mutually competitive, Na+-dependent, energy-dependent, concentrative and electrogenic 24. More recent studies 5,68,81 using membrane vesicles from fish intestine not only have confirmed results from those early studies, but also have shown that the D-glucose transport system in fish intestine is phlorizin-inhibitable and exhibits steric requirements similar to those exhibited by mammalian small intestine. The o-glucose transport system can be inhibited by D-galactose and o-glucose analogs like t~- and ~l-methyl-glucoside, but not by 2-deoxy-D-glucose, a o-glucose analog transported only by the basolateral, Na+-independent glucose transport system, nor by inositol, a cyclohexanol sugar which is also a vitamin. Thus, the functional characteristics of the D-glucose transport system in fishes closely parallels those found in mammals. The molecular basis for these properties is the Na+/o-glucose transporter, whose ion dependency, substrate specificity and phlorizin inhibition characterize those of the brush-border transport of D-glucose in fish intestines.

2. Molecular biology of the Na +~o-glucose transporter An intestinal Na+ /D -glucose transporter has been recently cloned from rabbit ileum 38. The properties of the protein deduced from the eDNA are identical to those of the native brush-border membrane protein identified in biochemical experiments 44,47. Coady and coworkers 12 then used the eDNA encoding the Na+/

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D-glucose transporter to examine the distribution of homologous mRNA in various classes of animals. The mRNA extracted from pyloric ceca of rainbow trout hybridized with radiolabeled cDNA from the cloned rabbit Na+/D-glucose transporter. Trout mRNA showed a distinct but relatively faint single band at 2.9 kb; the faint band may reflect a relatively low abundance of mRNA coding for the transporter. This is not surprising because v-glucose absorption rates 7.23 and site density of Na +/D-glucose transporters (see below) are much lower in fish compared to mammalian intestine. Thus, the mRNA encoding the Na+/D-glucose transporter is widely distributed among vertebrates, has been conserved in evolution, and may even be structurally related to the Na +/proline transporter from Escherichia r 39. Based on its predicted amino acid sequence, the Na+/D-glucose transporter is thought to traverse the plasma membrane 11 times 3s. The amino terminu~ is located intracellularly, the carboxyl terminus, extracellularly. The protein consists of 662 amino acid residues, and has a molecular weight of about 75 kDa. A long, hydrophilic, intracellular region linking membrane spans 10 and 11, and another long, highly polar, extracellular region linking spans 7 and 8 are believed involved in D-glucose binding. The activity of the Na+/D-glucose transporter in situ in the brushborder membrane requires the simultaneous presence of four intact, independent, identical subunits arranged as a homotetramer 7s. The gene encoding the human intestinal Na+ /D -glucose transporter is found on chromosome 22 (ref. 37). 3. Site-density o f Na +~D-glucose transporters in fish and other vertebrates

Because the D-glucose carrier of fish intestine is phlorizin-sensitive, we have attempted to measure the site density of these transporters by D-glucose-inhibitable, specific phlorizin binding (Ferraris and Vinnakota, unpublished observations). Ligand-binding analysis of specific phlorizin binding by isolated evened sleeves of catfish intestine reveals that the apparent atfmity (apparent because of unstirred layer effects) of specific phlorizin binding to the transporter is similar to that found in desert iguanas and laboratory mice (Table 1). Site density of the fish transporter is 3-6 times less than in mammals, but 3 times greater than in the iguana. The Ql0 TABLE 1 Site density of Na+/D-glucose cotransporters in the small intestine of fish, reptiles and mammals

Site density (pmol/mg) Total site number (pmol) Total glucose uptake (mmol/min) Turnover number/site (min-i) Apparent Kd (m)

Catfish 0.190 131 0.24c 1800c 0.33

Iguanaa 0.056 62 2 32,300 0.19

Mouseb "1.08 2100 17 8100 0.51

Woodrat* 0.52 1450 10 6900 3.03

"From Ferraris et al.29 b Ferraris et al.28 c 24oc; iguana, mouse and woodrat glucose uptake were determined at 37~ Turnover number was calculated by dividingtotal glucoseuptake by total site number.

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values for carrier-mediated glucose uptake is about 2.3, and is similar in the three species (turtle, iguana and woodrat) 56 studied so far. If this Q10 value were applied to the catfish results in Table 1, then catfish glucose uptake would be 0.72/zmol/min, and turnover number would be 5400 min -1. Thus, low glucose absorption rates in fish intestine may be due to a combination of lower site density of Na+ /D -glucose transporters as well as lesser amounts of absorptive tissue, even when body size differences are taken into account. There are a number of questions about the Na+/o-glucose transporter that remain unresolved. First, the amino acid sequence, molecular weight, and glycosylation sites of the transporter remain unknown for any fish species. Second, the sequence homology among classes of fish (cyclostomes, elasmobranchs and bony fishes) and among vertebrates may yield interesting information about the evolution of nutrient transport mechanisms. Third, the relative contribution of biochemical factors (i.e. site density of transporters, turnover number) as well as anatomical factors (mucosal folds, villi, microvilli) to species differences in glucose absorption should be investigated to explain the observed variation among vertebrates.

4. Basolateral transport The basolateral D-glucose transport system in fish is also functionally similar to that described for mammals. Transport of D-glucose and o-galactose in tilapia intestine (Oreochromis mossambicus) occurs by stereospecific, facilitated diffusion 69, and transport rate is quantitatively less than that in mammals and birds. It is not known whether differences in absorption rate is due to differences in site number or in turnover number per site. Basolateral glucose transport is also osmotically reactive, Na+-independent, and can be inhibited by phloretin and cytochalasin B, but not by phlorizin 69. Preferred substrates are D-galactose and 2-deoxy-o-glucose. Thus, these characteristics typify both the fish and mammalian glucose transporter. Unlike the Na+/D-glucose transporter found only in specialized epithelial cells (usually the small intestine and kidney of vertebrates, the nutrient absorbing gills6s and integument 92 of certain invertebrates), facilitative glucose transporters are present on the surface of almost all cells in organisms as diverse as cyanobacteria, algae, yeasts, protozoa, mouse, rat and man 4~ Its main function is to accelerate the translocation of glucose across the lipid bilayer along its concentration gradient. Facilitative D-glucose transporters are found in almost all cells because of the central importance of D-glucose delivery to cell metabolism. In certain specialized epithelial tissues like the small intestine, transporters can also function to transport D-glucose from the cell to extraceUular fluids.

4.1. Basolateral transporter isoforms There are five isoforms of the facilitated glucose transporter which differ in their tissue distribution, hormonal control, kinetic properties and substrate regulation 2. Two isoforms which predominate in the small intestine are thought to contain 501 or 524 amino acid residues. The facilitated glucose transporter traverses the plasma membrane 12 times, and both the amino and carboxyl termini are intracellular 6~

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The conserved polar amino acids found in membrane span number 7 are thought to be involved in glucose transloeation. For fish basolateral glucose transporters, the number of isoforms and their DNA and protein sequence have yet to be determined. Transporter site density, measured by eytoehalasin B binding, and turnover number of each site are important indices that will help explain species differences in basolateral glucose transport rates in fish and in other vertebrates.

IE'. Mechanisms of amino acid (AA) transport Following a meal intraluminal protein digestion releases small peptides and free AAs, which are then absorbed by several active and passive mechanisms. Multiple pathways handle the absorption of the more than 20 different AAs found in the fish gut lumen, taken up first across the brush-border membrane and then exiting across the basolateral membrane in series.

1. Amino acid entry across the brush-bordermembrane Three general mechanisms of AA uptake exist in most cells, including fish enteroeytes: (1) passive diffusion; (2) passive, Na+-independent transporters; and (3) active, Na+-dependent transporters. The latter two mechanisms involve recognition of specific transported solutes by membrane proteins and, hence, can be further subdivided into uptake pathways based on the classes of AA they transport. In mammals where the pathways have been systematically studied (e.g. mouse, rabbit, and rat; compared in ref. 55), at least four separate Na+-dependent transporters handle neutral, basic (cationic), acidic (anionic), and imino acids, plus 2 additional Na+-independent carriers (one for neutral and basic and one for acidic AAs). In contrast, the data in fish intestine are less dear, because the uptake pathways studied are divided among diverse species, fed different diets, in various developmental states, under assorted environmental conditions.

1.1. Na +-dependent uptakepathways Studies on AA uptake specificity reveal several possible pathways in different fish species (Table 2). In killifish (Fundulus heteroclitus) anterior intestine, Miller and Kinter 59 described one diffusive and two Na+-dependent components of eyeloleueine (a nonmetabolizable leueine analog) transport. Of the latter two, the primary pathway (60% of the total uptake) was common to all neutral AAs tested, whereas the second (20%) recognized both neutral o- and L-aaminocarboxylic acids, but not fl-alanine or taurine. Lack of stereospeeifieity is not uncommon among AA transporters. Christensen ~ points out that the 'weak' specificity may simply represent the best compromise for a limited number of transporters that must handle all AAs, 'our largest group of mutually analogous nutrients'. In contrast, rainbow trout (Oncorhynchus mykiss) intestine exhibited stereospeeific leueine uptake 49. Only L-form neutral AAs inhibited [14C]-leueine

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Brush-border membrane amino acid uptake pathways in fish intestine Species

Pathway

Accepted substrates

Excluded substrates

Comments

Leu, Ala, Pro, AIB, taurine

-

Only neutral AAs tested; D- and L-isomers accepted

Na +.dependent Killifish 59

Neutral AAs

#-Ala,

Killifish 59

Neutral

Leu, Ala, Pro a-amino-carboxylic acids

fl-Ala, taurine

Rainbow trout 49

Neutral

Leu, Met, Val

Acidic and basic AAs, D-Leu

Possibly 2 neutral pathways

European eel sl,89 Neutral

Ala, Gly

MeAIB

European eel s7,89 Imino

Pro, MeAIB

Basic, acidic

Ala acts as non-competitive inhibitor

European eel aa

Basic

Lys, Arg

Gly, BCH, MeAIB, Ala, ~l-Ala

Accepts Leu, Met, Cys, Pro

Tilapia 7~

Phenylalanine Phe, Ala, Met

MeAIB

European eel a~

Basic/Neutral

Lys, Arg, Gly, Cys, Ser, Ala, Leu, Met, Phe

pro, MeAIB, BCH, fl-Ala

Tilapia70

Dipeptide

Glycyl-L-Phenylala- Phe, Gly, Ala, nine Met, Pro

Na +-independent Similar to mammalian "L-system" a

All substrate AAs are listed using standard 3-letter abbreviations and refer to the L-isomer unless otherwise noted, fl-Ala = ~-Alanine; AIB = a-aminoisobutyric acid; MeAIB = a-(methylamino)isobutyric acid; BCH = 2-amino-2-norbornane carboxylic acid. a The pathway in eels differs from the epithelial L-system (as defined in Reference 79) in excluding the model substrate, BCH. Species: European eel (AnguiUa anguUla); rainbow trout (Oncorhynchus myk/ss); tilapia (Oreochromis mossambicus); killifish (Fundulus heteroclitus).

transport. Hence, stereospecificity of analogous transporters may vary in different species. More recent competition studies have begun to refine multiple uptake pathways in additional species. As shown in Table 2, the most extensive work has been on European eel (Anguilla anguilla) 81,87-89 using brush-border membrane vesicles (BBMV). Na+-dependent uptake comprised 3 pathways in eel intestine. The first transported neutral AAs and excluded the archetypical imino acid, a-(methylamino)isobutyric acid (MeAIB) 89. A second transported imino acids (proline and MeAIB) but excluded basic and acidic AAs 87. Unlike the intestinal imino transporter in rabbit and guinea pig 6a,79, the eel pathway also accepted alanine. However, when proline and alanine interactions were re-examined in a second paper, Vilella et al. 89 found that alanine was a noncompetitive inhibitor of proline uptake. This may indicate an allosteric site on the Na+/proline transporter that binds alanine and modulates pro-

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line uptake. The third pathway was largely specific for basic AAs ~. However, since lysine uptake was inhibited strongly by long-chain and sulfur-containing AAs (e.g. leucine, methionine, and eysteine), one or more additional neutral AA transporters may exist that also accept lysine. Phenylalanine uptake has been characterized recently in tilapia (O. mossambicus) intestine 7~ The Na+-dependent pathway showed a preference for methionine and alanine, a moderate acceptance for proline and glycine, and a complete exclusion of MeAIB. This specificity pattern closely resembles the PHE carrier described for the rabbit ileum brush border 79.

1.2. Na +-independent uptake pathways Vilella and colleagues8s described a facilitated transport component for lysine uptake in eel BBMV. Basic AAs were the strongest competitors (75% inhibition), followed by long-chain and aromatic neutral AAs (60%), and then by short-chain neutral AAs (50%). Imino acids exhibited essentially no inhibition. This pattern was distinct from the Na+-dependent pathway, which showed only weak inhibition by aromatics, no inhibition for short-chain neutrals, but moderate inhibition by imino acids. An entirely different type of facilitated transporter, one for peptides in tilapia BBMV, has been reported by Reshkin and Ahearn 7~ Intestinal peptide uptake across the brush border has been known for some time in mammals 1,27, but was previously unstudied in fish intestine. Uptake of the dipeptide glycyl-L-phenylalanine proved to be Na+-independent, unlike the Na+-energized phenylalanine transport measured in the same tissue. None of the AAs accepted by the phenylalanine transporter caused inhibition of dipeptide uptake. Additional evidence that the peptide was carried by a transporter distinct from the phenylalanine uptake pathway was the markedly different kinetic constants exhibited for the two solutes. The Km and Vmax for glycyl-L-phenylalanine uptake were, respectively, 10- and 5-fold higher than the corresponding constants for phenylalanine transport. Despite the lower affinity of the dipeptide transporter, the authors suggest that dipeptide uptake may make a significant contribution to AA delivery to the blood particularly when luminal peptide levels are high after a meal. Unfortunately, little is known about peptide concentrations in the gut lumen during the feeding cycle. 1.3. Na + :AA coupling ratios in AA uptake pathways Few studies have examined the stoichiometry of Na+-coupled nutrient cotransport in fish intestine. When AA uptake is measured as a function of external Na + concentration, Scatchard or Hill plots of the data permit estimates of the number of Na+binding sites per AA translocated 86. Using this approach in eel BBMV, Vilella et ai.88 found a coupling ratio of two or more Na + ions per AA. In the same species, Na+-dependent proline cotransport exhibited a 1-1 stoichiometry for Na+/proline uptake, further supporting separate uptake pathways for lysine and proline ss. By comparison proline uptake in rabbit jejunum BBMV occurs by an imino carrier that binds two Na + for each proline translocated 8~ Taken together with the above-described carrier specificities, the data suggest that fish and mammals may

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possess functionally different transporters even for closely related AA uptake pathways.

2. Transport across the enterocyte basolateral membrane Once AAs cross the brush-border membrane, they are metabolized, used in protein synthesis, or diffuse unchanged to the basolateral membrane. It is clear that this membrane must have transport properties distinct from the brush border to support the net absorption of nutrients from lumen to blood. Only recently have cell membrane fractionation techniques been used to isolate basolateral membrane vesicles (BLMV) in fish intestine. Reshkin and coworkers 72 prepared BLMV from European eel intestine and identified three carrier-mediated AA transport pathways for six L-AAs surveyed: (1) Na+-dependent carriers for proline and glutamate; (2) Na+-independent transporters for alanine, lysine, and phenylalanine; and (3) a glycine transporter that required both an inwardly directed (blood-to-cell) Na + gradient and an outwardly directed K + gradient. All three basolateral pathways could serve as influx routes supplying the enterocyte with AAs for metabolism and protein synthesis, such as in the post-absorptive state or during fasting. The Na+-independent pathway presumably serves a dual role by also facilitating the exit of absorbed AAs into the blood. Since the specificity of these pathways was not tested, each component might be resolved further into multiple pathways serving specific AA classes. In summary, intestinal AA transporters are functionally different from those of non-polarized animal cells; and, intestinal transporters from divergent vertebrate groups, such as fish and mammals, are also dissimilar in their specificities for some related classes of AAs. This dissimilarity points out the need to characterize AA transport pathways more extensively in fish species. Currently, we have a detailed, though incomplete, description of AA entry and exit pathways for one fish species, the carnivorous eel. Information about nutrient transporter characteristics in other species would facilitate our understanding of the regulation of nutrient absorption and metabolism. We now turn to a consideration of known patterns and possible signals for transport regulation.

14. Regulation o f nutrient transport The adaptive regulation of nutrient uptake in vertebrates has been the subject of several recent reviews8,14,18-2~ so we summarize here the patterns for fish before focusing on hormones as regulatory signals.

1. Adaptations to diet Adaptations to diet can be categorized broadly as genetic (fixed, hard-wired) or phenotypic (reversible), and the mechanisms operating within these categories, as either specific or nonspecific.

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1.1. Genetic adaptations Nonspecific. Nonspecific mechanisms are those which, if altered, will affect the absorption of all nutrients. The tendency for herbivores to have long, thin intestines, and for carnivores to have thick mucosa or pyloric ceca are examples of genetically fixed adaptations of nonspecific mechanisms in fishes. Herbivorous fish like the common carp (Cyprinus carpio), grass carp (Ctenopharyngodon idella), milkfish (Chanos chanos) and tilapia (O. mossambicus) have intestinal length/fork length ratios ranging from 1.9 to 5.8; carnivorous fish like trout (Oncorhynchus mykiss) and striped bass (Morone saxatilis) have lesser ratios of 0.46-0.49; and omnivorous fish like sturgeon (Acipenser sp.) and catfish (Ictahm~ sp.) have typically intermediate ratios of 0.87-1.60 (refs. 7, 25). Another nonspecific mechanism, the passive permeability of the intestine to various nutrients does not seem to vary significantly among fish species and diet sl. Specific adaptations. Because sugars and AAs are transported by discrete membrane proteins, their uptake rates can be modulated specifically and independently. Among species with diverse feeding habits, there are genetically fixed differences in the expression of specific nutrient transporters, and in the ability of those species to regulate that expression. When each group is fed its natural diet, herbivorous fishes have faster rates of glucose transport per unit weight of intestine than carnivorous ones 23,51. Buddington and coworkers 7 affirmed the existence of these genetically fixed adaptations of specific mechanisms to diet by feeding eight species of fish the same diet (thereby eliminating possible phenotypic adaptations). They determined intestinal transport rates of D-glucose and L-proline and found that the intestinal uptake capacity for D-glucose was much higher in herbivores, intermediate for omnivores, and lowest for carnivores. Differences in proline uptake were less pronounced, perhaps because even species on diverse diets have roughly similar protein requirements 7. However, the latter conclusion does not suggest that AA uptake is not genetically regulated, for two reasons. First, smaller fish had higher proline uptake rates than did larger fish of the same species fed the same diet. Presumably, fish may be genetically hard-wired to manage the variable protein requirements of different developmental states (e.g. rapid growth or transformations in juveniles) 13. Second, the pattern for one AA (proline) may not be representative of changes occurring in other AA uptake pathways. In contrast to omnivores, carnivorous rainbow trout have a limited ability to increase glucose uptake in response to changes in dietary carbohydrate 9,43. Apparently, differences in regulatory ability among fish species on diverse natural diets may be fixed genetically. Nevertheless, a recent study using hormones to stimulate trout growth indicates that endocrine signals may override the genetic limits on glucose uptake even in carnivorous fish is (discussed below under Hormonal regulation).

1.2. Phenotypic adaptations Phenotypic, reversible adaptations to diet primarily involve specific mechanisms. Omnivorous fish like carp 6,7 and tilapia ss can adapt to changes in dietary sugar by alterations in Vmax of glucose uptake. Glucose uptake rate per mg of gut tissue were 1.73-fold higher in carp proximal intestine fed a 24% glucose diet compared

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with those on the glucose-free diet. Nonspecific increases in gut mass and length contributed a smaller fraction to the overall higher glucose uptake capacities in carp fed the high-glucose diet. Similarly, tilapia fed a 60% carbohydrate diet showed a two-fold increase in the Vmax for BBMV glucose uptake over values in fish fed a 17% carbohydrate diet s5. Unlike the experiments with carp, there was no effect of dietary carbohydrate on gut mass. Thus, reversible changes in glucose uptake may occur mainly at the level of transporter density in the brush border, since alterations in Vmax correlate well with glucose transporter site density (see Mechanisms of sugar absorption, section III). Phenotypic adaptations of AA uptake to diet are well-known from studies in mammals, but the patterns are more complex, befitting the greater diversity of AA transporters versus that for sugar uptake. In essence the gut has evolved an absorptive strategy that is a compromise between obtaining available AAs as a source of both calories and essential nitrogen, and avoiding the toxicity of essential AA at high dietary levels 18,26. For the nonessential and nontoxic AA proline, one would expect the induction of its transporter with increased dietary proline levels, as long as the benefits (either calories or essential nitrogen) exceeded the biosynthetic costs of maintaining the transport (e.g. transporter synthesis, cation driving forces). Indeed, this pattern proved to be the case for mouse intestine 57. Other AAs showed a complex relationship between dietary protein levels and uptake rate. Is AA uptake inducible by diet in fish, with a similarly complex pattern? With minor exceptions, the answer is unknown. Few studies have directly examined nutrient transport adaptations by varying diet in the same species. In the omnivore tilapia (O. mossambicus), proline uptake by BBMV was unaffected by feeding diets differing in protein (4% fish meal vs 65%) 85. The only report affirming proline transport induction by diet is paradoxical. When adult rainbow trout were fed diets differing not in protein but in carbohydrate (0 and 24%), only proline uptake capacity was stimulated 6. Glucose uptake was unchanged. Buddington 6 suggested that trout, perhaps unable to distinguish between energy supplied by AA or glucose, may respond with enhanced AA uptake to any dietary increase in digestible energy. This explanation and additional questions regarding dietary induction of other AA transport pathways await further testing. 2. Hormonal regulation

One of the most intriguing questions in regulatory biology concerns the signals that switch on or off adaptational responses. An obvious, logical choice for signals regulating nutrient transport would be luminal or blood concentrations of nutrients themselves. However, this has not been explored in fish directly, though the general topic has been reviewed for mammalian intestine (see refs. 18, 26 and 53). We focus here instead on endocrine signals, a second plausible choice as mediators of transport regulation. Hormones integrate diverse physiological processes that control growth, development, reproduction, and environmental adaptation. We limit our discussion to three types of hormones for which data support a regulatory role in nutrient transport by the gut: steroids, growth hormone (GH), and thyroid hormones.

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2.1. Steroids

The sex steroids 17a-methyltestosterone (MT) and 17/3-estradiol (E2) as well as the interrenal steroid cortisol have been implicated in the control of nutrient absorption. 2.1.1. Anabolic steroids. MT, a potent growth-promoter in fish, improves food conversion efficiency, protein digestibility, and protein assimilation 21,46. In a series of publications on rainbow trout intestine, Habibi and coworkers found that MT and, to a lesser extent, E2 stimulated leucine transport in vitro 34,3s and in vivo 33. MT and E2 were both effective stimulators if given by injection over a 10-day period. However, MT alone enhanced transport when added directly to everted gut sac incubations for periods as short as 20 rain 3s. Several methodological problems confounded a precise understanding of where and how theses steroids might influence leucine absorption. For example, the Tween 80 solvent (the vehicle for the steroids added directly to incubations) by itself caused significant changes in leucine transport. The mucosal uptake of leucine was measured under steady state, not initial rate, conditions and in the presence of unstirred water layers, both of which cause significant errors in transport rate calculations (see refs. 52 and 53). It is also unknown what effect restraining unanesthetized fish for in vivo perfusions might have on nutrient uptake, but is certain to elevate plasma cortisol levels7s,s4. More recently, observations of short-term MT actions have been extended to glucose absorption in tilapia intestine. Hazzard and Ahearn 36 added various doses of MT to the serosal side of upper intestine for 30 rain, before measuring transepithelial glucose fluxes under open-circuit conditions. MT caused a twofold increase in net glucose absorption, owing to stimulation of the mucosal-toserosal flux. The response was dose-dependent, being maximal at 15 ng MT/ml of Ringer solution. The short time-course of MT effects adds to mounting evidence that steroids exert nongenomic actions on a variety of target tissues, including intestine64.66, 73. Long-term treatments (5-11 months) of seawater-adapted tilapia with MTsupplemented diets were also shown to stimulate glucose uptake in BBMV. At a growth-promoting dietary dose (10 mg/kg food), MT elicited four distinct transport effects compared with controls: the maximal glucose transport rate (Vmax) increased 2 fold; glucose passive permeability increased 53%; Na+/K+-ATPase activity rose 2.5 fold; and, the coupling ratio of Na+-glucose cotransport increased from 1:1 to 2:1. These changes suggest that the hormonally mediated adaptations occur at several levels in glucose transport mechanisms. Transporter induction (implied by the elevated Vmax), higher permeability, and higher coupling ratio would all accelerate glucose entry into the enterocyte, especially at luminal glucose concentrations higher than those in the blood. Since Na+influx through the transporter would also increase, the elevated Na+-pump activity might prevent dissipation of the Na+-gradient. Thus, the driving force for nutrient uptake would be maintained even at high transport rates. Collectively, the trout and tilapia data suggest that stimulation of nutrient absorption represents one mechanism contributing to MT's anabolic actions in fish.

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2.1.2. Interrenal steroids. Glucocorticoids, which mobilize fuels during stress in vertebrates, also play a developmental role in modulating intestinal enzyme levels in suckling mammals (see ref. 41). In migratory salmonids, by comparison, cortisol serves a developmental function in preparing fresh water salmon parr for seawater adaptation during the parr-smolt metamorphosis 31,77. Collie and Stevens 16 found that cortisol stimulates L-proline uptake in pyloric ceca of coho salmon (Oncorhynchus kisutch) prior to seawater migration. When salmon were implanted with cortisol pellets for two weeks, the Vmax and Km of proline uptake were increased two-fold, concomitant with a significant rise in plasma cortisol, compared with controls receiving cholesterol implants. Hence in fish as well as in mammals, glucoeorticoids appear to regulate digestive adaptations during ontogeny. 2.2. Growthhormone (GH) Somatic growth in juvenile vertebrates is accelerated by GH, requiring a boost in dietary requirements for calories and protein. In salmon undergoing parr-smolt transformation, the energetic costs of rapid growth are compounded by caloric needs to fuel migration and seawater adaptation. Like anabolic steroids, GH stimulates food consumption and more efficient conversion of ingested nutrients into body tissue 21. Two studies offer evidence that a component of this enhanced digestive efficiency is stimulation of nutrient transport. Bovine GH pellets implanted into coho salmon (0. kisutch) increased the Vmax of intestinal proline uptake and stimulated mucosal mass per cm of gut length 16. Both specific and nonspecific mechanisms, therefore, combined to yield greater AA uptake in growth-stimulated salmon. A second study confirmed this effect, but in juvenile striped bass (M. saxatilis) a2. GH injections for 3-4 weeks elevated Na+-dependent and Na+-independent AA in tissue slices and BBMV. The earliest effect, observed two days after a single injection, was an increased Vmax of Na+-dependent uptake. An slower increase in mucosal mass per serosal surface area later augmented AA uptake further. Thus, enhanced AA uptake may be an early adaptation to growth stimulation, ensuring an adequate fuel supply for anabolic metabolism. 2.3. Thyroid hormones Nutrient transport is regulated under many divergent circumstances, so we might not expect to find one signal common to all of them, even when transport changes in the same direction. But for some related subset of conditions, such as accelerated growth, whether occurring naturally or artificially induced, a 'unifying' signal is plausible. Some evidence supports the thyroid hormone 3,5,3'-triiodo-L-thyronine (T3) as one candidate signal regulating energy balance in a variety of anabolic states 22. Above we discussed two examples of hormonal growth enhancement (MT and GH), which were accompanied by increased nutrient absorption. If T3 signals intestinal adaptation under these anabolic conditions, we could make two testable a priori predictions. First, T3 levels should be elevated in these conditions. Second, T3 treatment itself should reproduce some or all of the effects on nutrient transport elicited by MT and GH.

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The first prediction appears to hold in some species, because fish receiving androgens or GH show increased T3 levels (reviewed in ref. 22). Unfortunately, "1"3 levels were not reported in any of the above-mentioned papers examining nutrient transport in response to MT or GH. However, in a companion study to the Reshkin et al.7~ paper, tilapia from the same MT-fed treatment groups did in fact exhibit elevated T3 levels~. It appears likely, then, that both MT and GH increase plasma T3 concentrations and stimulate nutrient uptake. The second prediction has been tested in tilapia by Reshkin et a/. 71 and, more recently, in rainbow trout by Collie et al. is. In the study by Reshkin et a/. 71, which examined MT's effects on glucose transport, tilapia were also fed T3-supplemented diets in graded doses. All parameters stimulated by MT were similarly affected by the optimal T3 dose (1 mg/kg feed), including body mass, brush-border glucose Vmax and Km, passive glucose permeability, Na+/K+-ATPase activity, and the Na + "glucose coupling ratio. The striking match between MT and "['3 actions supports the prediction that "1"3can mimic MT's effects on glucose transport. Similarly, in rainbow trout intestine, dietary T3 was able to reproduce most of GH's effects on glucose and proline uptake across the brush border is. Two weeks of either ovine GH injections (0.2-2.0/zg/g bwt, every 4th day) or T3-supplemented diets (10-20 mg/kg feed) stimulated Na+-dependent glucose and proline uptake per mg of wet gut mass. After 2 weeks of treatment, there were as yet no effects on body size or on gut mueosal mass. Hence, the early effects of both treatments are consistent with a change in nutrient transporter activity, rather than a nonspeeific mechanism such as mueosal growth. By 6 weeks of treatment, however, both hormones caused significant increases in body mass and mueosal mass per cm of intestine. As in tilapia, our second prediction holds for trout intestine, that T3 can reproduce the intestinal nutrient changes elicited by a second growth-promoter, GH. It should be noted that neither study excludes (3H- and MT-speeitie effects that are independent of T3's actions. However, the results support the hypothesis of T3 as a common signal underlying enhanced nutrient absorption in growthstimulated fish.

Fl. Summary andperspectives We began by considering the nutrient transport mechanisms for glucose and AAs, asking if the transport pathways are sufficiently known to permit detailed studies about their regulation. Fish intestine shares many fundamental nutrient transport properties with mammalian intestine, but we cannot tacitly assume that this is uniformly the case. Particularly for AA transport, there are many unsolved questions about the number and specificity of the pathways involved. For both glucose and AA absorption, the basolateral membrane remains the 'dark side of the epithelium '91. Its transporters are beginning to yield to membrane isolation techniques that provide vesicles for transport characterization. Too infrequently have such techniques been used to investigate fish intestine.

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With the cloning of glucose transporters on both 'sides' of mammalian enterocytes, there is now much excitement over the prospect that a transporter's protein domains (i.e. AA sequences) can be linked with the transport functions they perform. The recent reports of cloned AA transporters offer the possibility of associating unambiguously a specific protein with the class of AAs it transports 5~176 Significant sequence homology appears to exist among transporters in different vertebrate groups 12,39,40. Hence, sequence information in mammals may be used to create eDNA probes and identify related transporters in fish intestine. As our understanding of transport mechanisms improves, so should our ability to answer questions of the regulation of intestinal transport. We conclude by emphasizing below several areas where future research might be directed. (1) Dietary carbohydrate and species. Herbivorous and omnivorous fish adapt to dietary carbohydrate levels with altered glucose uptake rates, but carnivores (e.g. trout) appear less able to up-regulate glucose transport 2~ However, certain hormone treatments (GH, T3) seem capable of increasing glucose absorption even in carnivores. Higgs and coworkers 42 report that dietary T3 treatment offsets the reduced growth rates of rainbow trout on high-carbohydrate diets. Is intestinal glucose transport induced by differing carbohydrate levels in T3-supplemented diets or is the gut responding directly to T3 itself as the signal? Which steps in glucose absorption are regulated by T3 ? (2) Induction of AA transport. Intestinal proline uptake varies less than does glucose uptake to changes in dietary composition 2~ However, there have been no direct tests of altering specific AA levels in the diet to look for induction of specific AA transport pathways. The results could provide important, practical input into the design of fish diets that may boost AA absorption and promote rapid growth. (3) Steroids and nutrient transport. Androgens and interrenal steroids promote glucose and AA transport, but the mechanism remains unclear. The rapid effects of androgens offers a fruitful model to explore nongenomic mechanisms of action on nutrient transport. In addition, the interactions of steroids with other endocrine signals, such as T3, needs clarification before the proximate signals for transport regulation can be identified. How are these signals transduced within the enterocyte? (4) Intestinal absorption and fish growth. Rapid growth and enhanced nutrient transport are correlated, but it is unclear whether intestinal adaptation leads or simply responds to the growth of other tissues. The early response of the gut to hormonal growth stimulation may suggest the former, but time-course studies of changes in absorptive function relative to the function and growth of other tissues seem especially warranted.

Acknowledgements. We wish to thank our mutual colleagues for generously sharing ideas and manuscripts during the preparation of this review. Portions of our work were supported by the California Sea Grant College Program, Project R/A-71, and NIH Grant DK 42973. The U.S. Government is authorized to produce and distribute reprints for governmental purposes notwithstanding any copyright notation that may appear hereon.

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VII. References 1. Alpers, D.H. Uptake and fate of absorbed amino acids and peptides in the mammalian intestine. Fed• Proc. 45: 2261-2267, 1986. 2. Bell, G.I., T. Kayano, J.B. Base, C.E Burant, J. Takeda, D. Lin, H. Fukumoto and S. Seino. Molecular biology of mammalian glucose transporters. Diabetes Care. 13: 3, 1990. 3. Black, E.C., A.C. Robertson and R.R. Parker. Some aspects of carbohydrate metabolism in fish. In: Comparative Physiology of Carbohydrate Metabolism in Heterothermic Animals, edited by A.W. Martin, Seattle, University of Washington Press, pp. 89-124, 1961. 4. Bogt, G. and A~ Rigal. A chloride requirement for Na+-dependent amino-acid transport by brush border membrane vesicles isolated from the intestine of a Mediterranean teleost (Boops salpa). Biochim. Biophys. Acta 649: 455-461, 1981. 5. Bog(~, G., A. Rigal and G. P~rb~s. Analysis of two chloride requirements for sodium-dependent amino acid and glucose transport by intestinal brush-border membrane vesicles of fish. Biochim. Biophys. Acta 729: 209-218, 1983. 6. Buddington, R.K. Does the natural diet influence the intestine's ability to regulate glucose absorption? J. Comp. Physiol. 157: 677-688, 1987. 7. Buddington, R.K., J.W. Chen and J. Diamond. Genetic and phenotypic adaptation of intestinal nutrient transport to diet in fish. J. Physiol. 393: 261-281, 1987. 8. Buddington, R.K. and J.M. Diamond. Ontogenic development of intestinal nutrient transporters. Annu. Rev. PhysioL 51: 601-619, 1989. 9. Buddington, R.K. and J.W. Hilton. Intestinal adaptations of rainbow trout to changes in dietary carbohydrate.Am. J. PhysioL 253: G489-G496, 1987. 10. Cheeseman, C.I. Molecular mechanisms involved in the regulation of amino acid transport. Prog. Biophys. Mol. BioL 55: 71-84, 1991. 11. Christensen, H.N. Role of amino acid transport and countertransport in nutrition and metabolism. PhysioL Rev. 70: 43-77, 1990. 12. Coady, M.J., A.M. Pajor and E.M. Wright. Sequence homologies among intestinal and renal Na +glucose cotransporters. Am. J. PhysioL 259: C605-C610, 1990. 13. Collie, N.L. Intestinal nutrient transport in coho salmon (Oncorhynchus kisutch) and the effects of development, starvation, and seawater adaptation. J. Comp. PhysioL 156: 163-174, 1985. 14. Collie, N.L. Hormonal regulation of intestinal nutrient absorption in vertebrates. Am. Zool., in press. 15. Collie, N.L., T. Kuo and J.M. Diamond. Intestinal nutrient and thyroid hormone uptake in growthenhanced rainbow trout (Oncorhynchus mykiss), in preparation. 16. Collie, N.L. and J.J. Stevens. Hormonal effects on L-proline transport in coho salmon (Oncorhynchus kisutch) intestine. Gen. Comp. Endocrinol. 59: 399-409, 1985. 17. Diamond, J.M. Adaptations of intestinal nutrient absorption in mammals. S. A~ J. Sci. 83: 590-594, 1987. 18. Diamond, J.M. Modern concepts of regulation of intestinal nutrient transport. In: Modern Concepts in Gastroenterology, Vol. 2, edited by E. Shaffer and A.B.R. Thomson, New York, Plenum, pp. 209-225, 1989. 19. Diamond, J.M. Evolutionary design of intestinal nutrient absorption: enough but not too much. News PhysioL Sci. 6: 92-96, 1991. 20. Diamond, J.M. and R.K. Buddington. Intestinal nutrient absorption in herbivores and carnivores. In: Comparative Physiology: Life in Water and on Land, edited by P. Dejours, L. Bolis, C.R. Taylor and E.R. Weibel, Padova, Italy, Liviana Press, pp. 193-203, 1987. 21. Donaldson, E.M., U.H. Fagerlund, D.A. Higgs and J.R. McBride. Hormonal enhancement of growth. In: Fish Physiology, Vol. VIII, edited by W.S. Hoar, D.J. Randall and J.R. Brett, New York, Academic Press, pp. 456-598, 1979. 22. Eales, J.G. and D.L. MacLatchy. The relationship between "1"3production and energy balance in salmonids and other teleosts. Fish Physiol. Biochem. 7: 289-293, 1989. 23. Ferraris, R.P. and G.A. Ahearn. Intestinal glucose transport in carnivorous and herbivorous marine fishes. J. Comp. Physiol. 152: 79-90, 1983. 24. Ferraris, R.P. and G.A. Ahearn. Sugar and amino acid transport in fish intestine. Comp. Biochem. PhysioL 77A: 397-413, 1984. 25. Ferraris, R.P., M.R. Catacutan, R.L. Mabelin and A.P. Jazul. Digestibility in milkfish, Chanos chanos (Forsskal): effects of protein source, fish size and salinity. Aquaculture 59: 93-105, 1986.

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26. Ferraris, R.P. and J.M. Diamond. Specific regulation of intestinal nutrient transporters by their dietary substrates.Annu. Rev. Physiol. 51: 125-141, 1989. 27. Ferraris, R.P., J.M. Diamond and W.W. Kwan. Dietary regulation of intestinal transport of the dipeptide carnosine.Am. J. Physiol. 255: G143-G150, 1988. 28. Ferraris, R.P., J. Hsiao, R. Hernandez and B.A. Hirayama. Site density of mouse intestinal glucose transporters declines with age. Am. J. Physiol. 264: G285-293, 1993. 29. Ferraris, R.P., P.P. Lee and J.M. Diamond. Origin of regional and species differences in intestinal glucose uptake.Am. J. Physiol. 257: G689-G697, 1989. 30. Ferraris, R.P., S. Yasharpour, K.C.K. Lloyd, R. Mirzayan and J.M. Diamond. Luminal glucose concentrations in the gut under normal conditions. Am. J. PhysioL 259: G822-G837, 1990. 31. Folmar, L.C. and W.W. Dickhoff. The parr-sm.olt transformation (smoltification) and seawater adaptation in salmonids. Aquaculture 21: 1-37, 1980. 32. Gilles-Baillien, M. Several compartments involved in intestinal transport. In: Intestinal Transport, edited by M. Gilles-Baillien and R. Gilles, Berlin, Springer-Verlag, pp. 103-117, 1983. 33. Habibi, H.R. and B.W. Ince. Effects of steroids and sex reversal on intestinal absorption of L[14C]Leucine in vivo, in rainbow trout, Salmo gairdneri. Gen. Comp. Endocrinol. 52: 438-444, 1983. 34. Habibi, H.R. and B.W. lnce. A study of androgen-stimulated L-leucine transport by the intestine of rainbow trout (Salmo gairdneri Richardson) in vitro. Comp. Biochem. Physiol. 79A: 143-149, 1984. 35. Habibi, H., B.W. Ince and A.J. Matty. Effects of 17-a-methyltestosterone and 17-fl-oestradiol on intestinal transport and absorption of L-[14C]-Leucine in vitro in rainbow trout (Salmo gairdneri). J. Comp. Physiol. 151: 247-252, 1983. 36. Hazzard C.E. and Ahearn G.A. Rapid stimulation of intestinal D-glucose transport in teleosts by 17c~-methyltestosterone. Am. J. Physiol. 262: R412-R418, 1992. 37. Hediger, M.A., M.L. Budard, B.S. Emanuel, TK. Mohandas and E.M. Wright. Assignment of the human intestinal Na+/glucose gene (SGLT1) to the q11.2. Genomics 4: 297-300. 38. Hediger, M.A., M.J. Coady, TS. Ikeda and E.M. Wright. Expression cloning and cDNA sequencing of the Na +/glucose co-transporter. Nature 330: 379-381, 1987. 39. Hediger, M.A., E. Turk and E.M. Wright. Homology of the human intestinal Na+/glucose and Escherichia coil Na +/proline cotransporters. Proc. Natl. Acad. Sci. USA 86: 5748-5752, 1989. 40. Henderson, P.J. The homologous glucose transport proteins of prokaryotes and eukaryotes. Res. Microbiol. 141: 316-328, 1990. 41. Henning, S.J. Ontogeny of enzymes in the small intestine. Annu. Rev. Physiol. 47: 231-245, 1985. 42. Higgs, D.A., B.S. Dosanjh, L.M. Uin, B.A. Himick and J.G. Eales.Aquaculture 105: 175-190, 1992. 43. Hilton, J.W. and J.L. Atkinson. Responses of rainbow trout (Salmo gairdneri) to increased levels of available carbohydrate in practical trout diets. Br. J. Nutr. 47: 597-607, 1982. 44. Hirayama, B.A., H.C. Wong, C.D. Smith, B.A. Hagenbuch, M.A. Hediger and E.M. Wright. Intestinal and renal Na+/glucose cotransporters share common structures. Am. J. Physiol. 261: C296-C304, 1991. 45. Hopfer, U. Membrane transport mechanisms for hexoses and amino acids in the small intestine. In: Physiology of the Gastrointestinal Tract, edited by L.R. Johnson, New York, Raven, pp. 1499-1526, 1987. 46. Howerton, R.D. The Effects of the Synthetic Steroid, 17t~-Methyltestosterone and the Thyroid Hormone, Triiodo-L-thyronine on Growth of the Euryhaline Tilapia, Oreochromis mossambicus. University of Hawaii at Manoa, Hawaii. M.S. Thesis, 79 pp., 1988. 47. Ikeda, T.S., E. Hwang, M.J. Coady, B.A. Hirayama, M.A. Hediger and E.M. Wright. Characterization of a Na+/glucose cotransporter cloned from rabbit small intestine. J. Memb. Biol. 110: 87-95, 1989. 48. Ince, B.W., K.P. Lone and A.J. Matty. Effect of dietary protein level, and an anabolic steroid, ethylestrenol, on the growth, food conversion efficiency, and protein et~ciency ratio of rainbow trout, Salmo gairdneri. Br. J. Nutr. 47: 615-624, 1982. 49. Ingham, L. and C. Arme. Intestinal absorption of amino acids by rainbow trout, Salmo gairdneri (Richardson).Z Comp. Physiol. 117: 323-334, 1977. 50. Kanai, Y. and M. Hediger. Primary structure and functional characterization of a high-affinity glutamate transporter. Nature 360: 467-471, 1992. 51. Karasov, W.H., R.K. Buddington and J.M. Diamond. Adaptation of intestinal sugar and amino acid transport in vertebrate evolution. In: Transport Processes, lono- and Osmoregulation, edited by R. Gilles and M. Gilles-Baillien, Berlin, Springer-Verlag, pp. 227-239, 1985. 52. Karasov, W.H. and J.M. Diamond. A simple method for measuring intestinal solute uptake in vitro. J. Comp. Physiol. 152: 105-116, 1983.

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53. Karasov, W.H. and J.M. Diamond. Adaptive regulation of sugar and amino acid transport by vertebrate intestine. Am. J. Physiol. 245: G443-G462, 1983. 54. Karasov, W.H. and J.M. Diamond. Interplay between physiology and ecology in digestion. Bioscience 38:602-611, 1988. 55. Karasov, W.H., D. Solberg, S. Carter, M. Hughes, D. Phan, E Zollman and J.M. Diamond. Uptake pathways for amino acids in mouse intestine.Am. J. Physiol. 251: G501-G508, 1986. 56. Karasov, W.H., D.H. Solberg and J.M. Diamond. What transport adaptations enable mammals to absorb sugars and amino acids faster than reptiles? Am. J. PhysioL 249: G271-G283, 1985. 57. Karasov, W.M., D.H. Solberg and J.M. Diamond. Dependence of intestinal amino acid uptake on dietary protein or amino acid levels. Am. J. Physiol. 252: G614-G625, 1987. 58. Kong, C., S. Yet and J. Lever. Cloning and expression of a mammalian Na+/amino acid cotransporter with sequence similarity to Na+/glucose cotransporters. I. Biol. Chem. 268: 1509-1512, 1993. 59. Miller, D.S. and W.B. Kinter. Pathways of cycloleucine transport in killifish small intestine. Am. J. Physiol. 237: E567-E572, 1979. 60. Mueckler, M., C. Caruso, S.A. Baldwin, M. Panico, M. Blench, H.R. Morris, W.J. Allard, G.E. Lienhard and H.E Lodish. Sequence and structure of a human glucose transporter. Science 299: 941-945, 1985. 61. Munck, B.G. Intestinal absorption of amino acids. In: Physiology of the Gastrointestinal Tract, edited by L.R. Johnson, New York, Raven, 1097-1122, 1981. 62. Munck, B.G. "II'ansport of imino acids and non-a-amino acids across the brush-border membrane of the rabbit ileum. 1. Membr. Biol. 83: 15-24, 1985. 63. National Academy of Sciences-National Research Council. Nutrient Requirements of Warmwater Fishes and SheUftshes, rev. ed., Washington, D.C., National Academy Press, 1983. 64. Norman, A.W. "I~anscaltachia (the rapid hormonal stimulation of intestinal calcium transport): a component of adaptation to calcium needs and calcium availability. Am. Zool., in press. 65. Pajor, A.M., B.A. Hirayama and E.M. Wright. Molecular biology approaches to the comparative study of Na+/glucose cotransport. Am. J. Physiol. 263: R489--495, 1992. 66. Redding, J. and R. Patifio. Reproductive physiology. In: Physiology of Fishes, edited by D. Evans, Boca Raton, FL, CRC Press, pp. 447-478, 1993. 67. Reichl, J.R. Absorption and metabolism of amino acids studied in vitro, in vivo, and with computer simulations. In: Absorption and Utilization of Amino Acids, Vol. I, edited by M. Friedman, Boca Raton, FL, CRC Press, pp. 93-156, 1989. 68. Reshkin, S.J. and G.A. Ahearn. Intestinal glucose transport and salinity adaptation in a euryhaline teleost. Am. J. Physiol. 252: R567-R578, 1987. 69. Reshkin, S.J. and G.A. Ahearn. Basolateral glucose transport by intestine of teleost, Oreochromis mossambicus. Am. J. Physiol. 252: R579-R586, 1987. 70. Reshkin, S.J. and G.A. Ahearn. Intestinal glycyl-L-phenylalanine and L-phenylalanine transport in a euryhaline teleost. Am. J. Physiol. 260: R563-R569, 1991. 71. Reshkin, S.J., M.L. Grover, R.D. Howerton, E.G. Grau and G.A. Ahearn. Dietary hormonal modification of growth, intestinal ATPase, and glucose transport in tilapia. Am. 1. Physiol. 256: E610-E618, 1989. 72. Reshkin, S.J., S. Vilella, G. Cassano, G.A. Ahearn and C. Storelli. Basolateral amino acid and glucose transport by the intestine of the teleost, Anguilla anguilla. Comp. Biochem. Physiol. 91A: 779-788, 1988. 73. Rommerts, E Cells surface actions of steroids: a complementary mechanism for regulation of spermatogenesis? In: Spermatogenesis, Fetilization, Contraception, Molecular, Cellular and Endocrine Events in Male Reproduction, edited by E. Nieshlag and U. Habenicht, Berlin, Springer-Verlag, pp. 1-19, 1992. 74. Saier, M.S., Jr., G.A. Daniels, P. Boerner and J. Lin. Neutral amino acid transport systems in animal cells: potential targets of oncogene action and regulators of cellular growth. J. Membr. Biol. 104: 1-20, 1988. 75. Schreck, C.B. Physiological, behavioral, and performance indicators of stress. Am. Fisheries Soc. Symp. 8: 29-37, 1990. 76. Smith, I..S. Digestive functions in teleost fish. In: Fish Nutrition, 2nd edn., edited by J.E. Halver, New York, Academic Press, pp. 331--421, 1989. 77. Specker, J.L. and C.B. Shreck. Changes in plasma corticosteroids during smoltification of coho salmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol. 46: 53-58, 1982.

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Hochachka and Mommsen (eds.), Biochemistry and molecular biology of fishes, vol. 4 O 1995 Elsevier Science B.V. All rights reserved. C H A P T E R 10

Metabolic organization of thermogenic tissues of fishes JAMES S. BALLANTYNE Department of Zoology, University of Guelph, Guelph, Ontario, N I G 2W1 Canada

I. II. III. IV. V.

Introduction Metabolism of endothermic muscle of sharks Metabolism of endothermic muscle of tuna Metabolism of billfish brain heater Summary and prospectus Acknowledgements VI. References

I. Introduction Living systems catalyze many of the exothermic chemical reactions found in nature. Although the amount of heat produced by a chemical reaction cannot be changed, by coupling one reaction with another, the amount of heat released can be reduced by conserving the energy in chemical bonds. Uncontrolled combustion yields few useful intermediate molecules and much energy is lost as heat, consequently, much of the early evolution of life on this planet must have been involved with optimizing systems to minimize heat loss. Only much later in the evolution of life did the controlled production of heat become a metabolic option and confer survival advantages on organisms. With metabolism optimized to trap as much energy as possible as ATP, two mechanisms of heat production are possible. One involves bypassing ATP synthesis and the other involves 'wasting' ATP through the action of ATPases. Bypassing ATP synthesis occurs in mammalian brown adipose tissue through the action of a proton channel, thermogenin. This protein allows protons to enter the mitochondria without participating in ATP synthesis. This leak partially collapses the membrane potential and proton gradient across the inner mitochondrial membrane. The leaks themselves are merely signals used to turn on the metabolic processes generating heat. Heat is produced when substrates are oxidized in Krebs cycle to pump protons out of the mitochondrial matrix to reestablish the membrane potential. This allows the mitochondria to respire at high rates without the need to phosphorylate ADP. Proton leaks probably occur in all mitochondrial membranes and may be a design feature of some endothermic mitochondria. Brand and

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colleagues 12 have shown the membranes of mitochondria from ectothermie reptiles to be less leaky than those of similar sized mammals at the same temperature. In these cases the leak is nompecific and a property of the membrane. Such 'basal' leaks may be important thermogenic mechanisms but, since they are properties of entire membranes not specific channels, regulation of the rates would be problematic requiting long-term changes in membrane properties. Such changes do occur and are mediated by thyroid hormones in mammals 13. Leaks mediated by specific channels, however, offer the advantage of being more rapidly modulated. In addition to bypassing mitochondrial ATP synthesis, another mechanism for generating heat is to release the energy trapped in ATP itself. A variety of ATPases exist that could be marshalled for this function. Two of the major ones are transport ATPases: Na+/K+-ATPase and Ca2+-ATPase. A third ATPase, myosin ATPase, is involved in energy transduetion during muscle contraction. In understanding the design of thermogenic mechanisms it is important to account for all of these processes. The use of heat production to elevate and control body temperature is most widespread among terrestrial organisms. Insects, birds and mammals all have representatives capable of sustaining higher than ambient body temperatures. Since the low thermal conductivity of air provides a favorable environment for the development of endothermy, some of these endotherms can be quite small (less than one gram for some insects). Endothermy in the aquatic environment is confined to larger organisms due to the high thermal conductivity of water. The smallest endothermie fish weigh about 1-2 kg or 1000 times more than the smallest terrestrial endothermic vertebrate (2 g). Endothermic tissues have been documented in the large species of three groups of fishes: the lamnid sharks, the billfishes, and the tunas. Large size alone is not a sufficient condition for endothermy in fishes. Specific anatomical features have been developed to enable warm-bodied fish to retain heat as well as shunt it to other tissues (see ref. 8 for review). Bone ~1 has suggested that the constancy of temperature, not simply elevated temperature, was the critical factor in the design of warm tissues. Constancy requires regulation to control the rate of heat production, the capacity for sustained heat production and the ability to control heat loss. The metabolic production of heat must occur at faster rates than it is dissipated if the tissue temperature is to rise above ambient. If the capacity to produce heat exceeds the rate at which it is dissipated, regulation of the reactions producing heat must be strict to prevent tissue temperature from rising to dangerous levels. The tissues of fish may be heated by intracellular as well as extracellular chemical reactions. The viscera of lamnid sharks 17 and tunas 16 have been shown to have higher than ambient temperatures. Heat production in these tissues is an extracellular phenomenon based on the exothermic hydrolysis of protein and triglyeerides 16. As an extraeellular process, the normal mechanisms of metabolic regulation do not operate to control the rates of heat production. Since digestion of food is the source of heat, regulation of heat production in these tissues may simply involve regulation of heat dissipation through control of the vascular heat exchangers. Nevertheless, the involvement of the cells of the visceral tissues in heat

Metabolic organization of thermogenic tissues o.ffishes

243

production cannot be ruled out and further studies of the metabolism of these tissues are required. Intracellular processes of heat production are better understood and will be the primary focus of this review. In fish, every tissue producing significant amounts of heat by intracellular processes is a muscle. These include a modified extraocular muscle in the head of billfish and the lateral red muscles of tunas and lamnid sharks. Insights into the organization of metabolism of thermogenie tissues of fishes is, therefore, dependent on an understanding of the metabolism of fish red muscle. The goal of this review is to summarize the current state or our knowledge relating to the organization of metabolism and its regulation in thermogenic tissues of fishes. This review will rely heavily on studies of the metabolism of nonthermogenic fish muscle to elucidate the function of the metabolism of thermogenic tissues.

II. Metabolism of warm muscle of sharks Among the elasmobranchs, endothermy occurs in one family, the Lamnidae, with only 5 species. These sharks are large, fast swimming predators that successfully compete with, and feed upon, marine mammals. While the metabolic rates of most elasmobranchs are lower than those of comparable teleost fishes 14 studies of one endothermie lamnid, the shortfin mako shark (Isurus oxyrhinchus), indicate it has a metabolic rate comparable to that of some Warm-bodied tunas 2s. The primary source of heat in lamnid sharks is the lateral red muscle 51. The heat produced by this tissue is used to warm the blood which passes through the brain and eyes 9 and viscera 17 as well as the muscle itself. The sources of heat in elasmobranch muscle are the processes associated with muscle contraction. The two major transport ATPases (Na+/K+-ATPase and Ca2+-ATPase) must be involved as well as the myosin ATPase. The oxidation of substrates to supply ATP for these enzymes produces most of the heat. The elasmobranchs differ from virtually all other groups of fish in that fatty acids are not important oxidative substrates in muscle. The levels of enzymes of oxidation of fatty acids, carnitine acyl transferases, are undetectable in the muscle of nonendothermic sharks 4~ and isolated red muscle mitochondria do not oxidize fatty acids or acyl carnitines 2,19,4~ The explanation for the absence of lipid oxidation in elasmobranch red muscle has been attributed to the absence of the fatty acid carrier protein in the blood 22. The levels of plasma nonesterified fatty acids in elasmobranchs are low by comparison with those of some species of fishes4,22,52 but are far higher in concentration than is possible based on their solubility. In the absence of albumin, it has been suggested 4 that fatty acids must be carried by other lipid fractions of the plasma and consequently may not be delivered to the tissues with sufficient rapidity to sustain muscle contraction. The large lipid reserves in the liver of elasmobranchs, could be an accessible energy source for red muscle provided the carbon is transported in another form. Ketone bodies have been suggested to be major fuels for elasmobranch red muscle 2.19,4~ Ketone body levels in elasmobranch plasma are high and increase

J.S. BaUantyne

244 TABLE 1

Substrate oxidation by mitochondria isolated from 'cold' and 'warm' elasmobranch red muscle Substrate Glutamine "" Glutamate Proline /~-Hydroxybutyrate Pyruvate a-Glycerophosphate Palmitoyl-L-carnitine Endogenous

State 3' rate of respir'ation

Raja erinacea 19

Squalus acanthias 19

19.18 9.32 8.16 8.85 9.70 ND ND

~- 1.77 (7) 4- 1.49 (7) + 0.99 (7) 4- 1.20 (6) 4- 1.65 (6)

45.65 4- 4.97 (5) 26.31 4- 7.61 (6) 28.47 4- 2.46 (4) 54.04 4- 5.15 (6) 27.51 4. 7.64 (4) ND ND

3.61 4- 0.43 (7)

3.71 4- 0.88 (5)

lsurus oxyrinchus 2 '

49.84 4- 2.21 (3) 45.94 4- 13.81 (3) 38.43 4- 41.16 (2) 46.53 -4- 3.92 (3) 34.34 4- 12.53 (3) ND ND 3.72 4- 2.24 (3)

Values are means 4- SEM with the number of mitochondrial preparations given in parentheses. Respiration is expressed in nmol O2/min/mg protein. Rates for little skate (Raja erinacea) and spiny dogfish (Squalus acanthias) were determined at 10~ and the rates for mako shark (Isutus oxyrinchus) were determined at 20~ ND = Not detected.

during starvation 52. The source of plasma ketone bodies is likely hepatic lipid since liver is capable of the oxidation of fatty acids in elasmobranchs 3,43,44 and Anderson 1 has shown ketone body formation from palmitoyl-CoA by liver mitochondria. Ketogenesis from amino acids has been demonstrated in elasmobranch hepatocytes38 and may play an important role in supplying substrates for muscle contraction. In addition to ketone bodies, amino acids may be important direct energy sources in thermogenic red muscle of elasmobranchs tg. Three of the major substrates oxidized by elasmobranch red muscle mitochondria are amino acids (glutamine, glutamate and proline; Table 1). The high protein diet of elasmobranchs would make reliance on amino acids as an energy source for muscle contraction a viable alternative to lipid. Of the amino acids oxidized, glutamine in particular, appears to be a major metabolic substrate in elasmobranch red muscle2,19. Plasma glutamine levels in the dogfish shark (Squalus acanthias) are about 12/~M and in the muscle itself levels are about 0.6 mM. This 50 fold gradient indicates active uptake of this amino acid 19 although nothing is known of the mechanism of transport of this amino acid across the sarcolemma in elasmobranchs. One possibility is a Na + coupled symport mechanism similar to that found in mammalian muscle 32. Once inside the cell, glutamine is converted to glutamate via phosphate dependent glutaminase (PDG). PDG levels in elasmobranch red muscle are higher than those found in muscle of any species (Table 2) and rival levels in the mammalian kidney 19. The pathway for oxidation of glutamine is thought to be autocatalytic, in that mitochondrial malic enzyme converts some of the amino acid carbon to pyruvate providing acetyl CoA to condense with the remaining carbon in oxaloacetate to form citrate. Since this reaction involves the production of NADH, there is no loss of ATP synthetic capacity when some of the malate is converted to pyruvate instead of oxaloacetate. Since glutamate is also oxidized by elasmobranch red muscle mitochondria, and glutamate is an intermediate in the oxidation of glutamine, some coordination of

Metabolic organization of thermogenic tissues of fishes

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TABLE 2 Activities of glutarninase and glutamine synthetase in red muscle of fishes Species

Glutaminase

Glutamine synthetase

Hagfish 19 Skate 19 Dogfish 19 Bowfin 18 Lake char 18

0.39 1.96 3.26 0.22 0.23

NA 0.27 NA 0.09 0.01

The units are/xmoles of substrate converted per minute per gram wet weight of tissue. All measurements were made at 10*C. Hagfish -- Petromyzon marinus, Skate = Raja erinacea, Dogfish ffi Squalus acanthias, Bowfin ffi Amia calva, Lake char ffi Salvelinus namaycush. NA ffi Not analyzed.

the catabolism of these two substrates is required especially if their oxidation serves two separate purposes. The oxidation of glutamine by elasmobranch red muscle mitochondria is regulated, in part, by a compartmentation of glutamate. Several lines of evidence support this conclusion. Chamberlin and Ballantyne 19 have shown that oxidation of exogenous glutamate is inhibited by aminooxyacetate, an inhibitor of pyridoxal phosphate-dependent transaminases while glutamine oxidation is not. This indicates that glutamate entering from outside the mitochondrion has different access to certain matrix enzymes than glutamate generated inside the mitochondrion from glutamine via PDG. Glutamate derived from glutamine is catabolized via glutamate dehydrogenase (GDH) not aspartate aminotransferase (AspAT). Studies of similar systems in teleost fishes indicate aspartate production from glutamate is much greater than that from glutamine 18 and CO2 production from glutamine is much higher than that from glutamate even though respiration rates with the two substrates are similar (M. Gerrits and J.S. Ballantyne, unpublished data). The physical nature of the compartmentation can only be speculated upon. In one possible configuration, the membrane carrier for glutamine is coupled to PDG which is, in turn, coupled to GDH (Fig. 1). Transfer of glutamate formed by PDG directly to GDH occurs without release of glutamate. This glutamate, therefore, does not have access to AspAT, the other mitochondrial enzyme using glutamate as a substrate. GDH converts glutamate to a-ketoglutarate which may enter Krebs cycle for oxidation. In the other glutamate pool, glutamate entering via the glutamate/aspartate carrier has immediate access to AspAT and a-ketoglutarate is again produced. This a-ketoglutarate pool may not be the same as that produced by GDH and, therefore, may not enter the Krebs cycle (region 7 in Fig. 1). Glutamate carbon must exit as ot-ketoglutarate to permit continuous operation of the malate-aspartate shuttle. Compartmentation of glutamate may occur simply to ensure the exit of its carbon as a-ketoglutarate. Thus by compartmentalizing these two functions of glutamate, cytoplasmic redox can be balanced while oxidation of amino acids such as glutamine and proline may proceed unaffected, in this scheme, cytosolic glutamate enters the mitochondria only for redox balance. Glutamine is oxidized to supply energy for ATP synthesis. Oxidation of glutamine in elasmobranch red muscle could potentially compete

246

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Fig. 1. Compartmentation of glutamate metabolism in thermogenic tissues of fishes. Some intermediates are omitted for clarity. 1 = glutamine carrier; 2 = phosphate-dependent glutaminase; 3 = glutamate dehydrogenase; 4 = malate/a-ketoglutarate carrier; 5 = glutamate aspartate carrier; 6 = aspartate amino transferase; 7 = segment of Krebs cycle. AAT = aspartate aminotransferase; MDH = malate dehydrogenase; ME = malic enzyme.

with oxidation of other substrates such as fl-hydroxybutyrate. As indicated above, oxidation of glutamine occurs via GDH in the mitochondrial matrix. Oxidation of/5-hydroxybutyrate also occurs in the mitochondrial matrix via the action of /3-hydroxybutyrate dehydrogenase. Both enzymes require NAD to convert their substrate into the compound entering Krebs cycle. Competition for mitochondrial matrix NAD may, therefore, occur (Fig. 2). A similar situation has been studied in the mammalian kidney, where it has been found that elevated levels of ketone bodies inhibit glutamine catabolism 3s. Inhibition is due to increased formation of citrate due to higher rates of acetyl CoA condensation with oxaloacetate during ketone body metabolism. The elevated levels of a-ketoglutarate that result inhibit GDH resulting in the accumulation of glutamate and the inhibition of PDG (Fig. 2). It has also been suggested that c~-ketoglutarate may inhibit the glutamine carder 24 in mammalian kidney. Similar studies are required to establish if the regulatory mechanism operating in the mammalian kidney applies to elasmobranch red muscle. fl-Hydroxybutyrate may compete with pyruvate for entry into the mitochondria since both substrates are transported on the monocarboxylate carrier. Since amino

Metabolic organization of thermogenic tissues of iishes

247

Fig. 2. Competition between ketone bodies and glutamine as oxidative substrates in thermogenic tissues of fishes. Some intermediates are omitted for clarity. 1 = mitochondrial monocarboxylate carrier; 2 = flhydroxybutyrate dehydrogenase; 3 = 3-oxoacid CoA transferase; 4 = acetoacetyl CoA acetyltransferase; 5 = pyruvate dehydrogenase; 6 = citrate synthase; 7 = aconitase; 8 = isocitrate dehydrogenase; 9 = glutamine transporter; 10 = phosphate-dependent glutaminase; 11 = glutamate dehydrogenase; 12 = ot-ketoglutarate dehydrogenase; 13 = succinyl CoA synthetase; 14 = succinate dehydrogenase; 15 = fumarase; 16 = malate dehydrogenase 17 = malic enzyme.

acids and ketone bodies are important energy sources for elasmobranch muscle, carbohydrate may thus be spared from oxidation. Little is known of the interaction between ketone body and carbohydrate metabolism in elasmobranchs. Plasma glucose levels are lower in elasmobranchs compared to teleost fishes s2 and muscle mitochondria oxidize pyruvate at lower rates than those of glutamine and flhydroxybutyrate (Table 1). Hexokinase levels in red muscle of elasmobranchs are relatively high 39 perhaps implying glucose can be taken up by the tissue to replenish carbohydrate depletion during anaerobic glycolysis. Cytoplasmic redox balance when carbohydrate is catabolized aerobically is achieved through the malateaspartate shuttle rather than the ct-glycerophosphate shuttle based on the absence of mitochondrial oxidation of t~-glycerophosphate (Table 1). The rates of substrate oxidation per mg mitochondrial protein of the red muscle mitochondria of the highly active shortfin mako shark are not higher than those of less active elasmobranchs such as the little skate or the dogfish shark (Table 1). The substrate preference for oxidation by mitochondria is similar (Table 1) and the degree of coupling of oxidation to phosphorylation as indicated by the respiratory

248

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control ratio is similar2 suggesting heat production is due to rapid oxidation of substrates and high myosin ATPase activity not through a high proton leak as occurs in the brown adipose tissue of mammals. The basal proton leak in warm-bodied elasmobranch muscle has, however, not been determined. The contributions of Na+/K+-ATPase and Ca2+-ATPase likewise have not been determined in this tissue but may be expected to resemble those of other muscles and thus contribute relatively little to heat production 2~

III. Metabolism of warm muscle of tuna The red muscle of tuna is involved in 'basal' swimmingr with heat production a by-product of locomotion. Tunas by contrast with elasmobranchs are representatives of a recently evolved group of fishes, the teleosts. One important difference in the metabolism of the endothermic red muscle of the tunas compared to that of the elasmobranchs is the use of lipid as an energy source. The presence of a fatty acid carrier protein, albumin, in the blood allows efficient transport of fatty acids to red muscle for oxidation. Red muscles of skipjack (Katsuwonus pelamis) 3~ and kawakawa tuna (Euthynnus affin/s)23 have been shown to contain significant amounts of fat and glycogen. Reliance on exogenous glucose as indicated by hexokinase levels is moderate in red muscle compared to white27. The studies of Weber and colleaguess~ indicate plasma glucose turnover rates for tuna are higher than those reported for any other fish species. The extent of utilizationof glucose by red muscle for thermogenesis remains to be determined. The studies of tuna red muscle by Moyes et al.42 indicate similar substrate preferences to other teleost red muscle mitochondria. Pyruvate and palmitoyl carnitine are oxidized at the highest rates indicating lipids and carbohydrates are preferred energy sources. Hulbert et al.31 report abundant lipid droplets in skipjack tuna (K. pelamis) but Mathieu-Costello and coworkers 36 find few lipid droplets in red muscle of the same species. Such differences likelyreflect seasonal changes in food abundance (cf.Chapter I, thisvolume). The role of amino acids as energy sources in tuna muscle, has not been examined. Since glutamine has been found to be a major oxidative substrate in aU other fish red muscle examined to date2,1s,19 the importance of glutamine metabolism in one of the most metabolically active fish tissueswould be of considerable interest. The regulation of amino acid catabolism is likelyto resemble that found in other teleosts~s as well as elasmobranchs 19. Although it is assumed that tunas lack the ability to use ketone bodies, this has never been demonstrated. Tissues of other teleostshave been found to oxidize ketone bodies including /3-hydroxybutyrate as energy sources2 and the enzyme /~-hydroxybutyrate dehydrogenase has been demonstrated in a variety of teleost tissues (ref.34a). The special regulatory requirements to control one of the most metabolically active fish tissues have been the subject of investigation for many years. Redox balance is required for aerobic glycolysis and although this may be supplied

Metabolic organization of thermogenic tissues of fishes

249

in part by the malate-aspartate shuttle, the u-glycerophosphate shuttle is also likely to be important since the levels of ct-glycerophosphate dehydrogenase are high in tuna red muscle 27 compared to other fish species such as carp 41. The rationale for a reliance on this hydrogen shuttle compared to the malate-aspartate shuttle is unknown but may be speculated upon. It may simply be faster to balance cytoplasmic redox without transporting substrates into the mitochondria. The malate-aspartate shuttle involves the transport of two metabolites into the mitochondrial matrix in exchange for two other metabolites (Fig. 1). The uglycerophosphate shuttle does not require the transport of u-glycerophosphate into the matrix the mitochondrial form of the enzyme is accessible to the cytoplasm. Transport mechanisms are frequently flux controlling and elimination of substrate transport may help enhance the rates of redox balance. Alternatively, the use of the a-glycerophosphate shuttle may be related to the mechanism of coupling the respiration rate of the mitochondria to the rate of muscle contraction and ADP supply. Mitochondrial respiration in vitro is controlled in part by availability of ADP. ADP levels in tuna red muscle are lower in steady-state swimming than in 'resting' fish 27. While regulation of mitochondrial respiration rates by ADP availability may suffice in low performance tissues such as liver, other tissues may require a more rapid initiation of respiration than can be provided by ADP. Calcium may provide such a signal. Calcium stimulates the mitochondrial oxidation of u-glycerophosphate in mammals (see ref. 28 for review), insects 29 and other fish 2. This has not been demonstrated in tuna red muscle mitochondria. Calcium release in muscle cells precedes the accumulation of ADP and may provide a more rapid signal for the initiation of mitochondrial respiration. Such a rapid response time may only be critical in warm-bodied fish pushing the limits of metabolism, t~-Glycerophosphate levels in red muscle do not change from rest to steady-state 27 perhaps implying stimulation by elevated calcium levels occurs allosterically with a decrease in the apparent Km for u-glycerophosphate as has been described for brown adipose tissue mitochondria 15. Although the kinetics of tuna red muscle t~-GPDH have been studied 3~ the effects of calcium remain to be examined. Competition between o~-glycerophosphate dehydrogenase and lactate dehydrogenase (LDH) for NADH is a function of pH and other factors 26. At low pH, LDH balances redox with lactate accumulation, while at high pH, u-GPDH balances redox 26. Another function of a-glycerophosphate dehydrogenase may be the in situ recycling of glycerol released during fatty acid oxidation. Glycerol may be used to re-esterify fatty acids or as a source of carbon for gluconeogenesis. Brown adipose tissue, another tissue using lipid as an energy source, has high levels of mitochondrial u-glycerophosphate dehydrogenase 15 and glycerol kinase 6. Gluconeogenesis from glycerol may be an important function of these enzymes. Re-esterification of fatty acids also requires glycerol-3-phosphate and may provide a mechanism to prevent excessive accumulation of free fatty acids. The levels of glycerol kinase in tuna red muscle have not been determined but should be high. Lactate may serve as an energy source for red muscle especially following periods of burst activity in white muscle. Lactate accumulation in white muscle of tuna may be as high as 90 mM 27. The levels of LDH in red muscle of tuna are low (500 units

250

J.$. Ballantyne

per gram tissue fresh weight; 1 unit defined as the amount of enzyme producing 1 /~mol of product per min at the given assay conditions) measured in the forward (pyruvate to lactate) direction 27. Since the reverse direction (lactate to pyruvate) direction is generally ten- to fifteen-fold lower in activity in tuna (J.S. Ballantyne, unpublished data) and functioning well below maximal rates, lactate to pyruvate flux would likely be low and not sufficient to maintain high rates of Krebs cycle activity. Several studies have attempted to explain the apparent high metabolic rate of this tissue. The red muscle fibres have a mitochondrial volume density of 28.5% 36. This is not substantially different from values obtained for a variety of nonendothermic fish muscles (e.g. 31% for trout red muscle34). The activities of citrate synthase per mg mitochondrial protein of tuna red muscle are similar to those of carp red muscle 42. The extreme aerobic capacity of tuna red muscle is apparently due to a combination of factors including the larger red muscle mass, the high mitochondrial density and an increased membrane surface area 42. Perhaps most importantly, the elevated temperatures of tuna red muscle plays a role in the enhancement of power production 33 and heat production. The studies of Moyes and colleagues 42 indicate the red muscle mitochondria of tuna are well coupled although as in the case of the elasmobranch endothermic red muscle the basal proton leak was not quantified.

IV. Metabolism of billfish brain heater The red muscle of tunas and lamnid sharks, generates excess heat as part of its normal function in locomotion. In these cases, the lateral red muscle is a very active tissue in very active fish. Heat is produced by ATP hydrolysis and mitochondrial oxidation of substrates. In contrast, the swordfish (Xiphiasgladius) and other billfish are relatively inactive and do not generate significant amounts of heat in their lateral red muscle. They do have warm brains and eyes due to the presence of a modified extraocular muscle s. Much of the anatomy and biology of this tissue has been extensively reviewed by Blocks. Of all thermogenic tissues of fishes, the brain heater is by far the best characterized, but the regulation of metabolism in this tissue has not been examined in detail. The brain heater organ has lost much of its contractile apparatus. That component of heat production derived from myosin ATPase activity is, therefore, lost. Accompanying the loss of contractile elements, is an enormous proliferation of mitochondfia with up to 70% of the cell volume being occupied by mitochondria 7. The lack of contractile function has lead to the speculation that heat production in this tissue is by a mechanism similar to that of mammalian brown adipose tissue. In brown adipose tissue, a proton leak v/a a specific proton channel 'thermogenin' effectively uncouples the mitochondria resulting in high rates of respiration with no requirement for the phosphorylation of ADE Substrate oxidation consequently proceeds at high rates to pump protons out of the mitochondria. The high rates of

Metabolic organization of thetmogenic tissues of lishes

251

TABLE 3 Substrate oxidation by mitochondria isolated from swordfish (Xiphias gladius) brain heater organ Substrate Endogenous Glutamine Glutamate Proline /~-Hydroxybutyrate Pyruvate Palmitoyl-L-carnitine a-Glycerophosphate (5 mM 4- 1 mM CaCI2)

State 3 rate of respiration 18.41 4- 8.97 (3) 148.81 4. 50.39 (4) 167.16 4. 48.10 (4) 37.48 -4- 16.56 (4) 60.32 4- 27.34 (4) 151.28 4. 61.80 (4) 143.70 4- 49.80 (4) 167.03 4- 91.16 (4)

Values are means 4- SEM with the number of mitochondrial preparations given in parentheses. Respiration is expressed in nmol O2/min/mg protein. Rates were determined at 20°C. Data are taken from reference 2.

substrate oxidation are the source of heat. Block7 reports being unable to detect the proton channel 'thermogenin' in this tissue. Freshly isolated mitochondria of the heater tissue of the swordfish are tightly couplede indicating the capability for ATP synthesis. As in the two other thermogenic fish tissues examined above, the basal proton leak has not been quantified in billfish brain heater mitochondria. In spite of the absence of a specific proton channel, the mitochondria of the biUfish brain heater resemble those of mammalian brown adipose tissue in several respects. The substrates oxidized are similar. Swordfish brain heater has high levels of citrate synthase, carnitine palmitoyl transferase and 3-hydroxyacyl CoA dehydrogenase49 and palmitate is readily oxidized by isolated mitochondria (Table 3) indicating substantial reliance on lipid as an energy source for thermogenesis. Brown adipose tissue mitochondria also rely on lipid as an energy source (see ref. 45 for review). Ketone bodies are also oxidized by swordfish heater (Table 3) and brown adipose tissue 21. Teleost fishes have been thought to be unable to oxidize ketone bodies 52 although more primitive fishes such as elasmobranchs 19,52, holosteans 4s and chondrosteans 47 do use ketone bodies as an energy source. A recent investigation has revealed the presence of fl-hydroxybutyrate dehydrogenase in several species of teleost fishes (ref. 34a). Obviously the role of ketone bodies in teleost fishes needs to be reexamined. Amino acids also serve as an energy source in binfish brain heater with glutamine playing a prominent role (Table 3) as occurs in other fish muscle. Glutamine is also an important oxidative substrate in brown adipose tissue where PDG levels are higher than in liver21. The role of glutamine oxidation in brown adipose tissue remains to be explained. Competition between ketone bodies and amino acids such as glutamine may occur as indicated above for elasmobranch red muscle. Carbohydrate oxidation may also play an important role in the energetics of the biUfish heater organ. Both Tullis et al. 49 and Ballantyne et al. 2 report high levels of hexokinase in this tissue and glycogen levels are highs. High rates of a-glycerophosphate oxidation in both bill heater organ and brown adipose tissue is another unusual parallel between these two tissues and may indicate similar mechanisms for controlling heat production.

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I.s. BaUantyne

The loss of the contractile apparatus removes another ATPase and its signal (ADP) that could be involved in heat production. In the absence of a specific mitochondrial proton leak channel, some other means must be provided to allow regulation of heat production. The brain heater has substantial amounts of sarcoplasmie reticulum (SR) and Block7 has suggested that the calcium ATPase associated with this organeUe provides the ADP required to initiate mitoehondrial respiration. Electrical or hormonal stimulation of the muscle cell initiates calcium release from the SR. The calcium pump in the membrane of the SR pumps calcium back into the SR at the cost of ATP hydrolysis. Continued release of calcium maintains a constant supply of ADP to maintain mitochondrial respiration at high rates. The signal for enhanced mitoehondrial respiration rates could be calcium itself rather than ADP. A more rapid response could be elicited from the mitoehondria if calcium were the primary signal. ATP is only hydrolyzed to ADP after calcium is released from the sareoplasmie retieulum. Calcium entry into the mitoehondria would, therefore, precede that of ADP. At least three key mitochondrial enzymes are activated by intramitoehondrial calcium (pyruvate dehydrogenase, isoeitrate dehydrogenase, a-ketoglutarate dehydrogenase) (Fig. 3). Calcium activation of , ketoglutarate dehydrogenase and isoeitrate dehydrogenase has been demonstrated in trout heart mitochondria 37. Although Ca 2+ activation of pyruvate dehydrogenase in fish muscle has not been demonstrated, this enzyme may also be Ca 2+ activated in brain heater organ mitoehondria since several mammalian forms of the enzyme are activated by mieromolar calcium~. Ballantyne et al. 2 also found substantial calcium-stimulated oxidation of a-glyeerophosphate in swordfish brain heater mitoehondria. High levels of t~-glycerophosphate dehydrogenase have been associated with redox balance during aerobic glycolysis. Calcium stimulation of respiration may be an important mechanism for stimulating mitoehondrial metabolism before ADP levels drop as discussed above. Such a coupling of intraeellular calcium levels to mitochondrial respiration provides a link between the proposed mechanism 7 for stimulation of heat production in the tissue. Brown adipose tissue mitoehondria also have substantial levels of calcium activated t~-glyeerophosphate 15. The site of action of calcium stimulation is a-glyeerophosphate dehydrogenase 15. Calcium acts allosterically to reduce the apparent Km for a-glycerophosphate 15. Regulation at the site of mitoehondrial a-glyeerophosphate dehydrogenase may be important in sparing glycogen or glucose as has been suggested for mammalian brown adipose tissue. Inhibition of this enzyme by aeyl CoA in mammalian brown adipose tissue reduces flux through glyeolysis15. This inhibition would also result in the accumulation of the glycerol 3-phosphate required for esterifieation of fatty acids to form triglyeerides. The levels of glycerol kinase in swordfish brain heater organ 2 indicate in situ re-esterifieation of fatty acids may occur in this tissue. A final similarity between mammalian brown adipose tissue and swordfish brain heater is the rate of heat production. Similar rates of heat production of swordfish heater organ have been calculated using either mitoehondrial respiration rates 2 or enzyme activities49. The rates of heat production of swordfish heater organ rivals that of mammalian brown adipose tissue (0.5 W/g) 2. The amount of heat produced varies with the substrate oxidized (0.08 W/g for palmitate versus 0.37 W/g for

Metabolic organization of thermogenic tissues of fishes

253

Fig. 3. Summary of the metabolic organization of swordfish brain heater organ. Dashed lines indicate potential sites of regulation due to calcium and other effectors. Some intermediates are omitted for clarity. 1 = plasma membrane sodium dependent glutamine carrier; 2 = plasma membrane sodium dependent monocarboxylate carrier; 3 = fatty acid transport; 4 = sodium dependent glucose transporter; 5 = Na +/K+-ATPase; 6 = acyl CoA synthetase; 7 = carnitine acyl transferase complex; 8 = mitochondrial monocarboxylate carrier; 9 = mitochondrial glutamine carrier; 10 = phosphate-dependent glutaminase; 11 = glutamate dehydrogenase; 12 ffi fl-hydroxybutyrate dehydrogenase; 13 = mitochondrial calcium efltux pathway; 14 = mitochondrial Na+/H+-exchanger; 15 ffi acetyl-CoA acetyltransferase; 16 = 3oxoacid CoA-transferase; 17 = malic enzyme; 18 = pyruvate dehydrogenase; 19 = citrate synthase; 20 = malate dehydrogenase; 21 = fumarase; 22 = succinate dehydrogenase; 23 = succinyl CoA synthetase; 24 = mitochondrial Ca2+/Na+-exchanger; 25 = electron transport chain with proton pumps; 26 = t~-ketoglutarate dehydrogenase; 27 = isocitrate dehydrogenase; 28 = aconitase; 29 = mitochondrial proton leak; 30 = ATP synthetase; 31 = adenylate exchanger; 32 = mitochondrial calcium influx; 33 = sarcoplasmic reticulum calcium ATPase; 34 = sarcoplasmic reticulum calcium channel; 35 = glycogen phosphorylase; 36 = phosphofructokinase-1; 37 = cytoplasmic glycerol 3-phosphate dehydrogenase; 38 = triacylglycerol lipase; 39 = glycerol kinase; 40 = mitochondrial glycerol 3-phosphate dehydrogenase; 41 = aldolase; 42 = glyceraldehyde phosphate dehydrogenase; 43 = phosphoglycerate kinase, phosphoglyceromutase, enolase, pyruvate kinase.

pyruvate) 2. Such considerations may explain the choice of substrates oxidized in this tissue but the similarities of the mechanisms regulating substrate oxidation remain to be determined. There are several sites of heat production in cells. The two major ATPases in the swordfish brain heater are the transport ATPases, Na+/K+-ATPase and the

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Ca2+-ATPase. Using ouabain binding studies Block and Franzini-Atmstrong 1~ have demonstrated high densities of Na+/K+-ATPase in billfish. Without quantitative estimates of the activity of these two ATPases, it is difficult to assess their respective contributions to thermogenesis. In mammalian muscle the ratio of CaZ+-ATPase to Na+/K+-ATPase is between 25 and 125 (see ref. 20 for review). At rest these two pumps are probably responsible for similar fractions (about 5%) of the total energy exchange of the muscle2~ During activation of mammalian muscle, calcium release from SR is enhanced 30 fold compared to Na + influx and K + efflux2~ increasing the contribution of calcium cycling to the total energy turnover. Under these conditions calcium cycling may stimulate 20-50% of total heat produced 2~ Of this the Ca2+-ATPase would contribute 1/3 of the heat with the remaining 2/3 due to heat release during substrate catabolism to produce ATP 2. In summary, heat production in billfish brain heater involves a variety of thermogenic processes (Fig. 3). Substrate (glutamine, ketone bodies and glucose; sites 1, 2, and 4 in Fig. 3 respectively) entry into the cell at the expense of the Na + gradient will increase Na+/K+-ATPase (site 5 in Fig. 3) activity as would electrical stimulation of the muscle. This would stimulate mitochondrial respiration by generating ADE The entry of ketone bodies and pyruvate (site 8 in Fig. 3) into the mitoehondrial matrix at the expense of the membrane potential and proton gradient would stimulate mitochondrial respiration to pump protons out and reestablish these gradients. Hormonal and/or electrical stimulation of the cell resulting in calcium release from the SR would activate glycogen phosphorylase (site 35 in Fig. 3) enhancing glycolysis. Redox balance for this pathway (the t~-glycerophosphate shuttle) would be simultaneously activated by cytoplasmic calcium stimulation of mitochondrial a-glycerophosphate oxidation (site 40 in Fig. 3). Ca2+-ATPase in the SR (site 33 in Fig. 3) would pump calcium back into the SR and produce ADP stimulating mitochondrial respiration. Calcium entry into the mitochondrial matrix would stimulate respiration in several ways. Activation of three mitochondrial dehydrogenases (pyruvate dehydrogenase, a-ketoglutarate dehydrogenase and isocitrate dehydrogenase; sites 18, 27, 26 in Fig. 3 respectively) may occur. Calcium efflux from mitochondria in exchange for Na + (site 24 in Fig. 3) would stimulate mitochondrial respiration when Na + efflux is coupled to proton influx (site 14 in Fig. 3). Proton influx collapses the proton gradient and must be redressed by pumping protons out of the mitochondrial matrix (site 25 in Fig. 3.) It is unlikely that all of these mechanisms operate simultaneously but more information is needed to establish which processes operate under specific conditions. The Similarities between mammalian brown adipose tissue and billfish heater organ in both metabolism and metabolic regulation are a remarkable parallel development. The marshalling of the existing metabolic potential of fish muscleto produce a similar thermogenir tissue with a design similar to that developed from white fat cells in the mammals indicates the constraints placed on the 'design' of a new tissue using 'hardwired' metabolic pathways.

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V. S u m m a r y a n d prospectus The thermogenic tissues of fishes are similar in many respects. All are highly aerobic muscles with high mitochondrial densities. In the tuna and elasmobranchs, myosin ATPase combines with the two major transport ATPases (Ca2+-ATPase and Na+/K+-ATPase) to provide ADP to stimulate mitochondrial respiration while in the swordfish brain heater organ the Na+/K+-ATPase and the Ca2+-ATPase assume most of this function. Measurements of all three ATPases in fish thermogenic tissues would help quantify the relative importance of each of these. In the brain heater tissue calcium may also provide a signal to initiate high rates of mitochondrial respiration. Although ATPases figure prominently in the mechanisms of heat production, ATP hydrolysis itself is only responsible for about one third of the heat produced, the majority being released during mitochondrial substrate oxidation 2. Redox is likely balanced by the malate-aspartate shuttle in all systems with the potential for redox balance via the t~-glycerophosphate shuttle operating in swordfish brain heater and tuna red muscle but not in the elasmobranch. Ketone bodies and amino acids are the major oxidative fuels of the endothermic red muscle of the lamnid sharks. Lipid, carbohydrate and amino acids are possible energy sources in the endothermic tissues of the teleosts. Some regulation presumably occurs when more than one substrate is available but these processes have not been investigated in any fish thermogenic tissues. Even though the current evidence suggests that mitochondria isolated from endothermic tissues of warm-bodied fishes are well coupled 2. the existence of a basal proton leak higher than found in nonendothermic tissues cannot be ruled out. Temperature may also influence proton permeability. In spite of the importance of temperature to the metabolism of these fishes there are very few studies of the effects of temperature on the metabolism of the relevant tissues. Future studies directed at the thermal sensitivity of enzymes and isolated mitochondria should be undertaken. Very little is known of the hormonal regulation of heat production. Block8 has suggested that catecholamines are involved in billfish heater organ and the role of calcium as a signal supports this view. It is also possible that thyroid hormone in involved in mediating heat production in these fish as occurs in mammals. Ballantyne and colleagues 5 have shown rapid stimulation of mitochondrial respiration in red muscle of fish after administration of triiodothyronine. The regulation of fish red muscle metabolism is poorly understood. Differences in apparent metabolic complexity exist between the more primitive elasmobranchs and the more advanced teleosts but the mechanisms of regulating even the simplest of these metabolic units have not been adequately addressed. The current evidence suggests that the mechanisms of metabolic regulation of the endothermic fishes do not differ from the nonendothermic counterparts. In spite of the difficulty in obtaining these largely pelagic fishes as experimental animals they will continue to be important models for the understanding of metabolism in fishes, because they push the limits of metabolism.

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Acknowledgements. I would like to acknowledge useful discussions with Mary Chamberlin, Tom Singer, Martin Gerrits and Chris Moyes. Studies from the author's laboratory are supported by the Natural Sciences and Engineering Research Council of Canada through research grants and by a University Research Fellowship.

1,7. References 1. Anderson, P.M. Ketone body and phosphoenolpyruvate formation by isolated hepatic mitochondria from Squalus acanthias (Spiny dogfish). J. Exp. Zool. 254: 144-154, 1990. 2. Ballantyne, J.S., M.E. Chamberlin and T.D. Singer. Oxidative metabolism in thermogenic tissues of the swordfish and mako shark. J. Exp. Zoo/. 261: 110-114. 1992. 3. Ballantyne, J.S. and T.W. Moon. The effects of urea, trimethylamine oxide and ionic strength on the oxidation of acyl carnitines by mitochondria isolated from the liver of the little skate Raja erinacea. J. Comp. Physiol. 156: 845--851, 1986. 4. Ballantyne, J.S., H.C~ Glemet, M.E. Chamberlin and T.D. Singer. Plasma nonesterified fatty acids of marine teleost and elasmobranch fishes. Mar. BioL, 116: 47-52, 1993. 5. Ballantyne, J.S., T.M. John, T.D. Singer and O.V. Oommen. Short-term effects of triiodothyronine on the bowfin, Amia calva (Holostei), and the lake char, Salvelinus namaycush (Teleostei). J. Exp. ZooL 261: 105-109, 1992. 6. Bertin, R. and R. Portet. Effect of ambient temperature on lipid metabolism in brown fat during perinatal period. Comp. Biochem. Physiol. 70B: 193-199, 1981. 7. Block, B.A. Billfish brain and eye heater: a new look at nonshivering heat production. NIPS 2: 208-212, 1987. 8. Block, B.A. Endothermy in fish: thermogenesis, ecology and evolution. In: Biochemistry and Molecular Biology of Fishes, Vol. 1, edited by P.W. Hochachka and T.P Mommsen, Amsterdam, Elsevier Science Publishers, pp. 269-311, 1991. 9. Block, B.A. and EG. CareT. Warm brain and eye temperatures in sharks. J. Comp. PhysioL 156B: 229-236, 1985. 10. Block, B.A. and C. Franzini.Armstrong. The structure of the membrane systems in a novel muscle cell modified for heat production. J. Cell Biol. 107: 1099-1112, 1988. 11. Bone, Q. III. Myotomal muscle fiber types in Scomber and Katsuwonus. In: The Physiological Ecology of Tunas, edited by G.D. Sharp and A.E. Dizon, New York, Academic Press, pp. 183-205, 1978. 12. Brand, M.D., P. Couture, P.L. Else, K.W. Withers and A.J. Hulbert. Evolution of energy metabolism. Proton permeability of the inner membrane of liver mitochondria is greater in a mammal than in a reptile. Biochem. J. 275:81-86, 1991. 13. Brand, M.D., D. Steverding, B. Kadenbach, P.M. Stevenson and R.P. Hafner. The mechanism of the increase in mitochondrial proton permeability induced by thyroid hormones. E ~ J. Biochem. 206: 775-781, 1992. 14. Brett, J.R. and J.M. Blackburn. Metabolic rate and energy expenditure of the spiny dogfish, Squalus acanthias. J. Fish. Res. Bd. Can. 35: 816--821, 1978. 15. Bukowiecki, L.J. and O. Lindberg. Control of sn-glycerol 3-phosphate oxidation in brown adipose tissue mitochondria by calcium and acyl-CoA. Biochim. Biophys. Acta 348:115-125, 1974. 16. Carey, EG., J.W. Kanwisher and E.D. Stevens. Bluefin tuna warm their viscera during digestion. J. Exp. BIOL 109: 1-20, 1984. 17. Carey, EG., J.M. Teal and J.W. Kanwisher. The visceral temperature of mackerel sharks. PhysioL ZooL 54: 334--344, 1981. 18. Chamberlin, M.E., H.C~ Glemet and J.S. Ballantyne. Glutamine metabolism in a holostean fish (Amia calva) and a teleost (Salvelinus namaycush).Am. J. Physiol. 260: R159-R166, 1991. 19. Chamberlin, M.E. and Ballantyne, J.S. Glutamine metabolism in elasmobranch and agnathan muscle. J. Exp. Zool. 264: 267-272, 1992. 20. Clausen, T., C. Van Hardeveld and M.E. Everts. Significance of cation transport in control of energy metabolism and thermogenesis. Physiol. Rev. 71: 733-774, 1991. 21. Cooney, G., A. Mitchelson, P. Newsholme, M. Simpson and E.A. Newsholme. Activities of some key enzymes of carbohydrate, ketone body, adenosine and glutamine metabolism of the liver and brown and white adipose tissues of the rat. Biochem. Biophys. Res. Commun. 138: 687--692, 1986.

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22. Fellows, EC.I., EJ.R. Hird, R.M. McLean and T.I. Walker. A survey of the non-esterified fatty acids and binding proteins in the plasma of selected animals. Comp. Biochent Physiol. 67B: 593-597, 1980. 23. George, J.C. and E.D. Stevens. Fine structure and metabolic adaptation of red and white muscle in tuna. Env. Biol. Fishes 3: 185-191, 1978. 24. Goldstein, L. and J.M. Boylan. Renal mitochondrial glutamine transport and metabolism: studies with a rapid mixing rapid fltration technique.Am. J. Physiol. 234: F514-F521, 1978. 25. Graham, J.B., H. Dewar, N.C. Lai, W.R. Lowell and S.M. Arce. Aspects of shark swimming performance determined using a large water tunnel. J. Exp. Biol. 151: 175-192, 1990. 26. Guppy, M. and Hochachka, P.W. II. Skipjack tuna white muscle: a blueprint for the integration of aerobic and anaerobic carbohydrate metabolism. In: The Physiological Ecology of Tunas, edited by G.D. Sharp and A.E. Dizon, New York. Academic Press, pp. 175-181, 1978. 27. Guppy, M. and P.W. Hochachka. Metabolic sources of heat and power in tuna muscles. II. Enzyme and metabolite profiles. J. Exp. Biol. 82: 303-320, 1979. 28. Hansford, R.G. Relationship between mitochondriai calcium transport and control of energy metabolism. Rev. Physiol. Biochem. Pharmacol. 102: 1-72, 1985. 29. Hansford, R.G. and J.B. Chappell. The effect of cae+on the oxidation of glycerol phosphate by blowfly flight muscle mitochondria. 8iochem. Biophys. Res. Commun. 27: 686-692, 1967. 30. Hochachka, P.W., Hulbert, W.C. and Guppy, M.I. The tuna power plant and furnace. In: The Physiological Ecology of Tunas, edited by G.D. Sharp and A.E. Dizon, New York, Academic Press, pp. 153-174, 1978. 31. Hulbert, W.C., M. Guppy, B. Murphy and P.W. Hochachka. Metabolic sources of heat and power in tuna muscles. I. Muscle fine structure. J. Exp. Biol. 82: 289-301, 1979. 32. Hundal, H.S., M.J. Rennie and P.W. Watt. Characteristics of L-glutamine transport in perfused rat-skeletal muscle. J. Physiol. 393: 283-305. 1987. 33. Johnston, I.A. and R.W. Brill. Thermal dependence of contractile properties of single skinned muscle fibres from Antarctic and various warm water marine fishes including skipjack tuna (Katsuwonus pelamis) and kawakawa (Euthynnus aflfinis). J. Comp. Physiol. 155B: 63-70, 1984. 34. Johnston, I.A. and T.W. Moon. Fine structure and metabolism of multiply innervated fast muscle fibres in teleost fish. Cell Tissue Res. 219: 93-109, 1981. 34a. LeBlanc, P.J. and J.S. Ballantyne./]-Hydroxybutyrate dehydrogehase in teleost fish. J. EXp. Zool. 267: 356-358, 1993. 35. Lemieux, G., C. Pichette, P. Vinay and A. Gougoux. Cellular mechanisms of the antiammoniagenic effect of ketone bodies in the dog. Am. J. Physiol. 239: F420-F426. 1980. 36. Mathieu-Costello, O., P.J. Agey, R.B. Logemann, R.W. Brill and P.W. Hochachka. Capillary-fiber geometrical relationships in tuna red muscle. Can. J. Zool. 70: 1218-1229. 1992. 37. McCormack, J.G. and R.M. Denton. A comparative study of the regulation by Ca 2+ of the activities of the 2-oxoglutarate dehydrogenase complex and NAD + isocitrate dehydrogenase from a variety of sources. Biochem. J. 196: 619-624, 1981. 38. Mommsen, TP. and TW. Moon. The metabolic potential of hepatocytes and kidney tissue in the little skate, Raja erinacea. J. EXp. Zool. 244: 1-8, 1987. 39. Moon, TW. and TP. Mommsen. Enzymes of intermediary metabolism in tissues of the little skate, Raja erinacea. J. Exp. Zool. 244: 9-15, 1987. 40. Moyes, C.D., L.T Buck and P.W. Hochachka. Mitochondrial and peroxisomal fatty acid oxidation in elasmobranchs. Am. J. Physiol. 258: R756-R762, 1990. 41. Moyes, C.D., L.T Buck, P.W. Hochachka and R.K. Suarez. Oxidative properties of carp red and white muscle. J. EXp. Biol. 143: 321-331, 1989. 42. Moyes, C.D., O.A. Mathieu-Costello, R.W. Brill and P.W. Hochachka. Mitochondrial metabolism of cardiac and skeletal muscles from a fast (Katsuwonus pelamis) and a slow (Cyprinus carpio) fish. Can. J. Zool. 70: 1246-1253, 1992. 43. Moyes, C.D., T.W. Moon and J.S. Ballantyne. Oxidation of amino acids, Krebs cycle intermediates, lipid and ketone bodies by mitochondria from the liver of Raja erinacea. J. Exp. Zool. 237: 119-128, 1986. 44. Moyes, C.D., R.K. Suarez, EW. Hochachka and J.S. Ballantyne. A comparison of fuel preferences of mitochondria from vertebrates and invertebrates. Can. J. Zool. 68: 1337-1349, 1990. 45. Nicholls, D.G. and R.M. Locke. Thermogenic mechanisms in brown fat. Physiol. Rev. 64: 1-64, 1984. 46. Rayner, M.D. and M.J. Keenan. Role of red and white muscles in the swimming of the skipjack tuna. Nature 214: 392-393, 1967.

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47. Singer, T.D. and V.G. Mahadevappa and J.S. Ballantyne. Aspects of the energy metabolism in the lake sturgeon, Acipenser fluvescens: with special emphasis on lipid and ketone body metabolism. Can. J. Fish. Aquat. Sci. 47: 873-881, 1990. 48. Singer, T.D. and J.S. Ballantyne. Metabolic organization of a primitive fish, the bowfin (Amia calva). Can. J. Fish. Aquat. ScL 48: 611-618, 1991. 49. Tullis, A., B.A. Block and B.D. Sidell. Activities of key metabolic enzymes in the heater organs of scombroid fishes. J. Exp. BioL 161: 383-403, 1991. 50. Weber, J.-M., R.W. Brill and P.W. Hochachka. Mammalian metabolic flux rates in a teleost: lactate and glucose turnover in tuna. Am. J. PhysioL 250: R452-R458, 1986. 51. Wolf, N.G., P.R. Swift and EG. Carey. Swimming muscle helps warm the brain of lamnid sharks. J. Comp. Physiol. 157B: 709-715, 1988. 52. Zammit, V.A. and E.A. Newsholme. Activities of enzymes of fat and ketone body metabolism and effects of starvation on blood concentrations of glucose and fat fuels in teleost and elasmobranch fish. Biochem. J. 184: 313-322, 1979.

Hochachka and Mommsen (eds.), Biochemistry and molecular biology of fishes, vol. 4 9 1995 Elsevier Science B.V. All rights reserved. CHAPTER

11

Electric organs: structure, physiology, hormone-sensitivity, and biochemistry HAROLD H. ZAZON Department of Zoology, Patterson Laboratory, The University of Texas, Austin, TX 78712, U.S.A.

I. Introduction II. Distribution III. Structureand neural control IV. EOD variation and species communication V. Functionalorganization of the EO in weaklyelectric pulse-type fish VI. Functionalorganization of the EO in weaklyelectric wave-typefish VII. Sex differences in and hormonal modulation of the EOD VIII. Biochemistryof electric organs: acetylcholine receptor and related molecules IX. Biochemistryof electric organs: muscle-specific proteins X. Biochemistryof electric organs: intermediate filaments Xl. Proteinsorting and targeting XII. Futuredirections XIII. References

I. Introduction Electric organs (EOs) are a uniquely piscine adaptation. Strongly electric fish, whose discharges are used to stun prey or attackers, were familiar to the ancient Mediterranean civilizations and native tribes of South America and Africa. The EOs of these fish have played key roles in the neurosciences from demonstrating to eighteenth century scientists that animals could generate electricity to being the tissue of choice for recent biochemical identification and molecular cloning of the acetylcholine receptor, the sodium channel, and other important molecules 59 .66 - 68 . Beside the more familiar strongly electric eel, Electrophorus, or the Torpedo ray, a far greater number of fish possess weak EOs the discharges of which cannot be sensed by us without the aid of electronic amplification. These fish, notably the gymnotiform and rnormyriform teleosts, are nocturnally active and frequently live in murky habitats. They use their EOs, often in preference to visual cues 18, to locate objects around them and to communicate with each other (for reviews on electroreception see refs. 17, 36 and 42). In this chapter, I review the electric organs of both strongly and weakly electric fish in terms of their distribution, functional organization, hormone-sensitivity, and biochemistry. The reader is encouraged to consult previous reviews on the electric organ 5,9,1~

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Fig. 1. Summary diagram of known groups of electric fish. A line drawing of a representative fish from each group, the structure of its electrocytes, and a sample EOD are given. The dots on the electrocytes indicate sites of electromotoneuron termination. The s or w near each fish indicates whether the electric organ generates a strong or weak discharge. The EODs are shown with positive polarity up. From ref. 5.

II. Distribution EOs are found in seven unrelated groups of extant fishes suggesting that they have probably evolved at least that many times. (Fig. 1). In every group but one the EO is embryonically derived from a muscle precursor 2325444647 .... . EOs develop from different muscles in the different taxa, however, strengthening the argument for their multiple origins. The fact that EOs are all myogenic argues less for a common ancestor than for the idea that muscle tissue is easily converted into EO.

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Among the elasmobranchs strong EOs are found in two families of rays (Torpedinoidae, Rajidae) which are not closely related. In addition, the muscles that give rise to the EO and the source of the motor innervation differ between these two families further suggesting an independent evolution of the EO within elasmobranchs 2,t~ The strong EO of the Torpenoidae, best studied in the genus Torpedo, is apparently defensive. The weaker EO of the Rajidae is too weak to be defensive but may function in social communication 14. A strong EO is found in the catfish family Malapteruridae. The EO of this species derives from muscles of the body wall and is a thin jacket of cells that entirely surrounds the body from head to tail fin44. Weak EOs have evolved independently in the members of the genus Synodontis (the upside-down catfish). The EO of Synodontis is the most muscle-like of all EOs so far described. As well as possessing sarcomeres, the electrocytes have a distinct but poorly organized T-tubule system, and retain origination and insertion points on bone and swim bladder 31. While not well-studied, the EOD of this group is thought to function in social communication and electrolocation 31. The independent origin of the EOs in these 2 groups of catfish is further suggested by their innervation: the EO of Malapterurus is innervated by 2 giant motoneurons one on each side of the rostral spinal cord, while a pool of smaller medullary motoneurons innervate the EO of the synodontids 15,31. Well-known to neuroethologists, weak EOs are found in the two independently derived lineages of electric fish (Gymnotiformes, Mormyriformes). Many elegant behavioral studies have elucidated the role of the EO in social communication and electrolocation in these fish 17,35,36. One member of the gymnotiforms, the electric eel (Electrophorus electricus), possess a strong electric organ as well. The EO of one exceptional family of gymnotiforms, the Apteronotidae, is comprised of the axons of the electromotoneurons in adult fish 1~ (see section VI). However, even these fish are the exception that proves the rule as they possess a muscle-derived EO as larvae but lose it as they mature 47. In fact, all weakly electric gymnotiforms and mormyriforms may possess a myogenic larval EO for the first 2 months of life after which time it degenerates as the adult EO develops46,47,8~ Last, the perciform teleosts, the Uranoscopidae, have evolved an EO from the oculomotor muscles. The EO of these fish is innervated by a large cluster of electrotonically coupled motoneurons, formerly of the oculomotor nucleus, which have been co-opted for a new function 54. The uranoscopid EO is not used for communication or electrolocation since they have no electrosensory system. They are believed to discharge the EO into their mouth to aid in immobilizing captured but struggling prey.

IlL Structure and neural control Generally EOs are composed of columns of large cells oriented along the same axis and ensheathed in connective tissue. This arrangement channels the flow of current along the axis of the organ and out into the water. Myogenically derived

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eleetrocytes are flattened and wafer-like, long and tubular, or barrel-shaped (Fig. 1). The electroeytes in a column are usually innervated on the same face, so that when that face is depolarized by the release of neurotransmitter, current flows into the innervated face and out of the uninnervated face of each eleetroeyte along the column 8. The EO in all species is innervated by a distinct pool of motoneurons (eleetromotoneurons) which, like somatomotoneurons, are nicotinic eholinergie neurons. These may be spinal or cranial motoneurons depending on the group. A welldefined medullary pacemaker nucleus controls the electromotoneurons in both gymnotiform and mormyriform electric fish 13. The pool of pacemaker neurons are electrotonically coupled to each other and to the output neurons of the pacemaker nucleus which typically make extensive diverging contacts with a large number of motoneurons. This tight coupling and high degree of divergence optimizes synchronous firing of the electrocytes. Pacemaker cells have been identified in some other groups as well and they may well be a general feature of eleetromotor systems to insure coordinate activation of the eleetromotoneurons and electrocytes.

IV. EOD variation and species communication The EODs of gymnotiform and mormyriform teleosts are species-specific 41'42'49. In addition, there may be sex differences, age differences (larva v e r s u s adult), status differences (dominant v e r s u s submissive individuals), and individual differences in the waveform of the d i s c h a r g e 22'32'37'39'58'80'81 (Fig. 2). While only a few studies have investigated the potential behavioral relevance of this variation, the ones that have suggest that they are discriminable to conspecifics and may convey behaviorally relevant information 39'49'5~ EODs can be classified as either pulse-type, in which the pulses are generated irregularly and pulse duration is short compared to the interval between pulses, or wave-type, in which the EO is driven with extreme regularity and where the pulse duration is about equal to the interval between pulses (Fig. 2). Pulse- and wave-type EODs are found in both orders of weakly electric fish.

A

13

w Fig. 2. The EODs of a male and female (A) pulse fish (Sternopygusmacrurus). From ref. 84.

(Hypopomus brevirostris) and (B) wave

fish

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I4. Functional organization of the EO in weakly electric pulse-type fish Pulse-type EODs differ between species in the number and polarity of their phases and by differences in the amplitude and duration of each phase. Sex differences within a species depend on variations in the duration and amplitudes of each phase (Fig. 2). As will be described below, the number of phases and their polarities are determined by fixed parameters as the site of innervation, the number of excitable faces and, in the mormyrids, the geometry of the electrocyte. The duration and amplitude of each phase, however, depend on more subtle properties like the duration and amplitude of the action potentials generated by each face. While the generation of a mono- or di-phasic EOD is essentially identical in gymnotid and mormyrid fish, the mechanisms by which EODs with three or four phases are produced is achieved differently. Gymnotiform electrocytes are structurally simple and generate only mono- or biphasic pulses. The multiphasic gymnotiform EOD is built from the discharges of a number of functionally distinct electric organs that fire with phase delays. Diversity in the multiphasic mormyrid EOD, on the other hand, comes from the synchronous activation of a single population of electrocytes each of which possesses complex morphological and physiological membrane specializations. The simplest EOD is a monophasic pulse as given by Electrophorus. The Electrophorus electrocyte is a simple rectangular cell with a posterior face that is innervated and generates action potentials, and a highly invaginated anterior face that is electrically inexcitable 45,64. Upon being depolarized by synpatic input the posterior face fires an action potential during which positive current (Na + ions) enters. The head-positive monophasic EOD pulse is generated when this current flows headward along each column of electrocytes during the posterior membrane's spike. This occurs in both the main electric organ, which generates the eel's high-voltage pulse, as well as in Sach's organ, which generates the low voltage pulse. Intracellular recordings of electrocytes from other gymnotiforms and mormyrids which generate monophasic EOD pulses have shown this to be generally true of them all. Both faces of the electrocytes are excitable in those species that produce a diphasic discharge. First, the posterior face fires an action potential during which current flows toward the head. The flow of current through the anterior membrane depolarizes it causing it to spike and current then flows tailward. The alternate flow of current headward and tailward gives theEOD its diphasic shape. For an electrocyte with two excitable faces to generate a diphasic discharge it is imperative that the two faces do not spike simultaneously or the currents flowing in opposite directions will cancel out. It seems to be a general property of such electrocytes that the innervated membrane has a lower threshold than the uninnervated side (such that the uninnervated side is not brought to threshold by the synaptic input) allowing the innervated face to fire first. The two faces may differ in other excitable properties as well. In some instances the two faces produce spikes of different duration; if there is an asymmetry in spike duration it is the face which fires second and which does not have to worry about colliding with a spike, that usually produces the longer duration spike.

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Some gymnotiforms possess so-called accessory organs which are physically distinct from the main organ. The EOD of Steatogenys elegans, for example, is diphasir over most of its body but triphasir with an early negative wave, over its head. Bennett 9,1~ has shown that the small head-negative component is generated by a small EO running along the lower jaw (the submental organ). The electrocytes of this organ are innervated on their anterior faces which are the only spiking face and thus produce monophasir head negative potentials. The discharge of the submental organ is in phase with the rest of the EOD but is activated slightly earlier making the EOD triphasir Similar accessory organs have been described in other gymnotiform species 9,1~ The most complex gymnotiform EOD thus far studied is that of Uymnoms carapo which has four distinct components ~1,19,~,s7,78. In this species, the EO runs from the pectoral girdle to the tip of the tail; it appears as a continuous organ but it includes three functionally distinct regions. The most rostral portion of the organ contains electrocytes which are innervated on both anterior and posterior faces. The posterior face generates an action potential but the anterior face gives only a synaptir potential. The electrocytes in the midbody region are also innervated on both faces. Here, both faces are capable of spiking, but the anterior face has a lower threshold and fires first. The largest part of the EO, that found in the tail, follows the typical gymnotiform pattern of innervation only on the posterior face. Both faces generate action potentials and the posterior face has the lower threshold. When the EO is normally activated, the anterior face of the most rostral set of electrocytes produces an EPSP first, which generates a small slow head negative component. Then, the anterior faces of the midbody electrocytes spike causing a larger head negative wave. The posterior faces of the midbody electrocytes fire at about the same time as the posterior faces of the electrocytes in the tail and these generate a large head positive component, the most pronounced component of the EOD. Finally, the anterior faces of the electrocytes in the tail fire generating the last head negative component. The electrocytes of mormyrids, on the other hand, are flattened, thin and bear prominent stalks (Fig. 1). The organization of the stalks is species-dependent and is critical to the generation of an EOD with the proper number of phases 6'12. The simplest electrocyte bears a single stalk that fuses with one face (usually the posterior) of the electrocyte. The action potential is generated in the stalk and propagated into the posterior membrane, which causes headward current flow, followed by a depolarization of the anterior membrane and tailward current flow. This results in a diphasir spike according to the principles discussed above for the gymnotiform electrocyte. In other species, a complex network of stalks may penetrate the main body of the electrocyte and fuse with it on the other side. For example, the stalk may appear on the anterior side of the electrocyte where it is innervated, it may then penetrate the main body of the electrocyte where it fuses with the posterior face. In these electrocytes a spike is generated in the stalk and proceeds along the stalk in a tailward direction resulting in a head-negative EOD phase. The spike then propagates into the posterior membrane of the electrocyte depolarizing it and

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causing it to generate an action potential followed by the generation of an action potential from the anterior face. These events result in a triphasic action potential with an initial head-negative wave. Perhaps the most impressive electrocytes are those of the mormyrid Stomatorhinus corneti. In this species the stalk penetrates the body of the electrocyte from the posterior side, emerges from the anterior face and repenetrates it, finally emerging again from and fusing with the posterior face. The movement of currents alternately in a headward, then tailward direction in the stalks followed by the usual headward and tailward movement when the posterior and anterior membranes are sequentially depolarized results in an EOD with 4 phases.

VI. Functional organization of the EO in weakly electric wave-type fish Wave-type EODs differ between species and individuals within a species by their frequency. Stemopygus, the species with the lowest EOD frequency discharges at 50-200 Hz while some of the apteronotids discharge in excess of 1 kHz. The sole wave-type mormyriform fish, Gymnarchus niloticus, discharges around 300 Hz. The resting EOD frequency of wave-type fish is extremely regular varying only by a few tenths of a percent 16. Yet, EOD frequency may be modulated by up to 25% during social interactions. The production of the EOD in wave-type fish is relatively simple and is similar in both gymnotids and mormyrids. It is easiest to consider a wave-type EOD as a pulse-type EOD that is extremely regular (Figs. 1 and 2). If the durations of the EO pulses are about the same as those of the inter-pulse interval, the EOD will be periodic and nearly sinusoidal. In the wave-type fish with myogenic EOs (Eigenmannia, Stemopygus, Gymnarchus) the EO generates a monophasic pulse as occurs in Electrophorus. The EOD frequency of a wave-type EOD is determined by the firing frequency of the pacemaker nucleus. However, in order for the waveform to approximate a sinewave, the EO pulse duration must also vary. So, for example, the EOD frequency of Stemopygus sp. varies from 50 to 200 Hz and EO pulse duration varies from 4 to 14 ms (Fig. 2). These two parameters covary so that the fish with the lowest EOD frequency has the longest pulse duration 62,63. These two independent parameters may be manipulated by hormonal modulation (see below). The Apteronotidae, the sole group of electric fish without a myogenic EO, also generate a wave-type discharge. Some members of the Apteronotidae generate EOD frequencies in excess of 1 kHz. The electromotor system is electrotonically coupled from the PMN to the electromotoneurons 69. It is thought that their myogenic EO is lost in order to eliminate the one obligatory chemical synapse in the electromotor circuitry that might fail at these high frequencies. The unique EO of the Apteronotidae is composed of bundles of electromotoneuron axons which project forward within the EO and then make a hairpin turn posteriorly; they end blindly within the bundle since their muscle-derived target cells have degenerated (Fig. 1). Each axon possess three to half a dozen specialized

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nodes of Ranvier just before the hairpin turn and at the blind end. The specialized node is 5-20/zm in length (which is one or two orders of magnitude longer than a normal node of Ranvier) and has no Na+channels 69,79. The absence of excitable channels at this node near the hairpin turn prevents current flowing into the axon from the previous node from depolarizing this patch of membrane. Instead, current flows outward, eapaeitatively coupled to the extraeellular space through the extensive membrane area. The summation of the outward currents from large numbers of simultaneously active nodes generates the first phase of the EOD. The movement of the action potential into the blind end of the axon and of current out of the specialized nodes into extraeellular space generates the second phase of the EOD 79. It would be interesting to determine the mechanisms by which Na+ehannels are selectively targeted to most nodes of Ranvier, but selectively eliminated from the large specialized nodes.

VII. Sex differences in and hormonal modulation of the EOD Sex differences in the EOD waveform have been reported for a large enough sample of weakly electric fish, that it may turn out to be the rule. These sex differences are under the control of gonadal steroids: they disappear after gonadeetomy or in captivity under conditions when sex steroid titers fall, and they are induced by treatment with sex steroids or gonadotropin 4,7'51'52'62'63'sS-sT. In both gymnotiform and mormyriform pulse fish the duration of some or all components of the pulse are longer in males than in females and juveniles captured in the field during breeding season 3s,51. The mormyrids Brienomyrus brachyistius and Gnathonemuspetersii have been particularly well studied. While the EOD of Brienomyrus is triphasie and that of Gnathonemus has four phases 3,7,51. In both species the major components of the EOD pulses of females and juveniles are broadened after implantation with a variety of androgens, including the non-aromatizable androgen 5-a-dihydrotestosterone (DHT). On the other hand implantation with an estrogen, 17/5-estradiol, has little effect. Similar results have been obtained with two species of the pulse gymnotiform Hypopomus. In both eases the EOD pulse is diphasie and the sex difference is primarily in the second phase 29'4~ (Fig. 2). As in mormyrids, the EOD pulse of Hypopomus occidentalis can be influenced with DHT treatment but not by estrogen 29. Along with the sex difference in the EOD pulse, the EO itself usually shows morphological sex differences in pulse fishs,6. In both groups the eleetroeytes of males are larger than those of females and they may have additional membrane infoldings. In the hypopomids tail size is also sexually dimorphie with males having longer or thicker tails 29,4~ In both groups of pulse fish androgen treatment of females and juveniles increases the size of the electroeytes. Presumably as a consequence of increases in eleetroeyte size, tail size also increases in the Hypopomids. It is not obvious whether this is strictly a byproduct of mechanisms underlying the production of the EOD or whether sex differences in tail morphology may provide additional visual cues.

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A

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,

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Ooy Fig. 3. Changes in EOD pulse duration (A) and frequency (B) in Stemopygus implanted with dihydrotestosterone (solid lines) or empty control (dashed lines) capsules. Note the concurrent lowering of EOD frequency and broadening of EOD pulse. From ref. 63.

Sex differences in EOD frequency occur in three species of gymnotiform wave fish commonly studied. In Stemopygus and Eigenmannia sexually mature males have lower EOD frequencies than females 3~176 There is a negative correlation between androgen level and EOD frequency in gonadally recrudescing Stemopygus in the field and gonadotropin hormone-treated fish in the laboratory 85,s7. Additionally, a variety of androgens lower EOD frequency and broaden the EO pulse in female and juvenile Stemopygus (Fig. 3) and 17fl-estradiol is reported to raise EOD frequency 6~ The apteronotids show interesting species differences in sexual dimorphism of EOD frequency. The EOD frequencies of sexually mature female Apteronotus leptorhynchus are lower than those of males 61. When A. leptorhynchus of either sex are treated with testosterone, their EOD frequency is lowered and this effect is antagonized by inhibitors of the aromatase enzyme (M. Zucker and H. Zakon, unpublished results). This result suggests that testosterone acts via its conversion to an estrogen. In support of this hypothesis, EOD frequency is lowered in this species by treatment with 17~l-estradiol 61 (M. Zucker and H. Zakon, unpublished results). On the other hand, the congeneric species A. albifrons shows a sexual dimorphism of EOD frequency that is opposite and more similar to that seen in the other gymnotid wave fish: male EOD frequency is lower than that of females (M. Zucker and H. Zakon, unpublished results). The detailed endocrinology is still being studied in this species.

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[ 200 nA

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300 ~ t

Fig. 4. Variation is sodium current kinetics of electrocytes from unsexed juvenile Sternopygus (EOD frequency of each fish is given next to the voltage-clamp traces). Each family of curves was generated by voltage-clamping the electrocytes from -50 to 0 mV (unpublished data courtesy of M.B. Ferrari, and from ref. 84).

A knowledge of the action of steroids on the ion currents of the EO may prove useful in understanding how steroids act on ion currents in central neurons or other excitable cellss4. While intracellular recordings before and after androgen treatment have been made from the EO of fish from 3 genera (Brienomyrus, Sternopygus, Hypopomus), the identification of the ion currents underlying the action potential has only been done in Sternopygus. In this species, an inward rectifying K +current sets the resting potential, the rising phase of the action potential depends entirely on a Na+current, and the falling phase of the spike depends primarily on the inactivation rate of the Na+current and, in some cells, a delayed rectifying K + current 24. Interestingly, the inactivation kinetics of the Na +current varies among electrocytes of fish with high versus low EOD frequencies. In other words, in fish with a low frequency EOD and broad action potentials, the Na+current inactivates slowly whereas in those fish that generate higher frequency EODs with short action potentials (Fig. 4). Long-term (3 week) androgen treatment slows down the inactivation rate of the Na+current, which apparently is the mechanism by which the EOD pulse becomes broader after androgen treatment (Ferrari and Zakon, in preparation).

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VIII. Biochemistry of electric organs: acetylcholine receptor and related molecules The rich innervation and large size of the EOs of Torpedo and Electrophorus have made them critical in providing large amounts of purified synaptic membranes for efforts to elucidate the structure of molecules involved in cholinergic synaptic transmission55, 59,67, 68, 72. Based on molecular weight and antigenic profile, the acetylcholine receptor (AChR) of the Torpedo EO has been shown to comprise 4 subunits in the same stoichiometry as that of mammalian muscle (approximately 40 (c~), 50 (/I), 57 (y), 65 (8) kDa in a 2" 1" 1" 1 ratio) 67,68,72 (Fig. 5). The AChR of Electrophorus, while less well studied, has also been isolated 55. When extracted under conditions that normally isolate all subunits in Torpedoor mammalian muscle, only 3 subunits (c~, fl, y) appear. Under altered conditions, however, a 8 subunit is recovered. While this suggests subtle differences in the organization of the AChR between these species, the similarity of subunit composition, and antigenicity across Torpedo,Electrophorus and mammals highlights the highly conserved nature of the AChR. A distinct AChR subunit type (E) is expressed in mature mammalian muscle (the 8 subunit is only expressed early in muscle development and in the extrajunctional region of mature mammalian muscle after denervation) 34. Such a subunit does not seem to occur in Torpedo EO nor is there any indication of denervation-induced

Fig. 5. Autoradiograms of antibodies against various proteins of Torpedo EO. The name of the antibodies are given below each lane. The leftmost lane contains antibodies against subunits of the ACh receptor, followed by lanes with antibodies against the 43 and 53 kDa proteins, which are associated with the ACh receptor, and a 90 kDa protein, which is likely to be the Na +/K+-ATPase. From ref. 26.

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changes in the TorpedoEO AChR 82'83. However, since the developmental dynamics of AChR subunits in Torpedomuscle are not known it is not yet clear whether these are tissue or species differences. In addition to the AChR, a number of other proteins are enriched in the postsynaptie membrane fraction of Torpedo EO and have also been found at the mammalian neuromuscular junction (Fig. 5). The most prominent of these is a 43 kDa molecule which is found in approximately equimolar concentrations as the AChR and is co-localized with it in the membrane 26,27,s2,a3. Attention was initially drawn to this protein when it was shown that after it, and other, proteins were extracted from Torpedo membranes, the AChRs no longer remained clustered but diffused throughout the membrane. It has since been shown that this molecule induces clustering of the AChR when messenger RNA for it and the AChR are injected into Xenopus oocytes 28. Interestingly, the 43 kDa protein is regulated differently during development than the AChR: both molecules are markedly increased in abundance following innervation of immature electroc~es, but while AChR mRNA is up-regulated by 30-fold following denervation of adult organs, the 43 kDa protein is increased by only 2 to 3 fold 27. Torpedo EO contains a number of other proteins most likely important to regulation of the synaptic membrane as, for example, dystrophin and dystrophin-related protein 2~ Both of these molecules, which are localized to the synaptic membrane are also found in mammalian muscle (Fig. 6). While the distribution of dystrophin at the mammalian neuromuscular junction and within Torpedo EO is quite similar, the highly similar protein, dystrophin-related protein is found in high levels in both mammalian and Torpedo neuromuscular junctions, but only at low density in the synaptic membrane of the electric organ 2~ The lack of dystrophin has been implicated as causal in Duchenne muscular dystrophy although it is unclear what role it plays in normal muscle physiology, or how muscle pathology results from its absence. At the present the function of dystrophin-related protein is also unclear.

IX. Biochemistry of electric organs: muscle-specific proteins Sarcomeres often form in the developing EO. While well-formed sarcomeres are maintained in the mature EOs of some species, they disappear in others. It would be informative to study the molecular processes underlying differentiation of the electrocytes in a range of species to determine where along the pathway of differentiation muscle development is halted or compromised. An understanding of the molecular events controlling the suppression of the muscle phenotype in electrocytes could be helpful in discerning the causes of muscle wasting in various human dystrophies. It is known, for example, that two of the muscle regulatory genes (MyoD and my5) are expressed in Torpedoelectrocytes 65. Mature electrocytes of the gymnotiform Sternopygushave no sarcomeres and do not express proteins like myosin and tropomyosin 7~ On the other hand, during regeneration of the electric organ the developing electrocytes express these protein for about a week and then these proteins gradually becomes undetectable (J.M.

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Fig. 6. Localization of dystrophin to the Torpedo electrocyte. A cryosectioned electrocyte is doublelabeled with a-bungarotoxin to localize the ACh receptor (A) and an anti-dystrophin antibody (A'). Using immunoelectron microscopy, dystrophin can be localized just below the plasma membrane on the innervated surface of the electrocyte. From ref. 74.

Patterson and H.H. Zakon, unpublished results). In addition, after the terminal portions of the EO are amputated to induce regeneration, the intact electrocytes near the wound transiently express myosin. This last observation indicates that at least portions of the suppressed muscle program may be reactivated in a differentiated electrocyte. F-actin is observed in the EO of the gymnotiforms Stemopygus (J. Patterson and H. Zakon, unpublished results) and Electrophorus77 where it is in highest density at the innervated end of the electrocyte. Two isoforms of actin are expressed in mammalian muscle and these come from separate genes. One form is sarcomeric actin and the other form, cytoplasmic actin, participates in the subsynaptic cytoskeletal framework 33. Both forms are believed to exist in the Electrophorus electrocyte based upon 2 dimensional gel analysis 73. It would be interesting to know whether the sarcomeric actin is functional or merely a vestige of the electrocyte's myogenic lineage. F-actin is also abundant in electrocytes from early embryonic Torpedo but falls to low levels thereafter. This shift in abundance is paralleled by a disruption and disappearance of organized myofibrils 25. In fact, with the exception of some F-actin along the non-innervated face, most of the F-actin detected after homogenization of the adult EO may actually be from nerve terminals rather than electrocytes 4s,53,83. It is interesting that no F-actin is localized to the postsynaptic cytoplasmic surface of

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the neuro-electroeyte junction as has been reported for mammalian neuromuscular junctions 33. Since the distribution of F-action in Torpedo muscle is not known, it is not clear whether this reflects a difference between the postsynaptie membranes of muscles and eleetroeytes in this species or whether this is a species difference.

X. Biochemistry of electric organs: intermediate filaments A number of intermediate filament proteins have been observed in the EO. The cytoplasm of the Torpedo electrocyte contains a meshwork of intermediate filaments which have been identified as desmin 48,75. These filaments extend throughout the cytoplasm and beneath both innervated and uninnervated membranes. In this it differs from desmin in rat muscle which is concentrated at the synaptic membrane and, less so, at Z bands 75. The desmin from the EO and muscle of Torpedo are believed to be identical 7s. Desmin has been biochemically or immunocytochemically identified in the EO and muscle of Electrophorus, Stemopygus and Hypopomus pinnicaudatus 21 (J. Patterson and H. Zakon, in preparation). It is possible that EO and muscle express different isoforms of the protein as muscle and EO are differentially stained by a range of monoclonal antibodies against mammalian desmin (J. M. Patterson and H.H. Zakon, in preparation). Five isoforms of desmin have been identified in the Electrophorus EO, but since Electrophorus muscle has not yet been studied, it is not known whether any of them are unique to the electric organ 21. At this time it is not certain whether these electrophoretie variants represent the products of different genes, different splice products of a single gene, or the same protein with differing degrees of phosphorylation 21. Certainly, the fact that there is a single desmin gene in mammalian muscle 71 and that the isoforms from the Electrophorus EO have identical molecular weights and protease digestion patterns argues for the last alternative. Electrocytes are usually typified by the proteins they lack when compared to muscle (i.e. myosin). However, one recently identified keratin-like protein is found in EO, but not muscle, of Stemopygus7~ Both the EO and skin label intensely with an antibody to acidic keratins (AE1). Western blots indicate that this antibody recognizes a molecule of the same molecular weight in skin and EO (J.M. Patterson, personal communication). While the definitive identification of this molecule awaits its sequencing, these results suggest that it is a keratin. This result is intriguing since it is the first example of extensive production of a keratin in a cell from a myogenic lineage. It is not known how widely this keratin-like molecule is found among the gymnotiforms.

XI. Protein sorting and targeting One of the most striking aspects of electrocytes is the organization of their plasmalemmas into distinct faces. For example, all electrocytes possess innervated

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faces that are rich in molecules involved with cholinergic transmission (see above) and uninnervated faces laden with Na+/K+-ATPase 1,26. This cellular feature and the electrocyte's tractable size make them potentially fruitful cells in which to study how proteins are differentially sorted and inserted into membranes. Recent studies on the EO of Torpedo have discovered an asymmetry in the organization of microtubules within the electrocytes resulting in selective associations with the subsynaptic space 43. Furthermore, a particular guanosine triphosphatebinding protein (Rab6p) which is believed to be involved in exocytosis has been localized within the Golgi apparatus and, most abundantly, within post-Golgi vesicles that are localized beneath the synaptic membrane. These vesicles are no longer localized there if the microtubular network underlying the synaptic membrane is disrupted 43. These, and other, results suggest that there is a specific intracellular network composed of microtubules which associate with the intermediate filament proteins of the subsynaptic membrane and convey proteins specifically bound to this membrane. The question of subcellular localization of membrane constituents is just beginning to receive attention using the electrocytes as a model system. Beside the elegant work already done future questions include detailing how the Na+/ K+-ATPase molecules are selectively ferried to the non-innervated membrane. In addition, those electrocytes that generate action potentials from only a single face must have a mechanism whereby ion channel proteins are delivered to and inserted into only the active face. The process by which ion channels are sorted and targeted cannot be pursued in the EO of Torpedo since these cells do not generate action potentials. Instead, these questions can be addressed using the electrocytes of other spedes such as Electrophorus or Stemopygus.

XII. Future directions EOs have played important roles in understanding the mechanisms of synaptic activation and electrical excitability. I would like to emphasize three areas in which EOs can continue to make key contributions to the cellular and molecular organization of excitable cells in the future. First, the extensive variation in the excitability properties of electrocytes across, or even within, species allows one to study how membrane excitability is regulated and how proteins are targeted to a particular membrane. For example, how is the AChR with its entourage of other proteins selectively targeted to a particular membrane in those species with a single active face in the electrocyte? The dorsal row of electrocytes of Gymnotus is innervated on both faces but only the posterior face generates action potentials. What causes the AChR to be present at both faces, but Na +channels only to one? Second, how do steroid hormones modulate the ion currents of the EO? The kinetics of the Na+current is modulated after androgen treatment in Sternopygus. This is the first example of androgen action on any ion current and the first example of steroidal modulation of Na+current kinetics. Is the Na+current also modulated

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by androgens in the mormyrids which have independently evolved EOs? If so, are the cellular and molecular processes underlying these modifications similar in the 2 taxa? Last, the independent evolution of a myogenic EO in seven taxa offers a series of interesting natural experiments in the molecular processes by which muscle can selectively suppress parts of the muscle program while retaining others.

XIII. References 1. Ariyasu, R.G., T.J. Deerinck, S.R. Levinson and M.H. Ellisman. Distribution of (Na+/K +) ATl'ase and sodium channels in skeletal muscle and electroplax. J. Neurocytol. 16: 511-522, 1987. 2. Baron, V.D., L.S. Sokolova and N.A. Mikhailenko. Identification of spinal electromotoneurons in the ray Raja clavata (Rajidae). Neuroscience 48: 397--403, 1992. 3. Bass, A.H. Species differences in electric organs of mormyrids: substrates for species-typical electric organ discharge waveforms. J. Comp. Neurol. 244: 313-330, 1986. 4. Bass, A.H. A hormone-sensitive communication system in an electric fish. J. Neurobiol. 17: 131-156, 1986. 5. Bass, A.H. Electric Organs Revisited. Electroreception, New York, John Wiley and Sons, 1986. 6. Bass, A.H., J.-P. Denizot and M.A. Marchaterre. Ultrastructural features and hormone-dependent sex differences of mormyrid electric organs. J. Comp. NeuroL 254: 511-528, 1986. 7. Bass, A.H. and C.D. Hopkins. Hormonal control of sexual differentiation: changes in electric organ discharge waveform. Science 220: 971-974, 1983. 8. Bell, C.C., J. Bradbury and C.J. Russell. The electric organ of a mormyrid as a current and voltage source. J. Comp. PhysioL l l0A: 65-88, 1976. 9. Bennett, M.V.L. Modes of operation of electric organs.Ann. New York Acad. Sci. 54: 458-494, 1961. 10. Bennett, M.V.L. Electric Organs. Fish Physiology, New York, Academic Press, 1971. 11. Bennett, M.V.L. and H. Grundfest. Electrophysiology of electric organ in Gymnotus carapo. J. Gen. Physiol. 42: 1067-1104, 1959. 12. Bennett, M.V.L. and H. Grundfe~t. Studies on morphology and electrophysiology of electric organs. III. Electrophysiology in mormyrids. Bioelectrogenesis, London, Elsevier, 1961. 13. Bennett, M.V.L., G.D. Pappas, M. Gimenez and Y. Nakajima. Physiology and ultrastructure of electrotonic junctions. IV. Medullary electromotor nuclei in gymnotid fish. J. Neurophysiol. 30: 236300, 1967. 14. Bratton, B. and J. Ayers. Observations on the organ discharge of the skate species (Chondrichthyes, Rajidae) and its relationship to behaviour. Environ. Biol. Fish. 4: 241-254, 1987. 15. Braun, N., T. Schikorski and H. Zimmermann. Cytoplasmic segregation and cytoskeletal organization in the electric catfish giant electromotoneuron with special reference to the axon hillock region. Neuroscience 52: 745-756, 1993. 16. Bullock, T.H. Species differences in effect of electroreceptor input on electric organ pacemakers and other aspects of behavior in electric fish. Brain Behav. Evol. 2: 85-118, 1969. 17. Bullock, T.H. and W. Heiligenberg. Electroreception. Wiley Series in Neurobiology, New York, John Wiley and Sons, 1986. 18. Cain, P., W. Gerin and P. Moiler. Short range navigation of the weakly electric fish Gnathonemus petersii L. (Mormyridae, Teleostei) in novel and familiar environments. Ethology., in press. 19. Caputi, A., O. Macadar and O. Trujillo-Cen6z. Waveform generation of the electric organ discharge in Gymnotus carapo. III. Analysis of the fish body as an electric source. J. Comp. Physiol. 165A: 361-370, 1989. 20. Cartaud, A., M.A. Ludoscky, EM.S. Tom(~, H. Collin, E Stetzkowski-Marden, T.S. Khurana, L.M. Kunkel, M. Fardeau, J.P. Changeux and J. Cartaud. Localization of dystrophin and dystrophinrelated protein at the electromotor synapse and neuromuscular junction in Torpedo marmorata. Neuroscience 48: 995-1003, 1992. 21. Costa, M.L., V. Moura Neto and C. Chagas. Desmin heterogeneity in the main electric organ of Electrophorus electricus. Biochimie 70: 783-789, 1988. 22. Crawford, J.D. Individual and sex specificity in the electric organ discharges of breeding mormyrid fish (Pollimyrus isidori). J. F~. Biol. 164: 79-102, 1992.

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23. Dahlgren, U. The origin of the electricity tissues in fishes. Am. Nat. XLIV: 193-202, 1910. 24. Ferrari, M.B. and H.H. Zakon. Conductances contributing to the action potential of Sternopygus electrocytes. J. Comp. Physiol. 173A: 281-292, 1993. 25. Fox, G.O. and G.P. Richardson. The development morphology of Torpedo marmorata: electric organ-myogenic phase. J. Comp. Neurol. 185: 293-316, 1978. 26. Froehner, S.C. Peripheral proteins of postsynaptic membranes from Torpedo electric organ identified with monoclonal antibodies. J. Cell Biol. 99: 88-96, 1984. 27. Froehner, S.C. Expression of RNA transcripts for the postsynaptic 43 kDa protein in innervated and denervated rat skeletal muscle. FEBS Lett. 249: 229-233, 1989. 28. Froehner, S.C., C.W. Luetje, P.B. Scotland and J. Patrick. The postsynaptic 43K protein clusters muscle nicotinic acetylcholine receptors in Xenopus oocytes. Neuron 5: 403-410, 1990. 29. Hagedorn, M. and C. Carr. Single electrocytes produce a sexually dimorphic signal in South American electric fish Hypopomus occidentalis (Gymnotiformes, Hypopomidae). J. Comp. Physiol. 156A: 511-523, 1985. 30. Hagedorn, M. and W. Heiligenberg. Court and spark: electric signals in the courtship and mating of gymnotid fish. Anim. Behav. 32: 254-265, 1985. 31. Hagedorn, M., M. Womble and TE. Finger. Synodontid catfish: a new group of weakly electric fish. Brain Behav. Evol. 35: 268-277, 1990. 32. Hagedorn, M. and R. Zelick. Relative dominance among males is expressed in the electric organ discharge characteristics of a weakly electric fish. Anim. Behav. 38: 520-525, 1989. 33. Hall, Z.W., B.W. Lubit and J.H. Schwarts. Cytoplasmic actin in postsynaptic structures at the neuromuscular junction. J. Cell Biol. 90: 798-792, 1981. 34. Hall, Z.W. and J.R. Sanes. Synaptic structure and development: the neuromuscular junction. Cell 72: 99-121, 1993. 35. Heiligenberg, W. The neural basis of behavior: a neuroethological view. Annu. Rev. Neurosci. 14: 247-267, 1991. 36. Heiligenberg, W. Neural Nets in Electric Fish. Computational Neuroscience Series, Cambridge, MIT Press, 1991. 37. Hopkins, C.D. Electric communication in the reproductive behavior of Sternopygus macrums (Gymnotoidei). Z. Tierpsychol. 35: 518-535, 1974. 38. Hopkins, C.D. On the diversity of electric signals in a community of mormyrid electric fish in West Africa.Am. Zool. 21: 211-222, 1981. 39. Hopkins, C.D. and A.H. Bass. Temporal coding of species recognition signals in an electric fish. Science 212: 85-87, 1981. 40. Hopkins, C.D., N. Comfort, J. Bastian and A.H. Bass. Functional analysis of sexual dimorphism in an electric fish, Hypopomus pinnicaudatus, order Gymnotiformes. Brain Behav. Evol. 35: 350-367, 1990. 41. Hopkins, C.D. and W.E Heiligenberg. Evolutionary designs for electric signals and electroreceptors in gymnotoid fishes of Surinam. Behav. Ecol. Sociobiol. 3:113-134, 1978. 42. Hopkins, C.D. Neuroethology of electric communication. Annu. Rev. Neurosci. 11: 497-535, 1988. 43. Jasmin, B.J., B. Goud, G. Camus and J. Cartaud. The low molecular weight guanosine triphosphatebinding protein Rab6p associates with distinct post-Golgi vesicles in Torpedo marmorata electrocytes. Neuroscience 49: 849-855, 1992. 44. Johneis, A.G. On the origin of the electric organ in Malapterurus electricus. Q. J. Microscop. Sci. 97: 455-464, 1956. 45. Keynes, R.D. and J. Martins-Ferreira. Membrane potentials in the elecroplates of the electric eel. J. Physiol. 119: 315-351, 1953. 46. Kirschbaum, E Electric-organ ontogeny: distinct larval organ precedes the adult organ in weakly electric fish. Naturwissenschaften 64: 387-388, 1977. 47. Kirschbaum, E Myogenic electric organ precedes the neurogenic organ in Apteronotid fish. Naturwissenschaften 70: 205-206, 1983. 48. Kordeli, E., J. Cartaud, H.-O. Nghiem, L.-A. Pradel, C. Dubreuil, D. Paulin and J.-P. Changeux. Evidence for a polarity in the distribution of proteins from the cytoskeleton in Torpedo marmorata electrocytes. J. Cell Biol. 102: 748-761, 1986. 49. Kramer, B. Sexual signals in electric fishes. Trends Ecol. Evol. 5" 247-250, 1990. 50. Kramer, B. and B. Otto. Waveform discrimination in the electric fish Eigenmannia: sensitivity for the phase differences between the spectral components of a stimulus wave. J. Exp. Biol. 159: 1-22, 1991. 51. Landsman, R.E. Captivity affects behavioral physiology: plasticity in signaling sexual identity.

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F~erientia 47: 31-38, 1991. 52. Landsman, R.E. and P. Moiler. Testosterone changes the electric organ discharge and external morphology of the mormyrid fish, Gnathonemus petersii (Mormyriformes). Erperientia 44: 900-903, 1988. 53. LaRochelle, W.J. and E. Ralston. Clusters of 43-kDa protein are absent from genetic variants of C2 muscle cells with reduced acetylcholine receptor expression. Dev. Biol. 132: 130-138, 1989. 54. Leonard, R.D. and W.D. Willis. The organization of the electromotor nucleus and extraocular motor nuclei in the stargazer (Astroscopus y-graecum).J. Comp. Neurol. 183: 397--414, 1979. 55. Lindstrom, J., B. Walter (Nave) and B. Einarson. Immunochemical similarities between subunits of acteylcholine receptors from Torpedo, Electrophorus, and mammalian muscle. Biochemistry 18: 4470--4480, 1979. 56. Lorenzo, D., J.C. Velluti and O. Macadar. Electrophysiological properties of abdominal electrocytes in the weakly electric fish Gymnotus carapo. J. Comp. Physiol. 162A: 141-144, 1988. 57. Macadar, O., D. Lorenzo and J.C. Velluti. Waveform generation of the electric organ discharge II. Electrophysiological properties of single electrocytes. J. Comp. Physiol. 165A: 353-360, 1989. 58. McGregor, P.IL and G.W.M. Westby. Discrimination of individually characteristic electric organ discharges by a weakly electric fish. Anim. Behav. 43: 977-986, 1992. 59. McMahan, U.J. The agrin hypothesis. Cold Spring Harbor Symp. Quant. Biol. 55: 407-418. 1990. 60. Meyer, J.H. Steroid influences upon the discharge frequency of a weakly electric fish. J. Comp. PhysioL 153A: 29-38, 1983. 61. Meyer, J.H., M. Leong and C.H. Keller. Hormone-induced and ontogenetic changes in electric organ discharge and electroreceptor tuning in the weakly electric fish Apteronotus. J. Comp. Physiol. 160A: 385-394, 1987. 62. Mills, A. and H.H. Zakon. Chronic androgen treatment increases action potential duration in the electric organ of Sternopygus. J. Neurosci. 11: 2349-2361, 1991. 63. Mills, A.C. and H.H. Zakon. Coordination of EOD frequency and pulse duration in a weakly electric wave fish: the influence of androgens. J. Comp. Physiol. 161: 417--430, 1987. 64. Nakamura, Y., S. Nakajima and H. Grundfest. Analysis of spike electrogenesis and depolarizing K inactivation in electroplaques of Electrophorus electricus. J. Gen. Physiol. 49: 321-349, 1965. 65. Neville, C_.M. and J. Schmidt. Expression of myogenic factors in skeletal muscle and electric organ of Torpedo californica. FEBS Lett. 305: 23-26, 1992. 66. Noda, M., S. Shimizu, T. Tanabe, T. Takai, T. Kayano, T. Ikeda, H. Takahashi, H. Nakayama, Y. Kana0ka, N. Minamino, K. Kangawa, H. Matsuo, M.A. Raftery, T. Hirose, S. lnayama, H. Hayashida, T. Miyata and S. Numa. Primary structure of Electrophorus electricus sodium channel deduced from cDNA sequence. Nature 312: 121-127, 1984. 67. Noda, M., H. Takahashi, T. Tanabe, M. Toyosata, Y. Furutani, T. Hirose, M. Asai, S. Inayama, T. Miyata and S. Numa. Primary structure of the alpha subunit precursor of Torpedo californica acetylcholine receptor deduced from cDNA sequence. Nature 299: 793-797, 1982. 68. Noda, M., H. Takahashi, T. Tanabe, M. Toyosata, S. Kikyotani, T. Hirose, M. Asai, H. Takashima, S. Inayama, T. Miyata and S. Numa. Primary structures of beta and delta subunit precursors of Torpedo californica acetylcholine receptor deduced from cDNA sequences. Nature 301:251-255, 1983. 69. Pappas, G.D., S.G. Waxman and M.V.L. Bennett. Morphology of spinal electromotoneurons and presynaptic coupling in the gymnotid Stemarchus albifrons. J. Neurocytol. 4: 469-478, 1975. 70. Patterson, J.M. and H.H. Zakon. Bromodeoxyuridine labeling reveals a class of satellite-like cells within the electric organ. J. Neurobiol. 24: 660-674, 1993. 71. Quax, W., L. Van den Brok, W. Vree Eghets, E Ramaekers and H. Bloemendal. Characterization of the hamster desmin gene: expression and formation of desmin filaments in nonmuscle cells after gene transfer. Cell 43: 327-338, 1985. 72. Raftery, M.A., M.W. Hunkapillar, C.D. Strader and L.E. Hood. Acetylcholine receptor complex of homologous subunits. Science 208: 1454-1457, 1980. 73. S,~, L.A., V.M. Neto, M.M.D. Oliveira and C. Chagas. Heterogeneity of purified actin in the electric organ of the electric eel Electrophorus electricus. J. Exp. Zool. 257: 43-50, 1991. 74. Sealock, R., M.H. Butler, N.R. Kramarcy, I~-X. Gao, A.A. Murnane, K. Douville and S.C. Froehner. Localization of dystrophin relative to acetylcholine receptor domains in electric tissue and adult and cultured skeletal muscle. J. Cell Biol. 113: 1133-1144, 1991. 75. Sealock, R., A.A. Murnane, D. Paulin and S.C. Froehner. Immunochemical identification of desmin in Torpedo postsynaptic membranes and at the rat neuromuscular junction. Synapse 3: 315-324, 1989. 76. Shumway, C.A. and R.D. Zelick. Sex recognition and neuronal coding of electric organ discharge

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waveform in the pulse-type weakly electric fish, Hypopomus occidentalis. J. Comp. Physiol. 163A: 465-478, 1988. Taffarel, M., M.E de Souza, R.D. Machado and W. de Souza. Localization of actin in the electrocyte of Electrophorus electricus L. Cell Tissue Res. 242: 453-455, 1985. Trujillo-Cen6z, O. and J.A. Echagiie. Waveform generation of the electric organ discharge in Gymnotus carapo. I. Morphology and innervation of the electric organ. J. Comp. Physiol. 165A: 343-351, 1989. Waxman, S.G., G.D. Pappas and M.V.L. Bennett. Morphological correlates of functional differentiation of nodes of Ranvier along single fibres in the neurogenic electric organ of the knife fish Sternarchus. J. Cell Biol. 53: 210-224, 1972. Westby, G.W.M. and E Kirschbaum. Emergence and development of the electric organ discharge in the mormyrid fish, Pollirnyrus isidori. I. The larval discharge. J. Comp. Physiol. 122A: 251-271, 1977. Westby, G.W.M. and E Kirschbaum. Emergence and development of the electric organ discharge in the mormyrid fish, PoUimyrus isidori. If. Replacement of the larval by the adult discharge. J. Comp. Physiol. 127A: 45-59, 1978. Witzemann, V., G. Richardson and C. Boustead. Characterization and distribution of acetylcholine receptors and acetylcholinesterase during electric organ development in Torpedo marmorata. Neuroscience 8: 333-349, 1983. Witzemann, V., D. Schmid and C. Boustead. Differentiation-dependent changes of nicotinic synapse-associated proteins. Eur. J. Biochem. 131: 235-245, 1983. Zakon, H.H. Weakly electric fish as model systems for studying long-term steroid action on neural circuits. Brain Behav. Evol. 42:242-251, 1993. Zakon, H.H., A.C. Mills and M.B. Ferrari. Androgen-dependent modulation of the electrosensory and electromotor systems of a weakly electric fish. Semin. Neurosci. 3: 449-457, 1991. Zakon, H.H., P. Thomas and H.Y. Yan. Electric organ discharge frequency and plasma sex steroid levels during gonadal recrudescence in a natural population of the weakly electric fish Sternopygus macrurus. J. Comp. Physiol. 169A: 493-499, 1991. Zakon, H.H., H.-Y. Yah and P. Thomas. Human chorionic gonadotropin-induced shifts in the electrosensory system of the weakly electric fish, Sternopygus. J. Neurobiol. 21: 826-833, 1990.

Hochachka and Mommsen (eds.), Biochemistry and molecular biology offishes, vol. 4 9 1995 Elsevier Science B.V. All rights reserved. CHAPTER 12

Biochemical and molecular aspects of singing in batrachoidid fishes PATRICK J. WALSH, THOMAS P. MOMMSEN * AND ANDREW H . BASS **

Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Science, 4600 Rickenbacker Causeway, Miami, FL 33149, U.S.A., *Department of Biochemistry and Microbiology, University of Victoria, P.O Box 3055, Victoria, British Columbia V8W 3P6, Canada and ** Section of Neurobiology and Behavior, Cornell University, Ithaca, IVY 14853, U.S.A.

I. II. III. IV. V. VI.

Introduction Contraction mechanics Cell ultrastructure and contractile proteins Metabolism Hormonal regulation Prospects for future research Acknowledgement VII. References

I. Introduction Fishes of the family Batrachoididae (toadfish and midshipman) have a rather simple, but none the less remarkable, neuromuscular system for generating sounds. Central to the production of sound in these species is a heart-shaped swimbladder which has two large strips of muscle, one on each side. Sound is produced when neural signals, originating in the brain, trigger simultaneous contraction of the muscles against the taut surface of the swimbladder. The finely tuned (pun fully intended) neuromuscular system of this family presents researchers with several interesting avenues of inquiry. First, the muscle is among the fastest in the animal kingdom. Thus comparative study of this tissue will yield interesting insights into muscle structure/function relationships in general. The sonic muscle is used to generate defensive sounds (grunts) by both sexes, as well as a mate call (boatwhistle of toadfish, hum of midshipman) used exclusively by males to attract females to their nest for mating. This differential use of the muscle in the sexes has led to a clear sexual dichotomy (even a trichotomy in one species) in several facets of the neuromuscular set up, presenting a perfect opportunity for a second line of research into the neuroendocrine basis of sex-related vocal traits. In both of these areas, scant attention has been paid to the metabolic adaptations underlying muscle function. It is the aim of this chapter to summarize what is known about the metabolic biochemistry of this unique muscle system, set upon a

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foundation of the only slightly larger body of information on ultrastructure of the muscle and biochemistry of the contractile apparatus. In an effort to come to a unifying picture of sonic muscle function we may meld information from different species and genera within this family. However, because this organ has such direct and obvious consequences to reproduction (and hence future composition of the gene pool), we caution the reader to be mindful that the species differences may ultimately yield more clues to the importance of sonic muscle function than species similarities. In essence, we view comparative metabolic study of the neuromuscular system within this family (and perhaps comparison with singing muscles in other phyla), in direct conjunction with study of behavioral ecology, as a third fruitful avenue for investigation. Indeed, populations within the same species may turn out to be the most appropriate comparative unit for study of this system.

II. Contraction mechanics The extremely rapid contraction mechanics of the 'superfast' Batrachoidid sonic muscles were first documented in the oyster toadfish (Opsanus tau) by Skoglund30,31. Upon electrical stimulation of the motor nerve, he detected contractions within 0.5 ms of the action potential, which reached a peak within 5-8 ms, and muscle relaxation was complete within an additional 5-7 ms. We now know that this cycle of contraction/relaxation is among the fastest in the animal kingdom. By way of comparison, peak contraction is reached in insect flight muscle in 15 ms, and in vertebrate fast muscle in 20 ms25,zs. The paired muscles contract in synchrony and there is less than a 0.5 ms delay in the onset of depolarization between the cranial and caudal ends of the muscle. The number of vibrations per second determines the fundamental frequency of vocalizations and is established by: (1) the sonic motor pathway in the brain: and (2) a temperature dependent phenomenon with frequency increasing along with temperature 4,s. Thus, at about 15~ the fundamental frequency for vocalizations is in the range of 100 s-1 (Hz). Duration does not seem to be temperature dependent 16 and for grunts ranges from 70 to 200 ms and for boatwhistles from 500 to 700 ms ~~ The hum vocalization was first described by Ibara and coworkers 27 who estimated the duration of single hums to be up to 60 rain. Remarkably, during mating season, toadfish and midshipman males can produce these sounds for hours at a time. Both intra- (male-male) and inter- (male-female) sex differences exist for sound duration (grunts versus hums) as well as for the fundamental frequency of vocalizations in the plainfin midshipman, Porichthys notatus. Midshipman have three classes of sexually mature adults: females and two male reproductive morphs referred to as Type I and II males (for review, see ref. 2). "I~e I males are about 40% larger in body size, have a 9-fold smaller gonad weight to body weight ratio (gonadosomatic index), and build and defend nest sites in the intertidal zone. Type I males also have the most extensive vocal repertoire; they generate the long duration hums to attract females to their nest and trains of grunts during agonistic

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encounters with conspecifics 1~ Type II males 'sneak' or 'satellite' spawn when females are in a nest with Type I males 7,1~ Type II males, like females, have never been observed to build nests; nor do they hum or appear to generate any sounds during prespawning behavior. Type II males and females do however infrequently generate low amplitude, isolated grunts when they are housed together in crowded conditions. The vocalizations of Type II males and females are also about 20% lower in fundamental frequency than those of Type I males (hums and grunts) 1~ which is determined by the rhythmic firing properties of the central sonic motor pathway 4,5. Although only male toadfish generate boatwhistles during reproductive encounters, comparable sex differences do not appear to exist in the fundamental frequency of vocalizations between males and females (see ref. 5 for discussion).

III. Cell ultrastructure and contractile proteins The innervation of sonic muscle fibers appears to be adapted for rapid and synchronous contraction. Gainer and Klancher 22 first noted polyaxonal and multiple innervation along the entire length of the muscle in O. tau, allowing simultaneous and distributed action potentials. Sonic motoneurons located in the caudal brainstem and rostral spinal cord form a pair of sonic motor nuclei that are contiguous along the midline. Sonic motoneurons are cholinergic, i.e. they contain acetylcholine, as most other vertebrate motoneurons 8. The axons of sonic motoneurons exit the brain via ventral occipital nerve roots to ipsilateraUy innervate each sonic muscle where they form terminal boutons along the surface of muscle fibers 3,4. The ultrastructure of sonic nerve terminals (or boutons) and neuromuscular junctions (NMJs) has been described in midshipman 2~ Sonic NMJs resemble those of other vertebrates in having distinct pre- and postsynaptic membranes separated by a basal lamina, and active zones with clusters of clear, round synaptic vesicles (--~0.04-0.06 /zm diameter). Mitochondria and glycogen deposits are scattered throughout terminal boutons and, more infrequently, dense core vesicles (--,0.08/zm diameter). Unlike tetrapods, the NMJs lack postjunctional folds as is the case for other fish striated muscle, excepting perhaps the sonic muscle of weakfish, Cynoscion regalis29. There are intra- (Type I versus Type II males) and inter- (females versus Type I males) sexual dimorphisms in the sonic nerve terminals. The terminal boutons of Type I males are larger and lie in a trough along a muscle fiber's surface. The larger terminal size of Type I males parallels the increased size of their muscle fibers. There are also sex dimorphisms in synaptic vesicle density, glycogen content, myelination and the number of terminal boutons per innervation site. Interestingly, the terminals of Type I males had the fewest number of synaptic vesicles which correlates with the higher level of activity of the sonic motor system in the mate calling Type I males. Preliminary findings also indicated that for all three adult morphs, specimens maintained in captivity beyond the breeding season had elevated vesicle densities which would correlate with what we assume to be the lessened activity of the motor system outside the breeding season (a decrease in vocalizations has never been directly assessed since midshipman move far offshore to deep water during the nonbreeding season).

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Sonic muscle fibers have a number of ultrastructural features that distinguish them from other striated muscle fibers 1,6,14,21. For example, the sarcoplasmic reticulum is greatly exaggerated, e.g. constituting nearly 30% of the muscle fiber volume in O. tau 21, and very rich in calcium content 32. Other distinguishing features include the small diameter of their myofibrils (300-400/~m), reduced area of the T-tubule system, the confinement of mitochondria to a central core and a thin peripheral rim of sarcoplasm, and the almost perfect alignment across adjacent myofibrils of the Z, A, H, and I bands and of triads (T-tubule plus terminal cisternae of sarcoplasmic reticulum). Fawcett and Revel 14 considered these features to be related to the physiological properties of the sonic muscle, namely their high speed of contraction (also see ref. 1). Sex dimorphisms have also been reported for the ultrastructural features of sonic muscle fibers. In R notatus, the sonic muscle fibers and myofibrils of Type I males are distinct from those of females and Type II males, which are similar to each other. First, the sonic muscle mass of l~pe I males, when corrected for body size, is almost 600% greater than either females or Type II males. Second, this is paralleled by a 300-400% increase in the number, and a 500% increase in the diameter, of muscle fibers. Third, there is a dramatic dimorphism in the phenotype of individual fibers. Thus, the sonic fibers of Type I males have a central donut-shaped ring of myofibrils bordered centrally and peripherally by a zone of sarcoplasm that is densely filled with mitochondria 6. The cross sectional area of the myofibril-containing zone is 60100% greater in 7 ~ e I males; the sarcoplasm-containing zone is 900% greater n. The sarcoplasmic reticulum which separates adjacent myofibrils also appears to be more highly branched in Type I males, while their Z-lines are nearly 20-fold wider6! The special features of the Type I male fibers and myofibrils are assumed to be adaptations related to their humming abilities 6. The muscle fibers of females and Type II males resemble those of male and female toadfish, as well as other species of sonic fish 6. More modest sex dimorphisms have been reported for muscle mass in toadfish 15.34. In O. tau, the larger muscle mass in males (~40%) is paralleled by a 47% increased fiber number 18. One report states that sonic muscle fibers are 15% larger in females18; another that fiber diameter is the same for females and males 1. Other sex dimorphisms include the number of mitochondria (although still relatively low compared to Type I male midshipman fibers), and the surface area of components of the T-tubule system (see ref. 1 for details). The specialized contractile and related proteins have been studied to some extent in the sonic muscle of O. tau. In accordance with the exaggerated sarcoplasmic reticulum, Appclt et al. 1 have recently demonstrated a high content of Ca 2+ ATPase (responsible for calcium sequestration in the SR) and of foot protein (part of the functional calcium-release channel at the SR-T-tubule junction) in the sonic muscle as compared with other muscles. ATPase values were not different between males and females, and density of feet was slightly higher in females than in males (although this was a calculated parameter with no measured error). Notable differences in other sonic muscle proteins also are apparent. Hamoir et al. 25 found that parvalbumin content in O. tau is about twice as high as that

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found in typical teleost skeletal muscle. The high parvalbumin content appears to correlate with the speed of the contraction cycle, and it is believed that parvalbumin serves as a cytoplasm/SR calcium shuttle and is involved in the rapid relaxation times. Interestingly, parvalbumin subtype Illf predominates in the sonic muscle instead of the typical pattern of three subtypes (including lllf) in fish trunk muscle. This parvalbumin has been purified from sonic muscle, its crystallography examined 24,35, and its amino acid sequence determined 13. It does not appear to be unusual compared to other fish IIIf parvmodulins 13, so we may conclude that the differential expression of parvmodulin types, rather than a fundamental change in amino acid sequence, is sufficient to adapt this molecule to the requirements of superfast contraction. The myosin light chains (LC2) (i.e. the 'head' of the thick filaments which contains the ATPase activity and which mechanically interacts with the thin actin filaments) of the sonic muscle migrate much faster in 8 M urea PAGE than do corresponding proteins from the trunk fast muscle 23,2s, and have a heavier molecular weight 26. However, the significance of these differences is still unknown. Clearly, the molecular architecture of this muscle is unique. Further molecular study will undoubtedly yield interesting insights. Unfortunately in many of these other protein studies, no mention of sex of fish was made, If these authors have recorded these observations it would be useful for them to reexamine their data for sex differences. Given the other manifold sex-related differences in this muscle, it would be interesting to see if these proteins also exhibited sex differences.

114. Metabolism Details on the metabolic machinery which powers the remarkable contractile abilities of the sonic muscle are far from complete. Fine and Pennypaeker 17 examined the histoehemical properties of the sonic muscle in O. tau in order to type the muscle. Based on their observations, a positive stain for ATPase after alkaline preincubation, and positive NAD diaphorase, they characterized the muscle as Type lla, or biochemically speaking as fast oxidative glycolytic (FOG). This is distinguished from two other typical muscle types: in fish Type I or slow oxidative (SO) (e.g. lateral line red muscle), and q'~,pe lib or fast glycolytic (FG) (e.g. caudal white muscle). Fine and Pennypacker 17 also noted several features of the muscle structure which appear to favor short diffusion distances for oxygen (e.g. small fiber size, large number of sarcoplasmic pockets where mitochondria are clustered). Glycogen content in the sonic muscle is about twice that in the body muscle, and fat content of over six times greater, with no apparent differences between sexes 19. Although one would expect high glycogen in either FOG or FG fibers, the presence of the high lipid content (which can only be metabolized aerobically) strongly supports the typing as FOG. These authors further suggest that the sonic muscle is a fatigue resistant fiber (FR) and this clearly fits with the abilities of males to produce sounds for hours at a time. In the Gulf toadfish (O. beta), sonic muscle mass increases with body mass, but

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Fig. 1. Correlation between sonic muscle mass and body mass in males (open squares) and females (closed diamonds) of the Gulf toadfish (Opsanus beta). Double logarithmic plot. Toadfish were collected by shrimp trawl around Miami, Florida. Linear regressions are: males, y -- -1.732+0.912x, r = 0.99; females, y - -1.882+0.913x, r - 0.97. The mean weight for males was 143.24-14.2 g (n - 50) and for females 164.64-17.2 (n = 47), t - 0.96. The y-intercepts are significantly different at p <0.001. From ref. 34, with permission.

as mentioned above, the mass of sonic muscle is higher in males than in females. Interestingly, the slope of the two log-log regression plots is identical for the two sexes (Fig. 1), but the two lines have significantly different y-intercepts. This indicates that the relative sex difference in sonic muscle mass is determined at a point outside of the fish range analyzed in our data; and this difference was already apparent in the smallest specimen sampled (7.3 g total weight). From this point on, sonic muscle mass increases to a similar degree in both sexes. Remarkably, mitochondrial densities in O. tau are rather lower than typical white muscle 1, and activities of the mitochondrial matrix enzyme, citrate synthase, are lower than skeletal muscle for O. beta 33'34. However for both parameters, males of O. beta have significantly higher values than females. In addition, when we measured the mass-specific aerobic capacity of the sonic muscle, as indicated by the activity of citrate synthase (EC 4.1.3.7), we noticed that the activity scales differently in males and in females. The activity of citrate synthase per gram of tissue wet weight, which can be taken as an approximate reflection of the abundance of mitochondria in the tissue, increases in males, but clearly decreases in females, while in the smaller (largely immature) fish, the y-intercepts are similar (Fig. 2). It can be concluded that, in contrast to sonic muscle mass, the differentiation of CS activities occurs during later stages in development and maturation. Therefore it seems that it is the degree of aerobic potential that enables males to vocalize for extended periods 34. While the picture for the toadfish can be summarized as 'fast, more oxidative fibers in males than in females with immediate mass differentiation and later developmental differentiation in oxidative potential', the situation is quite different in the plainfm midshipman (Porichthys notatus). In this species, the male sonic muscles are much more oxidatively oriented than the female counterparts. Table 1

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Fig. 2. Correlation between mass specific activity of citrate synthase (units per gram sonic muscle mass) and body mass in males (open squares) and females (closed diamonds) of the Gulf toadfish (Opsanus beta). Toadfish were collected by shrimp trawl around Miami, Florida. Linear regressions are: males, y = 1.159+0.006x, r ffi 0.51; females, y = 1.477+0.001x, r = 0.26. The slopes are significantly different at p <0.001. From ref. 34, with permission. TABLE 1 Enzyme activities in muscles of plainfin midshipman (Porichthys notatus) Enzyme

Sonic (females)

Sonic (males)

White (males)

Heart (males)

Citrate synthase Malate dehydrogenase Lactate dehydrogenase Aspartate aminotransferase Glucose 6-phosphate dehydrogenase Malic enzyme

7.5 4- 1.1 254 4- 53 56 4. 3.2 48 4. 9.6 0.224- 0.1

44 4. 5.6a 984 4- 115 a 94 4- 5.9 253 4. 34 a 0.164- 0.03

1.8 123 115 14 0.03

21 995 491 130 0.45

0.72 4- 0.1

1.23 4-

0.18

4. 0.5 4. 15 4- 4.0 4. 3.2 4. 0.01

0.15 4. 0.03

4. 2.2 4- 64 4"66 4- 14 + 0.12

1.394. 0.07

Enzymes were measured under Vmax conditions at room temperature, as outlined in reference 33. Animals were collected subtidally at the Bamfield Marine Station, British Columbia. Activities are given as units (mean 4- SEM) per gram tissue fresh weight for 4 or 5 independent observations. a Significantly different (p < 0.05) than the corresponding value in the female sonic muscle (Student's t-test). 1 Unit is defined as the amount of enzyme producing 1 /xmol of product per min. Enzymes are: citrate synthase (EC 4.1.3.7); Malate dehydrogenase (EC 1.1.1.37); Lactate dehydrogenase (EC 1.1.1.27); Aspartate aminotransferase (EC 2.6.1.1); Glucose 6-phosphate dehydrogenase (NADP-linked, EC 1.1.1.49) and Malic enzyme (malate dehydrogenase-decarboxylating (EC 1.1.1.40). Data from TP. Mommsen, K. Nickolichuck and A.H. Bass (unpublished).

gives a short overview over enzymatic capacities of some of the muscles in question, while Table 2 presents the corresponding values for some of these muscles in toadfish (O. beta). At this point, we have no indication about size and maturationdependent differentiation in P. notatus sonic muscle, but it is clear from the data in Table 1 that much larger sex-dependent differences exist between males and females. Judging from the enzymatic machinery, the female sonic muscle could be classified together with the white skeletal muscle, while the male sonic muscle is clearly more oxidative that the generally 'most-oxidative' muscular tissue in fishes, the heart. Obviously, toadfish and midshipmen have taken different metabolic

286

P.J. Walsh, T.P. Mommsen and A.H. Bass TABLE 2 Enzyme activities in muscles of Gulf toadfish (Opsanus beta)

Enzyme

Sonic (females)

Sonic (males)

White (females)

White (males)

Citrate synthase Malate dehydrogenase Lactate dehydrogenase Aspartate aminotransferase

1.51 4- 0.14 202 4- 11 482 4- 47 22.3 4- 1.6

2.68 399 333 26.2

0.44 4- 0.06 35.3 4- 3.3 292 4- 23 ND

0.42 4- 0.05 41.5 4- 5.4 356 4- 23.4 24.1

4- 0.31 a 4. 24a 4- 36 a 4- 2.9

Enzymes were measured under Vmax conditions at room temperature, as outlined in references 33 and 34. Animals were collected by shrimp-trawl around Miami, Florida. Activities are given as units (mean 4- SEM) per gram tissue fresh weight for 6 or 7 independent observations (sonic muscles) and 13 to 19 observations for white muscles. 1 Unit is defined as the amount of enzyme producing 1/~mol of product per min. For enzyme classification, see legend to Table 1. Data from reference 33. ND - not determined. a Indicates significantly different from female sonic muscle (p < 0.05). Enzymes in white muscles were not significantly different from each other. Data recalculated from references 33 and 34.

routes to achieve similar physiological goals, the production of sound. While in both cases, clearly a superfast, generally oxidative, fiber was committed to the job, a large latitude is apparent in the degree of oxidative potential. Again, an in depth analysis of calling behavior of toadfish versus midshipman may help to shed light on these different biochemical strategies. In an initial study on muscle size in mature; male midshipmen, we also noticed that size, expressed as sonic muscle somatic index (mass of sonic muscle expressed as a percentage of body weight), undergoes systematic changes in the course of the annual cycle, with a clear peak at about 1.4% of body weight during spawning/guarding time in June (T.R Mommsen and K. Nickolichuck, unpublished observations). Since this peak coincides further with a peak in oxidative capacity of the tissue, it is clear that the fish control not only muscle size but also metabolic machinery. Given that white muscle in toadfish (or other teleosts for that matter) is not an aerobic tissue, and that mitochondrial densities and enzyme activities are even lower in toadfish sonic muscle, how is prolonged contraction possible? One potential mechanism is that the muscle fibers, although capable of repeated superfast contraction, may have a much lower work load than a typical skeletal muscle used in an escape response to power a fish against the drag of water. A reduced work load probably is only a partial answer to this paradox. As described earlier, the sarcoplasmic zone of the sonic muscle fibers of R notatus is densely filled with mitochondria and is 900% larger in Type I males compared to either Type II males or females. Mitochondrial content in Type I males is also clearly far greater than in either male or female O. tau (compare Figs. 2 and 3 from ref. 6 to Figs. 1 and 2 from ref. 14). Appelt and colleagues I comment on the relatively small volume of mitochondria in toadfish sonic fibers (1% in females and 4% in males), which seems low given the continuous activity levels during the breeding season. Thus, it appears that in ~ e I male midshipman, there may be a better correlation between mitochondrial content and extended periods of vocal activity than in the toadfish, and also, given the interesting trimorphism in midshipman, this

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species is perhaps a more interesting candidate for biochemical and metabolic study than the toadfishes. More than likely the sonic muscle will exhibit unique mechanisms and further species differences for metabolic regulation in the areas of substrate preference during different types of vocalizations, rapidity of substrate mobilization, transport of metabolites across mitochondrial membranes, etc. A wide range of experimental approaches are possible. We recommend that initial experiments focus on classical metabolic analysis in which enzyme activities, equilibrium constants, and mass action ratios of metabolic intermediates are measured under different physiological circumstances. Two physiological systems might show special promise. Neurophysiologists have worked with induced sound production in these species for many years by stimulating regions of the brain. One could perform such stimuli for varying lengths, on different sexes, in different seasons, etc., and freeze clamp muscles for biochemical measurements and analyses (e.g. cross-over plots). Laboratory or field fish for which degree of sound production can be documented would also make excellent subjects. Finally, in an evolutionary sense, successfully mated males (which conveniently stay with the young in their nests for several weeks) could be biochemically compared to their less successful counterparts.

V. Hormonal regulation It is obvious from the above discussions that there are differences between males, females, and sneaker males in a variety of morphological and biochemical aspects. In midshipman, recent light and electron microscopic studies demonstrate the hormonal sensitivity of the sonic muscle. Androgens (testosterone, l lketotestosterone), but not estrogens (17fl-estradiol) induce nearly a 100% increase in sonic muscle weight in juvenile males and juvenile females, but only a 50% increase in Type II males (adult females have not been studied). The increase in muscle mass is most closely paralleled by an increase in the cross sectional area of the mitochondria-filled sarcoplasm9,11. The results suggest that sarcoplasm/ mitochondrial volume may vary seasonally in Type I males depending on circulating plasma levels of steroid hormones. This suggestion is supported by the above mentioned observation of a clear circannual change in sonic muscle mass in a Northern population of midshipmen (T.P. Mommsen and K. Nickolichuck, unpublished observation). The hormone-sensitivity of muscle mass was confirmed for adult females and extended to include the oxidative capacity of the sonic muscle. In a northern population of P. notatus, intraperitoneal injection of 17-methyltestosterone deposits leads to significant increases in sonic muscle mass, coupled with an increase in mass-specific aerobic capacity (T.P. Mommsen and A. Bass, unpublished results). Conversely, treatment of adult Type I males with estradiol for a few weeks resulted in significant decreases in the overall mass of the sonic muscle. Obviously, a number of different muscle parameters are under hormonal control in midshipmen, including muscle mass, oxidative machinery, sarcoplasm/mitochondrial volume, and every single one of these would make an interesting object of study.

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VI. Prospectsfor future research The sonic muscles of batraehoidid fishes represent excellent systems with which to study metabolic regulation and adaptation over both short and long time courses. The study of the mechanisms of rapid mobilization of enzymes and substrates which fuel contraction will certainly yield insights into basic metabolic control theory. The unique 'female-type' sonic muscle in mature 'sneaker' males certainly present researchers with an ideal to study hormone receptors (expression and function) and control of muscle differentiation by sex hormones. Additionally, study of the role of sex hormones in control of metabolism on seasonal and life-history time scales will yield insights into mechanisms of endocrine action and the evolutionary biology of sexual dimorphisms.

Acknowledgement. The authors' research is supported by NSF, NIH (U.S.A.) and NSERC (Canada).

VII. References 1. Appelt, D., V. Shen and C. Franzini-Armstrong. Quantitation of Ca ATPase, feet and mitochondria in superfast muscle fibres from the toadfish, Opsanus tau. J. Muscle Res. Cell Motil. 12: 543-552, 1991. 2. Bass, A.H. Dimorphic male brains and alternative reproductive tactics in a vocalizing fish. Trends Neurosci. 15: 139-145, 1992. 3. Bass, A~ and K. Andersen. Inter- and intrasexual dimorphisms in the vocal control system of a teleost fish: motor axon number and size. Brain Behav. Evol. 37: 204-214, 1991. 4. Bass, A.H. and R. Baker. Sexual dimorphisms in the vocal control system of a teleost fish: morphology of physiologically identified neurons. J. Neurobiol. 21: 1155-1168, 1990. 5. Bass, A.H. and R. Baker. Evolution of homologous vocal control traits. Brain Behau Ecol. 38: 240-254, 1991. 6. Bass, A.H. and M.A~ Marchaterre. Sound-generating (sonic) motor system in a teleost fish (Porichthys notatus): sexual polymorphism in the ultrastructure of myofibrils. J. Comp. Neurol. 286: 141-153, 1989. 7. Brantley, R.K. Ontogeny of Inter- and Intra-Specific Sexual Dimorphism in a Vocalizing Fish: Behavioral, Morphological and Endocrine Correlates. Ph.D. dissertation, Cornell University, 161 pp., 1992. 8. Brantley, R.IL and A.H. Bass. lntrasexual dimorphisms in a sound producing fish: alternative reproductive morphs? Soc. Neurosci. Abstr. 14: 691, 1988. 9. Brantley, R. and A. Bass. Sexual polymorphisms and androgen sensitivity of sound-generating muscle in a vocalizing fish. $oc. NeuroscL Abstr. 16: 323, 1990. 10. Brantley, R.IL and A.H. Bass. Alternative male spawning tactics and acoustic signals in the plainfin midshipman fish Porichtys notatus Girard Teleostei Batrachoididae. Ethology 96: 213-232, 1994. 11. Brantley, R.K., M.A. Marchaterre and A.H. Bass. Androgen effects on vocal muscle structure in a teleost fish with inter- and intra-sexual dimorphism. J. MorphoL 216: 305-318, 1993. 12. Brantley, R., J. Tseng and A. Bass. The ontogeny of inter- and intrasexual vocal muscle dimorphism in a sound-producing fish. Brain Behav. Evol. 42: 336-349, 1993. 13. Collin, C.G.S and N. Gerardin-Otthiers. The amino acid sequence of the parvalbumin from the very fast swimbladder muscle of the toadfish (Opsanus tau). Comp. Biochem. Physiol. 93B: 49-55, 1989. 14. Fawcett, D.W. and J.P. Revel. The sarcoplasmic reticulum of a fast-acting fish muscle. J. Biophys. Biochem. Cytol. 10: 89-102, 1961. 15. Fine, M.L. Sexual dimorphism of the growth rate of the swimbladder of the toadfish, Opsanus tau. Copeia 3: 483-490, 1975.

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16. Fine, M.L. Seasonal and geographic variation of the mating call of the oyster toadfish. Oecologia 36: 45-57, 1978. 17. Fine, M.L. and K.R. Pennypacker. Histochemical typing of sonic muscle from the oyster toadfish. Copeia 1: 130-134, 1988. 18. Fine, M.L., N.M. Burns and TM. Harris. Ontogeny and sexual dimorphism of sonic muscle in the oyster toadfish. Can. J. Zool. 68: 1374-1381, 1990. 19. Fine, M.L., K.R. Pennypacker, K.A. Drummond and C.R. Blem. Concentration and location of metabolic substrates in fast toadfish sonic muscle. Copeia 4: 910-915, 1986. 20. Fluet, A. and A. Bass. Sexual dimorphisms in the vocal control system of a teleost fish: ultrastructure and neuromuscular junctions. Brain Res. 531: 312-317, 1990. 21. Franzini-Armstrong, C. and G. Nunzi. Junctional feet and particles in the triads of a fast-twitch muscle fibre. J. Muscle Res. Cell Motil. 4: 233-252, 1983. 22. Gainer, H. and Klancher, J.E. Neuromuscular junctions in a fast-contracting fish muscle. Comp. Biochem. Physiol. 15: 159-165, 1965. 23. Hamoir, G. and B. Focant. Proteinic differences between the sarcoplasmic reticulums of the superfast swimbladder and the fast skeletal muscles of the toadfish Opsanus tau. Mol. Physiol. 1: 353-359, 1981. 24. Hamoir, G., O. Dideberg and P. Charlier. Crystallization and preliminary X-ray data for parvalbumin IIIf of Opsanus tau.J. Mol. Biol. 153: 487-489, 1981. 25. Hamoir, G., N. Gerardin-Otthiers and B. Focant. Protein differentiation of the superfast swimbladtier muscle of the toadfish, Opsanus tau. J. Mol. Biol. 143: 155-160, 1980. 26. Huriaux, E, E Lefebvre and B. Focant. Myosin polymorphism in muscles of the toadfish, Opsanus tau. J. Muscle Res. Cell Motil. 4: 223-232, 1983. 27. Ibara, R.M., L.T Penny, A.W. Ebeling, G. Van Dykhuizen and G. CaiUiet. The mating call of the plainfin midshipman fish, Porichthys notatus. In: Predators and Prey in Fishes, edited by D.L.G. Noakes et al., The Hague, Dr. W. Junk Publishers, pp. 205-212, 1983. 28. Kuffler, S.W. and E.M. Vaughan Williams. Properties of the 'slow' skeletal muscle of frog. J. Physiol. 121: 318-340, 1953. 29. Ono, R.D. and S.G. Poss. Structure and innervation of the swim bladder musculature in the weakfish, Cynoscion regalis (Teleostei: Scianenidae). Can. J. Zool. 60: 1955-1967, 1982. 30. Skoglund, C.R. Neuromuscular mechanisms of sound production in Opsanus tau. BioL Bull. 117: 438, 1959. 31. Skoglund, C.R. Functional analysis of swim bladder muscles engaged in sound production of the toadfish. J. Biophys. Biochem. Cytol. 10: 187-200, 1961. 32. Somlyo, A.V., H. Shuman and A.R Somlyo. Composition of sarcoplasmic reticulum in situ by electron probe X-ray microanalysis. Nature 268: 556-558, 1977. 33. Walsh, RJ., C. Bedolla and TP. Mommsen. Reexamination of metabolic potential in the toadfish sonic muscle. J. Exp. Zool. 241: 133-136, 1987. 34. Walsh, P.J., C. Bedolla and TP. Mommsen. Scaling and sex-related differences in toadfish (Opsanus beta) sonic muscle enzyme activities. Bull. Mar. Sci. 45: 68-75, 1989. 35. Wery, J.P., O. Dideberg, R Charlier and C. Gerday. Crystallization and structure at 3.2 A resolution of a terbium parvalbumin. FEBS Lett. 182: 103-106, 1985.

Hochachka and Mommsen (eds.), Biochemistry and molecular biology of fishes, voi. 4 9 1995 Elsevier Science B.V. All rights reserved. CHAPTER 13

Is glycolytic rate controlled by the reversible binding of enzymes to subcellular structures? STEPHEN P.J. BROOKS AND KENNETH B. STOREY * Nutrition Research Division, Health Canada, Tunney's Pasture, Ottawa, Ontario, Canada K1A OL2, 9Departments of Biology and Chemistry, Carleton University, Ottawa, Ontario, K1S 5B6 Canada

I. II. III. IV. V. VI. VII.

Introduction In vivo enzyme-subcellular binding experiments The search for a metabolic trigger In vitro measurement of enzyme binding In vitro kinetic studies Acknowledgements References

I. Introduction In order to maintain homeostasis under widely differing metabolic conditions cells must tightly regulate individual metabolic pathways. In the classical view of cellular biochemistry, the control of metabolic pathways is achieved through the reversible regulation of key enzymes of the pathway. These key enzymes are found at the beginning or the ends of pathways so that overall flux may be governed through control of only a few enzymes. For example, in the classical view, glycolytic flux is controlled primarily through allosteric regulation of four enzymes: glycogen phosphorylase (GP), hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK). Glycogen phosphorylase, HK and PFK are found at the start of the glycolytic sequence whereas PK is one of the terminal glycolytic enzymes. In fish, all four enzymes show several features common to many regulatory enzymes: they catalyze high energy steps that are (essentially) irreversible, they catalyze reactions which involve a high energy intermediate and they are multi-subunit enzymes. They are also allosterically regulated by several different compounds which serve to link enzyme activity to the energy state of the cell 34. The complexity of controls governing glycolytic enzyme activity (in this review we will confine ourselves to the enzymes of glycolysis) becomes apparent when one considers the widely different metabolic states that fish can endure. For example, muscle ATP production during the initiation of burst swimming in salmonid fish increases 10-15 fold when compared to slow swimming c o n t r o l s 22,49,56,63,65. On the

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other hand, metabolic rates in severely hypoxic and anoxic goldfish and carp drop to levels only 30% of their respective control values 23,ss-61. Both metabolic states demand a drastic reorganization of metabolism to balance energy-producing and energy-consuming reactions. It is this balance that is mediated through control of enzyme activities. Several different levels of enzyme control have been identified which can regulate glycolytic rates including: (1) changes in the concentration of the allosteric activators of GP, HI(, PFK and pK21'22'28'3~ (2) changes in cellular pH towards more acidic values during prolonged periods of exercise22,49; and (3) changes in enzyme phosphorylation (see Chapter 51, Volume 3 of this series). More recently a fourth method of glycolytic control has been proposed: the reversible binding of glycolytic enzymes to subcellular structures to form multienzyme complexes 7-1~ In situations where a rapid change in glycolytic rate occurs (during burst swimming in trout, for example) the formation of a multi-enzyme complex would represent a quick and easily reversible mechanism to control glycolytic rates. In mammals, several multi-enzyme complexes have been shown to occur. For example, the enzymes involved in DNA and RNA replication, protein biosynthesis, glycogen metabolism, amino acid metabolism, fatty acid metabolism, and the citric acid cycle have all been shown to exist in an aggregated state sS. In fish, a correlation between changes in glycolytic enzyme binding to cellular particulate matter (a crude preparation containing mitochondria, lysosomes, nuclei, F-actin and tubulin polymers) and changes in metabolic demand have also been observed (Fig. 1; refs. 7, 8, 23, 43). The potential kinetic advantages of enzyme-enzyme and enzyme-subcellular structure complexes are numerous and

100

["' i rested l exhausted

8 0 I-

.i0 :3 0 rn

6 0 I-

9 #0

I

l

*

-

20

0

PFK

ALD GAPDH PGK

PK

LDH

CK

Fig. 1. Changes in glycolytic enzyme binding in trout (Oncorhynchus myk/ss) skeletal muscle. Enzyme binding in rested animals (open bars) and animals exercised to exhaustion (closed bars) was measured using the Dilution method. *Significantly different from rested at the P <0.05 level. Adapted from ref. 7.

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include increased enzyme activity through localized regions of higher substrate concentrations and localized regions of higher allosteric activator concentrations: multi-enzyme complexes should catalyze reactions faster than soluble enzymes. However, a direct cause and effect relationship between increased glycolytic enzyme binding and increased flux through glycolysis has not been demonstrated. This chapter examines the evidence for and against the formation of a functional glycolytic complex. We will define the glycolytic complex as a multi-enzyme structure containing all the enzymes necessary to metabolize glucose to pyruvate (the complete glycolytic sequence). In order for the complex to be considered as functional, three criteria must be satisfied: (1) the enzymes must bind together to form a complex; (2) the enzymes must be kinetically active when bound in the multi-enzyme complex; and (3) the magnitude of the change in enzyme binding must account for the magnitude of the change in glycolytic flux. Over the past several years it has become clear that some glycolytic enzymes are bound to subcellular structures or to other enzymes in vivo. These associations may indeed regulate the rates of individual glycolytic enzymes. Why then is there so much resistance to the concept of a glycolytic 'metabolon'? As we shall see below, part of the answer comes from kinetic studies of bound glycolytic enzymes. Specifically, bound enzymes are often inhibited when compared with their soluble counterparts even though measurements of enzyme binding before and after metabolic stress indicate that the bound enzymes should be more active 12. Results such as these illustrate the sharp discrepancy between kinetic and binding data and suggest that a kinetically active enzyme complex does not exist (see criteria 1 and 2, above). In the present review, we will critically examine all the available data on glycolytic enzyme complex formation and present an overall view of the complex as it is presently understood. It is hoped that a balanced treatment has been achieved that will allow the reader to form his or her own conclusions regarding the existence of the glycolytic complex and the in vivo kinetic benefits that such a complex would exhibit.

II. In vivo enzyme-subcellular binding experiments We have chosen to call experiments that measured enzyme binding before and after a physiological stress 'in vivo' experiments although the actual bound/free measurements are usually performed after tissue homogenization and centrifugation. The following is a summary of procedures used to assess the in vivo bound/free status of glycolytic enzymes. The most convincing evidence in favor of enzyme-subcellular binding interactions in vivo has come from experiments using the Dilution method. In this technique 17, muscles are homogenized in an iso-osmotic sucrose solution (250 mM sucrose, zero ionic strength) and centrifuged to separate bound and free enzyme fractions 189 bound enzymes are found in the pellet, free enzymes in the supernatant (refer to Volume 3 of this series for a detailed protocol and analysis of the Dilution method). Because the technique is rapid, it provides a good method for assaying the distribution between free and bound enzymes under defined metabolic conditions (Fig. 1). Variations of this technique have incorporated changes in cen-

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trifugation time and speed24,3a,S2as well as changes in the ionic strength of the homogenization medium 16. Using this method, changes in glycolytic enzyme binding have been measured in exercised 7,a, starved43, and anoxic23 fish muscle as well as in other animals under a variety of metabolic states 9.1~ These studies demonstrated a positive correlation between increased enzyme association with particulate matter and increased energy demand in skeletal muscle7.8,17,23 as well as between decreased binding and decreased energy demand 9't~ By showing that: (1) enzyme binding changes in response to changes in metabolic demand; and (2) the direction of change correlates with the metabolic flux in situations of both increasing and decreasing metabolic rate, these data argue for both the existence and the functional significance of a glycolytic enzyme complex. The correlation between increased (or decreased) glycolytic demand and increased (or decreased) enzyme binding suggests that increased enzyme binding promotes an increase in glycolytic flux. This result predicts that glycolytic enzymes would be kinetically activated in the complex when compared with their soluble counterparts and limits us to considering only this possibility. This latter point is emphasized because it is essential that we remember it when examining the in vitro experiments on enzyme binding (see below). Although a majority of in vivo experiments have been performed with the Dilution methodology, questions about its validity rule out its use as an absolute identifier of enzyme complexes. Specifically, the Dilution method dilutes the cellular milieu 4-5 fold (depending on the procedure) prior to measurement of enzyme binding. This means that the cellular ionic strength and protein concentration have been diluted to a value that is 20-25% of the in vivo value before the identification of bound and free enzymes. This dilution may artificially increase (or promote) enzyme binding because enzymes bind largely through ionic interactions 8. The effects of diluting the cellular ionic strength on enzyme binding have been measured for rainbow trout (Oncorhynchus mykiss) white muscle PFK and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) by adding various salts and observing the outcome s. Fig. 2 shows this effect for PFK. Note that the binding is extremely sensitive to increasing salt concentrations so that at physiological concentrations of ATP, ADP, fructose 6-phosphate, lactate, Ca 2+, Mg2+, and KCI virtually 90% of the PFK and GAPDH activities are free s. Studies such as these have called the Dilution method into question and suggest that the absolute amount of enzyme bound may not reflect the true value in vivo (see Table 1). In order to better assess the enzyme bound/free distribution in vivo, several other methods have also been developed. These differ widely in their ability to reproduce cytoplasmic conditions so that results obtained with them must be interpreted accordingly. The other methods include: (1) (2) (3) (4) (5)

the Press Juice method1'2'4; the Minced and Spun procedure4; Glutaraldehyde Fixation39; Detergent Solubilization43'54; diffusion measurements with fluorescently labeled proteins *~.

295

Is glycolytic rate controlled by the reversible binding of enzymes to subcellular structures?

lO0 13

80 LLI

tu 60

0 MIIATP = F-6P ,., C~CI 2 = KCI

nil

o~ 40 20

0

20

40

60 [EFFECTOR]

80

I00

120

(rn_M)

Fig. 2. Release of rainbow trout PFK from particulate matter by salts and physiological metabolites. Increasing concentrations of a 1:1 mixture of MgCl2 and ATP (MgATP, open circles), fructose 6phosphate (F-6P, closed squares), CaCI2 (open squares) and KCI (closed circles) were added to trout muscle particulate matter suspended in 250 mM sucrose. Supernatant (Free) PFK activity was measured after centrifugation at 12,000 g for 5 rain. Adapted from ref. 8. TABLE 1 Comparison of glycolytic enzyme binding in rainbow trout (Oncorhynchus mykiss) white skeletal muscle estimated using three different methodologies Enzyme

Dilution method (% bound)

Quickly pressed method (% bound)

Rapid minced and spun (% bound)

PFK ALD GAPDH LDH

95.3 32.2 73.1 17.3

70.5 28.5 10.0 1.0

81.2 13.8 20.8 16.1

Percentage of bound enzyme was measured in exhausted trout white muscle. Description of methodologies is found in the text. Data modified from Brooks and Storey 8. Abbreviations: ALD = aldolase; PFK = phosphofructokinase-1; GAPDH = glyceraldehyde 3phosphate dehydrogenase; LDH = L-lactate dehydrogenase.

A short discussion on the merits of these techniques is provided below. The Minced and Spun method involves mincing tissue in a Waring blender and centrifuging the resulting homogenate for 20-24 h at 100,000 g to separate particulate matter and cellular juice fractions 1. This technique has the advantage that it does not dilute cell contents during the homogenization step; the ionic strength and the protein concentration of the cellular juice are maintained at values found in vivo. However, the lengthy centrifugation step allows changes in cellular metabolite levels to occur. Thus, enzyme binding is ultimately measured under conditions where ATP, creatine phosphate and cellular pH are lower than that found in vivo and the lactate concentration is higher because of anaerobic fermentation. Changes in ATP and phosphate concentrations and pH are known to alter the binding of aldolase (ALD) 4, GAPDH 2~ and PFK s (Fig. 2). A recent modification

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of this technique permits a more rapid measurement of enzyme binding. The tissue is minced finely with a razor blade and centrifuged for 2 h at 100,000 g (Rapid Minced and Spun method). The distribution of the enzyme activities between particulate matter and supernatant is calculated from the observed activities in each phase, and the known water content of the tissue (Table 1)a. Although this latter technique drastically reduces the length of time required to prepare the samples, it still involves a lengthy period of centrifugation which allows changes in cellular conditions to occur. The Pressed Juice technique is similar to that of the Minced and Spun method with the omission of the mincing step; muscle strips are centrifuged at 100,000 g for 24-48 h and enzyme activities in the supernatant (pressed juice) and pellet (dehydrated tissue) are measured. This method suffers the same drawbacks as the Minced and Spun method and also from a filter effect due to the presence of the plasma membrane. As a consequence of this, the free enzyme concentrations from pressed juice experiments are always significantly lower than those obtained by the Minced and Spun method 2,a. For example, more than 40% of the total ALD and lactate dehydrogenase (LDH) activity was not recovered after repeated washing of 'pressed muscle strips' in high ionic strength buffer2. A modified version of this method involves pressing muscle strips at 264 tons/in. 2 in a French Pressure Cell for 15 s (Quickly Pressed methodS). The free enzyme concentration is obtained from the pressed juice (taking into account the extracellular volume) and the percentage bound is calculated from the total activity measured in non-pressed strips. The large pressures employed in this technique should negate any filter effect caused by the presence of the plasma membrane (see Table 1). The measurement of enzyme binding by Glutaraldehyde Fixation of proteins has been used to determine the degree of enzyme association in synaptosomes 39. The procedure involves incubating an intact synaptosome preparation with increasing glutaraldehyde concentrations for 10 rain, washing the syaaptosomes, and measuring the activity that precipitates after centrifugation. Problems with this technique are: (1) whole tissues cannot be used as all cells must be equally exposed to the glutaraldehyde solution; (2) the time required to isolate cells often changes their metabolic profiles; and (3) since the cytoplasm is approximately 30% protein by weight (which corresponds to a protein concentration of 330 mg/ml) 27 the crowding of the cytoplasmic proteins in vivo may lead to glutaraldehyde-induced covalent protein-protein linkages between pairs of enzymes that may be physically close but may not be actually bound together. The Detergent Solubilization technique involves washing cells in Triton X-100 followed by centrifugation to identify bound enzymes43. Specifically, excised tissue is finely minced with a knife and stirred in 3 vol. of a high ionic strength buffer containing 0.05% Triton X-100. This suspension is centrifuged at 1000 g for 10 min and the pellet re-extracted twice. The bound fraction represents the enzyme activity remaining in the pellet after the third wash and the free enzyme activity is the total of the activities in the three supernatants. The rationale underlying the use of this procedure comes from a desire to minimize the mechanical disruption of the cytoplasmic matrix during the extraction stage 19. A plot of the percentage of activity

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TABLE 2 Comparison of the Dilution method and the Detergent solubilization method for measuring glycolytic enzyme binding in turtle (Pseudemys scripta) brain Enzyme

Dilution method (% bound)

Detergent solubilization (% bound)

PFK ALD GAPDH PGK PK LDH

18 21 21 31 19 27

53 46 42 40 46 49

Percentage of free enzyme was measured in brain from resting turtles. Data from Duncan and Storey 24. Abbreviations: ALD = aldolase; PFK = phosphofructokinase-1; GAPDH = glyceraldehyde 3phosphate dehydrogenase; PGK = phosphoglycerate kinase; PK -- pyruvate kinase; LDH -- L-lactate dehydrogenase.

solubilized at each wash showed that the third wash contributes only 10% to the total free activity suggesting that most of the free enzyme activity has been removed by this step. This procedure is rapid allowing the measurement of bound and free enzyme profiles in tissue samples from animals under a variety of metabolic conditions. However, the results obtained with this procedure are difficult to interpret because of two major problems: (1) the presence of the plasma membrane may inhibit free movement of enzyme from the cytosol to the buffer; and (2) cells situated at the center of the minced tissue are not likely to be exposed to Triton X-100. These problems may lead to an overestimation of the proportion of bound enzyme activity as illustrated in studies with anoxic turtle brain (Table 2). The Detergent method gives approximately twice the bound activity when compared with the Dilution method (which itself overestimates the degree of enzyme binding, Table 1). In general, the procedures used to measure enzyme binding vary widely in their ability to reproduce cytoplasmic conditions and, consequently, results obtained using them must be interpreted accordingly. Note that the limitations of these techniques all favor overestimation of the amount of bound activity. Note also that these techniques do not offer any information on the subcellular location of the bound enzymes: enzymes may be randomly distributed along one or several different organelles or bound together in a functional complex in vivo (particulate matter is composed of fractions of several different organelles). In the absence of any further experimentation, one may conclude that a glycolytic complex had been obtained which was bound to particulate matter. A recent study from our laboratory of enzyme complex formation in fish white muscle illustrates the problems inherent in many of these techniques. We showed that PFK, ALD, GAPDH and phosphoglycerate kinase (PGK) binding increased within 30 s of the initiation of burst swimming in trout when measured with the Dilution method 7. In a later report 8, we measured the binding of PFK and GAPDH using three other techniques: polyethylene glycol precipitation; a modification of the Minced and Spun method (Rapid Minced and Spun method); and a modification

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of the Pressed Juice method (Quickly Pressed method) to reduce the procedural limitations discussed above (Table 1). These newer techniques showed that the correlation between increased binding of GAPDH and increased length of exercise, as measured by the Dilution method, was an artifact; GAPDH was greater than 75% free when measured using the newer techniques as opposed to 25% free using the Dilution method. PFK binding was also reduced from 95% bound by the Dilution method to 70% bound by the Quickly Pressed method (Table 1). These results suggest that experimentally observed changes in enzyme binding may reflect limitations in the methodology and not actual in vivo changes in enzyme binding s.

I l L The search

for a m e t a b o l i c trigger

The identification of a metabolic trigger that mediates changes in enzyme binding in vivo is an important part of the identification of a functional glycolytic complex. The discovery of such a signal will immediately validate the complex by linking physiological changes in muscle metabolism to changes in enzyme binding through a known mediator. The absence of a signal compound does not, in and of itself, mean that an enzyme complex does not exist in muscle tissue. When combined with other data that suggest that enzyme binding is an artifact, however, the absence of a defined signal compound would argue against the existence of a complex. The experiments of this section were performed either with cell free homogenates or by the Dilution method. Despite the limitations of this method, it is effective for monitoring relative changes in the binding of glycolytic enzymes. This is because changes in the putative signal compound should reproduce changes in enzyme binding already measured using the Dilution method. Unfortunately, to date, a putative signal that mediates changes in glycolytic enzyme binding in vivo has not been identified. Cell-free studies with trout white muscle 7.s demonstrated that neither decreased pH, nor increased or decreased calcium concentrations could reproduce the changes in PFK, ALD and GAPDH binding observed after exercise. One can also show that changes in cellular pH occur much later than changes in enzyme binding in exercised trout muscle in vivo 7. Many of the in vivo studies on the putative metabolic trigger were performed with isolated tissues of the whelk (Bus)con caniculatum), a marine gastropod, because: (1) isolated tissues of this species respond to environmental anoxia in a fashion identical to that of the whole animal; and (2) it is relatively easy to alter cellular conditions in these tissues through manipulation of the incubation medium 1~ Results with isolated whelk tissues confirmed and extended the trout studies. In particular, these experiments showed that enzyme binding decreased when metabolic rates were depressed (during anoxia) but at the same time tissue pH had also decreased. This effect was exactly opposite to that observed when the cellular pH of the isolated tissues was artificially altered by incubating tissues in buffers of lower or higher pH (ref. 10). It is also opposite to the effect observed in exercised trout muscle 7 and to in vitro studies on pH and enzyme binding 8,20 ,36,53 ; in both these cases enzyme binding increased with decreasing cellular pH.

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299

Studies with isolated whelk tissues also demonstrated that changes in enzyme binding did not respond to changes in the concentration of protein kinase second messengers (cAMP, cGMP, Ca2+; see ref. 10) or to changes in the concentration of enzyme substrates and/or products. Enzyme substrates were altered by incubating isolated whelk tissues in the presence of iodoacetic acid which inhibited GAPDH activity. Addition of iodoacetic acid caused a 10-100 fold increase in PFK and ALD substrates and a two fold lowering of PK substrates but did not alter the binding of any of these enzymes when measured with the Dilution method 13. Ultimately, the fact that a cellular signal that regulates enzyme binding has not been identified could argue either for and against the existence of a functional glycolytic complex. On the one hand, the difficulty of characterizing a signal suggests that enzyme binding is specifically controlled by an intricate mechanism as yet to be identified. On the other hand, the lack of an identified cellular signal may indicate that enzyme binding is created by diluting a fragile cellular condition which cannot be reproduced in vitro.

IV. In vitro measurement o f enzyme binding In vitro experiments on enzyme binding can only provide a limited amount of information on the glycolytic complex. In general, such studies make use of purified systems often containing only two or three components. Therefore, although they provide details of the physical interaction between an enzyme and a single type of subcellular structure, they cannot, by themselves, show that a glycolytic complex exists. Problems with in vitro experiments also arise. Experiments measuring enzyme-particulate matter binding are often difficult to interpret because enzymes may be bound to one of several different subceUular structures including: the plasma membrane 5~ the microsomal fraction 14, F-actin polymers 6 and/or the outer mitochondrial membrane 64. Furthermore, enzymes may bind to different locations on the same subcellular structure. For example, PFK can bind either F-actin 62 or troponin C (a muscle fibril protein 4~) suggesting that a unique myofibril binding site does not exist for PFK. The localization of individual glycolytic enzymes to all of these structures makes it difficult to unequivocally conclude that all enzymes are binding to a single subcellular location; a unique subcellular binding site may not exist for each enzyme. The results obtained from reconstituted systems are more straightforward since defined species are present. They are, however, artificial by nature and do not give an idea of the true enzyme distribution in vivo. Most experimenters study enzyme binding to filamentous actin (F-actin) as the base structure for formation of a glycolytic enzyme complex. This is because: (1) F-actin binds the largest number of enzymes with the highest affinity; and (2) histochemical studies have localized most of the bound enzymes to the I-band of muscle tissue (see ref. 26 and references therein). Other problems with the in vitro analysis of enzyme binding include the fact that studies are conducted at low ionic strengths and neutral pH because physiological ionic strengths completely dissociate the complexes 4,8,2~ This shows that the

300

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interactions are largely ionic in nature and suggests that they do not occur in vivo (see also section II and Fig. 2). In vitro studies are also, by necessity, performed at protein concentrations well below those found in vivo. The effect of decreased protein concentration is unclear but mathematical calculations 11 suggest that the percentage of enzymes associated with F-actin will increase with increasing protein concentration. Both whole tissue extracts and isolated proteins have been used to quantify enzyme binding. These studies have shown that almost all glyeolytie enzymes bind to F-actin or particulate matter under low ionic strength conditions. Specificity of the enzyme-F-actin interactions is suggested by four different lines of evidence. Firstly, studies of the ALD-F-aetin interaction 62 and the PFK-F-aetin interaction 42,44 demonstrated that the binding phenomenon was saturable and apparently involved specific protein-protein interactions 47. Secondly, some enzymes are released from F-actin by relatively low concentrations of their substrates. This suggests the involvement of enzyme active sites (see section V) and may indicate the existence of a signal compound for enzyme release from F-actin. For example, the binding of PFK to F-actin was inhibited by low concentrations of ATP and ADP in rabbit muscle 42 and bass white muscle 53 but was not inhibited by fructose 6-phosphate 42,53. Binding of ALD to bovine skeletal muscle filaments and F-actin was competitively inhibited by its substrate fructose 1,6-bisphosphate 4,31. GAPDH binding was inhibited by increasing concentrations of fructose 1,6-bisphosphate 39. PK binding to mierotubules was decreased specifically by phosphoenolpyruvate 29 and LDH binding to particulate matter was reduced in the presence of NADH 36. Ca 2+ ions also inhibit the binding of PFK 4~ ALD 32 and GAPDH 8. The demonstration that specific inhibitors of the enzyme-F-aetin interaction exist (i.e. enzyme substrates and produets) argues for a physiologically relevant process. Thirdly, enzyme binding (specifically PFK) is dependent on the degree of enzyme phosphorylation suggesting that binding may be sensitive to cellular signals that change enzyme phosphorylation patterns 44. And fourthly, the binding of PFK 8,~, ALD 4, GAPDH 2~ and PK 1~ to particulate matter and F-actin is affected by changes in cellular pH with increased binding at lower pH values. Although several of the in vitro binding experiments suggest that glyeolytie enzymes bind specifically to F-actin, remember that it is extremely difficult to extrapolate these results to the situation in vivo. For example, although enzyme binding may be sensitive to low concentrations of substrates at low ionic strength, this sensitivity may disappear at the higher ionic strengths in vivo. Please refer to sections II and III for a complete discussion of the limitations of the binding experiments since many of the same caveats apply.

7. I n vitro kinetic studies

The kinetic studies of enzyme-F-actin complexes are perhaps the most important because they determine how a bound enzyme or a glyeolytie complex would function in vivo. Specifically, kinetic studies show whether an enzyme is activated

Is glycolytic rate controlled by the reversible binding of enzymes to subcellular structures?

301

or inhibited when bound to F-actin. If a glycolytic complex is bound to F-actin and if this complex is responsible for higher glycolytic flux, the enzymes in the complex should be at least as active as their soluble counterparts. Note that kinetic studies of individual enzyme-F-actin complexes cannot, by themselves, indicate exactly how a multi-enzyme complex would behave in vivo. This is because the juxtaposition of enzyme active sites in a multi-enzyme structure should create a localized compartment of higher substrate and product concentrations. The creation of a localized compartment will mean higher enzyme activities for bound enzymes because these enzymes 'see' higher substrate concentrations than the soluble enzyme. Such substrate effects are different, however, from the effects of enzyme binding on kinetic constants. In the latter case, changes in enzyme kinetic parameters are due solely to enzyme-F-actin binding. These changes may result either from a direct interaction between an active site (or an allosteric site) and F-actin, or from a conformational change in the enzyme induced by binding. In the former case, competitive inhibition with respect to a substrate (or activator) is observed whereas in the latter case, either the maximal activity and/or the affinity for substrate may be affected. Although the in vitro binding studies were criticized because the experiments were carried out at low ionic strength, this does not apply to the majority of kinetic experiments; as a general rule, the kinetic experiments were performed under conditions that more closely reflected the in vivo ionic strength. However, as with the binding experiments, the total protein concentration in the kinetic experiments was much lower than that found in vivo because of the difficulty in measuring enzyme activities at high concentrations. The effect of lower protein concentrations on the enzyme kinetic patterns is different for different enzymes. For example, PFK is more polymerized at higher protein concentrations 4s and the polymerized enzyme has a higher affinity for fructose 6-phosphate and a lower susceptibility to ATP inhibition 35. Allosteric activators which stimulate activity at low PFK concentrations do not work at high protein concentrations 5 or in permeabilized cells 3 because activity is already maximal. Studies on liver PK kinetics in the presence of polyethylene glycol indicate that PK would also be activated at higher protein concentrations 46. These studies illustrate the importance of protein concentration in determining overall kinetic effects and may indicate that activation of PFK by F-actin at low protein concentration in vitro may not be operative at the higher PFK concentrations in vivo. Table 3 presents the kinetic consequences of enzyme-F-actin binding for the enzymes of glycolysis. In only one case, that of the PFK-F-actin interaction, was the enzyme activated by enzyme binding. All other enzymes were inhibited when bound to F-actin. In the case of PFK, activation resulted from a lower Km for fructose 6phosphate and slightly higher inhibition constant for MgATP 42,44. The combination of a higher affinity for fructose 6-phosphate and a reduced ATP inhibition resulted in an overall PFK activation at low concentrations of fructose 6-phosphate in the presence of F-actin. Thus, the kinetic studies of PFK-F-actin complexes agree with the in vivo and in vitro binding data which suggest that PFK binding increases during periods of high glycolytic activity to increase glycolytic flux. In the case of ALD,

302

S.P..J.Brooksand K.R Storey TABLE 3 Kinetic effect of F-actin binding on selected glycolytic enzymes

Enzyme

Overall effect

Specific changes

PFK

Activation

ALD TPI GAPDH PGK PGM Enolase PK LDH

Inhibition Not known Inhibition Not known Not known Not known Inhibition Inhibition

ATP and citrate less effective inhibitors AMP and Pi better activators Higher affinity for substrate fructose 6-phosphate 42,44 Direct competition of F-actin for substrate TM Lower affinity for substrate glyceraldehyde 3-phosphate 2~

Lower affinityfor substrate phosphoenolpyruvate15 Lzwer affinityfor substrate NADH 36

Abbreviations: ALD = aldolase; PFK = phosphofructokinase-1; GAPDH = glyceraldehyde 3phosphate dehydrogenase; TPI = triosephosphate isomerase; PGK = phosphoglycerate kinase; PK = pyruvate kinase; LDH = L-lactate dehydrogenase; PGM = phosphoglucomutase.

F-actin competed directly with fructose 1,6-bisphosphate for ALD 4,31 so that the F-actin-bound ALD was completely inactive. The binding of GAPDH 2~ PK is and LDH 36 to F-actin also inhibited enzyme activity by increasing the Km value for the enzyme substrates. The anomalous kinetic responses of bound enzymes argue strongly against the concept of a functional glycolytic complex. If glycolytic flux increases as a result of an increase in enzyme binding (as expected from the in vivo studies) one would expect that all enzymes in the complex were, at least, as active as their soluble counterparts. The fact that the majority of bound enzymes are inhibited suggests that enzyme binding is not a mechanism of enzyme activation but rather a negative regulatory mechanism: binding reduces glycolytic flux. These conclusions are supported by a recent report which found that binding of glycolytic enzymes to subcellular structures in red blood cells was a negative control mechanism to s l o w glycolysis 33. Other studies showed that sonicated cells had a much higher rate of glycolysis than did intact or permeabilized cells indicating that the existence of an ordered cellular structure inhibited glycolysis37. The kinetic studies of F-actinbound enzymes suggests that a similar, inhibitory role for enzyme binding may exist in muscle tissue.

VI. Conclusion Do the enzyme-F-actin complexes identified in the present review meet the criteria initially set out for identifying a functional glycolytic complex? (1) The enzymes must readily associate under conditions of physiological ionic strength and protein concentrations. An abundance of physical evidence suggests that a proportion of many of the cytosolic glycolytic enzymes are bound to F-actin in

Is glycolytic rate controlled by the reversible binding of enzymes to subcellular structures ?

303

TABLE 4 Calculated estimates of the percentages of enzymes associated with F-actin in vivo Enzyme

% Bound

PFK ALD TPI GAPDH PGK PK LDH

27 3 7 7 0.04 60 2

Calculations based on in vitro dissociation constants and in vivo ionic strength and protein concentrations 11.

vivo. This is supported by in vitro studies demonstrating enzyme binding at low ionic strength as well as by in vivo studies using a wide variety of methodologies. Although it is extremely difficult to measure protein associations in vivo because most techniques dilute protein and/or ionic strength, the most reliable methods indicate that a defined percentage of some enzymes is associated with particulate matter (Table 1). Mathematical extrapolations using in vitro binding constants also suggest that a large percentage of PFK and PK would be bound to F-actin (Table 4). However, the fact that enzymes may associate with F-actin in vitro (or in vivo) does not necessarily indicate that a functional kinetic complex exists. F-actin is abundant in muscle and it is possible that enzymes are randomly distributed along the filament. This argument illustrates the major problem with the in vivo studies: they fail to localize bound enzymes to a single type of subceUular structure. Demonstrating that enzymes associate with particulate matter is not proof of a functional complex. If other (non-glycolytic) enzymes bind to F-actin a random distribution of several enzymes throughout the subcellular matrix will result. The percentage of enzyme binding may increase/decrease in response to increased/ decreased glycolytic flux but this does not necessarily indicate the formation of a functional glycolytic complex. Glycolytic enzymes must be localized to one specific structure before a functional complex formation is considered a possibility. (2) The enzymes must be kinetically active when bound and formation of the complex must confer a kinetic advantage to the bound enzymes. The kinetic studies of the enzyme-F-actin complexes show that neither condition was fulfilled for ALD, GAPDH, PK and LDH. In fact, ALD is completely inhibited when bound to F-actin. (3) The magnitude of the change in enzyme binding must account for the magnitude of the change in glycolytic flux. A consideration of the situation in trout skeletal muscle shows that this condition is not fulfilled. During the early stages of exercise, glycolytic flux in trout white muscle increased by 10-15 f o l d 21'49'56'63'65. Changes in glycolytic enzyme binding during exercise (Table 1) showed that the largest increases were on the order of 50% (PFK and GAPDH). Unless the activities of the bound enzymes are much higher than those of the soluble enzymes the relatively small increase in enzyme binding cannot support the large increase in

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glycolytic flux. Bound PFK may have a much higher activity than the soluble enzyme because binding changes PFK sensitivity to inhibitors and activators (the effects are synergistic). This is not true for ALD, GAPDH, PK and LDH: all these enzymes are inhibited when bound. These results suggest that changes in ALD, GAPDH, PK and LDH enzyme binding are not related to changes in glyeolytie flux in vivo. Does enzyme binding play a role in modulating glyeolytie rates? PFK binds tightly to F-aetin under in vivo conditions of high ionic strength, high protein concentrations, and in vivo pH. Binding of PFK to F-actin increases in response to increased glyeolytie flux. PFK is also activated by binding to F-aetin and this effect is modulated by reversible phosphorylation. All these factors suggest that binding of PFK is intimately linked to the energy state of the cell. In light of the fact that the other glycolytic enzymes are inhibited when bound to F-actin, what function could this binding have? It may serve to localize PFK in the vicinity of the myosin ATPase where it would be more susceptible to changes in the adenosine nueleotide tri-/diphosphate pool. A greater interaction between PFK and the myosin ATPase would exist because a shorter distance between these enzymes would lead to a localized higher nucleotide pool concentration. Thus, during periods of high glyeolytie flux, localized low ATP concentrations and high ADP and phosphate concentrations (brought about through muscle myosin ATPase activity) would activate PFK by: (1) reducing the concentration of the PFK inhibitor ATP; and (2) increasing the concentration of the PFK activators ADP and phosphate. Because PFK is a key regulatory enzyme of the cell, changes in its activity would result in large changes in the overall glyeolytie flux. A closer correspondence between PFK activity and the myosin ATPase activity would, therefore, provide a tighter link between glyeolytie flux and ATP demand resulting in quicker response in glycolytie rates to energy needs in the working muscle. Acknowledgements. Work on glycolytic complexes continues in the laboratory of K.B.S. thanks to research grants from NSERC (Canada) and NIH (U.S.A., GM 43796). The authors also wish to thank J.M. Storey for editorial criticisms.

VII. References 1. Amberson, W.R., A.C. Bauer, D.E. Philpott and E Roisen. Proteins and enzyme activities of press juices, obtained by ultracentrifugation of white, red and heart muscles of the rabbit. J. Comp. Cell. Physiol. 63: 7-21, 1964. 2. Amberson, W.R., E Roisen and A.C. Bauer. The attachment of glycolytic enzymes to muscle ultrastructure. J. Comp. Cell. Physiol. 66: 71-90, 1965. 3. Aragon, J.J., EE. Feliu, R. Frenkel and A. Sols. Permeabilization of animal cells for kinetic studies of intracellular enzymes: In situ behavior of the glycolytic enzymes of erythrocytes. Proc. Natl. Acad. Sci. USA 77: 6324-6328, 1980. 4. Arnold, H. and D. Pette. Binding of aldolase and triosephosphate dehydrogenase to F-actin and modification of catalytic properties of aldolase. Eur. J. Biochem. 15: 360-366, 1970. 5. Bosca, L., J.J. Aragon and A. Sols. Modulation of muscle phosphofructokinase at physiological concentration of enzyme. J. Biol. Chem. 260: 2100-2107, 1985. 6. Bronstein, W.W. and H.R. Knull. Interaction of muscle glycolytic enzymes with thin filament protein. Can. J. Biochem. 59: 494-499, 1981.

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7. Brooks, S.P.J. and K.B. Storey. Subcellular enzyme binding in glycolytic control: in vivo studies with fish muscle. Am. J. Physiol. 255: R289-R294, 1988. 8. Brooks, S.P.J. and K.B. Storey. Reevaluation of the "glycolytic complex" in muscle: a multitechnique approach using trout white muscle. Arch. Biochem. Biophys. 267: 13-22, 1988. 9. Brooks, S.P.J. and K.B. Storey. Glycolytic enzyme binding and metabolic control in estivation and anoxia in Otala lactea. J. Exp. Biol. 151: 193-204, 1990. 10. Brooks, S.P.J. and K.B. Storey. Studies on the regulation of enzyme binding during anoxia in isolated tissues of Busycon canaliculatum. J. Exp. Biol. 156: 467-481, 1991. 11. Brooks, S.P.J. and K.B. Storey. A quantitative evaluation of the effect of enzyme complexes on the glycolytic rate in vivo: mathematical modeling of the glycolytic complex. J. Theor. Biol. 149: 361-375, 1991. 12. Brooks, S.P.J. and K.B. Storey. Where is the glycolytic complex? A critical evaluation of present data from muscle tissue. FEBS Letts. 278: 135-138, 1991. 13. Brooks, S.P.J. and K.B. Storey. Control of glycolytic enzyme binding: effect of changing enzyme substrate concentrations on in vivo enzyme distributions. Mol. Cell. Biochem. 122: 1-7, 1993. 14. Caswell, A.H. and A.M. Corbett. Interaction of glyceraldehyde 3-phosphate dehydrogenase with isolated microsomal subfractions of skeletal muscle. J. Biol. Chem. 260: 6892-6898, 1985. 15. Chart, L.M., T. Hickmon, C.J. Cobbins and Y.Y. Davidson. The interaction of rabbit muscle pyruvate kinase with F-actin. Fed. Proc. 45: 1657, 1986. 16. Clarke, EM. and D.J. Morton. Glycolytic enzyme binding in fetal brain - the role of actin. Biochem. Biophys. Res. Comm. 109: 388-393, 1982. 17. Clarke, EM., ED. Shaw and D.J. Morton. Effect of electrical stimulation post-mortem of bovine muscle on the binding of glycolytic enzymes. Biochem. J. 186: 105-109, 1980. 18. Clarke, EM., P. Stephan, G. Huxham, D. Hamilton and D.J. Morton. Metabolic dependence of glycolytic enzyme binding in rat and sheep heart. Fur. J. Biochem. 138: 643-649, 1984. 19. Clegg, J.S. Properties and metabolism of the aqueous cytoplasm and its boundaries. Am. J. Physiol. 246: R133-R151, 1984. 20. Dagher, S.M. and H.O. Hultin. Association of glyceraldehyde 3-phosphate dehydrogenase with the particulate fraction of chicken skeletal muscle. Fur. J. Biochem. 55: 185-192, 1975. 21. Dobson, G.E, E. Yamamoto and P.W. Hochachka. Phosphofructokinase control in muscle: nature and reversal of pH-dependent ATP inhibition. Am. J. Physiol. 250: R71-R76, 1986. 22. Dobson, G.E, W.S. Parkhouse and P.W. Hochachka. Regulation of anaerobic ATP-generating pathways in trout fast-twitch skeletal muscle. Am. J. Physiol. 253: R186-R194, 1987. 23. Duncan, J.A. and K.B. Storey. Role of enzyme binding in muscle metabolism of the goldfish. Can. J. Zool. 69: 1571-1576, 1991. 24. Duncan, J.A. and K.B. Storey. Subcellular enzyme binding and the regulation of glycolysis in anoxic turtle brain. Am. J. Physiol. 262:R517-R523, 1992. 25. Durrieu, C., R. Bernier-Valentin and B. Rousset. Microtubules bind glyceraldehyde 3-phosphate dehydrogenase and modulate its enzyme activity and quaternary structure. Arch. Biochem. Biophys. 252: 32-40, 1987. 26. Freidrich, P. Supramolecular Enzyme Organization, Oxford, Pergamon Press, pp. 152-172, 1988. 27. Fulton, A.B. How crowded is the cytoplasm? Cell 30: 345-347, 1982. 28. Goldhammer, A.R. and H.H. Paradies. Phosphofructokinase: structure and function. Curr. Top. Cell. Reg. 15: 109-141, 1979. 29. Hackney, D.D. Pyruvate kinase binding to microtubules is dependent on the absence of PEP. Biophys. J. 57: 348a, 1990. 30. Hall, E.R. and G.L. Cottam. lsozymes of pyruvate kinase in vertebrates: their physical, chemical, kinetic and immunological properties. Int. J. Biochem. 9: 785-793, 1978. 31. Harris, S.J. and D.J. Winzor. Enzyme kinetic evidence of active-site involvement in the interaction between aldolase and muscle myofibrils. Biochim. Biophys. Acta 911: 121-126, 1987. 32. Harris, S.J. and D.J. Winzor. Effect of calcium on the interaction of aldolase with rabbit muscle myofibrils. Biochem. Biophys. Acta 999: 95-99, 1989. 33. Harrison, M.L., P. Rathinavelu, P. Arese, R.L. Geahlen and ES. Low. Role of band 3 tyrosine phosphorylation in the regulation of erythrocyte glycolysis. J. Biol. Chem. 266: 4106-4111, 1991. 34. Hochachka, P.W. Intermediary metabolism in fishes. In: Fish Physiology, Vol. 1, edited by W.S. Hoar and D.J. Randall, New York, Academic Press, pp. 351-390, 1965. 35. Hofer, H.W. Influence of enzyme concentration on the kinetic behavior of rabbit muscle phosphofructokinase. Hoppe-Seyler's Z. Physiol. Chem. 352: 997-1003, 1971.

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36. Hultin, H.O. Effect of environment on kinetic characteristics of chicken lactate dehydrogenase isozymes. In Isozymes II, Physiology and Function, edited by C.L. MarkeR, New York, Academic Press, pp. 69-85, 1975. 37. Jackson, S.A., M.J. Thomson and J.S. Clegg. Glycol)sis compared in intact, permeabilized and sonicated L-929 cells. FEBS Letts. 262: 212-214, 1990. 38. Knull, H.R. Association of glycolytic enzymes with particulate fractions from nerve endings. Biochem. Biophys. Acta 522: 1-9, 1978. 39. Knull, H.R. Compartmentation of glycol)tic enzyme in nerve endings as determined by glutaraldehyde fixation. J. Biol. Chem. 255: 6439-6444, 1980. 40. Lan, J. and R.E Steiner. Effects of calcium-binding proteins on phosphofructokinase and characteristics of their bindings. Biophys. J. 57: 1470, 1989. 41. Lan, J. and R.E Steiner. The interaction of troponin C with phosphofructokinase. Comparison with calmodulin, Biochem. J. 274:445-451, 1991. 42. Liou, R.S. and S. Anderson. Activation of rabbit muscle phosphofructokinase by F-actin and reconstituted thin filaments. Biochemistry 19: 2684-2688, 1983. 43. Lowery, M.S., S.J. Roberts and G.N. Somero. Effects of starvation on the activities and localization of glycol)tic enzymes in the white muscle of the barred sand bass Paralabrax nebulifer. Physiol. Zool. 60: 538-549, 1987. 44. Luther, M.A. and J.C. Lee. The role of phosphorylation in the interaction of rabbit muscle phosphofructokinase with F-actin. J. Biol. Chem. 261: 1753-1759, 1986. 45. Luther, M.A., H.E Gilbert and J.C. Lee. Self-association of rabbit muscle phosphofructokinase: role of subunit interaction in regulation of enzyme activity. Biochemistry 22: 5494-5403, 1983. 46. Medina, R., J.J. Aragon and A. SoB. Effect of polyethylene glycol on the kinetic behavior of pyruvate kinase and other potentially regulatory liver enzymes. FEBS Lefts. 180: 77-80, 1985. 47. Mejean, C., E Pons, Y. Benjamin and C. Roustan. Antigenic probes locate binding sites for the glycol)tic enzymes glyceraldehyde 3-phosphate dehydrogenase, aldolase and phosphofructokinase on the actin monomer in microfilaments. Biochem. J. 264: 671-677, 1989. 48. Pagliaro, L. and D.L. Taylor. Aldolase exists in both the fluid and solid phases of cytoplasm. J. Cell. Biol. 107: 981-991, 1988. 49. Parkhouse, W.S., G.P. Dobson, A.N. Belcastro and P.W. Hochachka. The role of intermediary metabolism in the maintenance of proton and charge balance during exercise. Mol. Cell. Biochem. 77: 37-47, 1987. 50. Pierce, G.N. and K.D. Philipson. Binding of glycolytic enzymes to cardiac sarcolemmal and sarcoplasmic reticular membranes. J. Biol. Chem. 260: 6862-6870, 1985. 51. Plaxton, W.C. and K.B. Storey. Glycol)tic enzyme binding and metabolic control in anaerobiosis. J. Comp. Physiol. 156B: 635-640, 1986. 52. Reid, S. and C.J. Masters. On the ontogeny and interactions of phosphofructokinase in mouse tissues. Int.J. Biochem. 18: 1097-1105, 1986. 53. Roberts, S.J., M.S. Lowery and G.N. Somero. Regulation of binding of phosphofructokinase to myofibrils in the red and white muscle of the Barred Sand Bass Paralabrax nebulifer (Serranidae). jr. Exp. Biol. 137: 13-27, 1988. 54. Shearwin, K., C. Nanhua and C. Masters. Interaction between glycol)tic enzymes and cytoskeletal structure - the influence of ionic strength and molecular crowding. Biochem. Int. 21: 53-60, 1990. 55. Srere, P.A. Complexes of sequential metabolic enzymes. Ann. Rev. Biochem. 56: 89-124, 1987. 56. Storey, K.B. Metabolic consequences of exercise in organs of rainbow trout. J. EXp. Zool. 260: 157-164, 1991. 57. Storey, ICB. and J.M. Storey. Facultative metabolic rate depression: molecular regulation and biochemical adaptation in anaerobiosis, hibernation, and estivation. 0.. Rev. Biol. 65: 145-174, 1990. 58. Van den Thillart, G. Adaptations of fish energy metabolism to hypoxia and anoxia. Mol. Physiol. 2: 49-61, 1982. 59. Van den Thillart, G., E Kesbeke and A. van Waarde. The influence of anoxia on the energy metabolism of goldfish Carassias auratus (L.) Comp. Biochem. Physiol. 59A: 329-336, 1976. 60. Van Waversveld, J., A.D.E Addink and G. van den Thillart. Simultaneous direct and indirect calorimetry on normoxic and anoxic goldfish. J. E ~ . Biol. 142: 325-335, 1989. 61. Van Waversveld, J., A.D.E Addink and G. van den Thillart. The anaerobic energy metabolism of goldfish determined by simultaneous direct and indirect calorimetry during anoxia and hypoxia. J. Comp. Physiol. 159B: 263-268, 1989. 62. Walsh, T.P., D.J. Winzor, EM. Clarke, C.J. Masters and D.J. Morton. Binding of aldolase to aetin-containing filaments. Biochem. J. 186: 89-98, 1980.

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63. Wieser, W., U. Platzer and S. Hinterleitner. Anaerobic and aerobic energy production of young rainbow trout (Salmo gairdneri) during and after bursts of activity. J. Comp. Physiol. 155B: 485-492, 1985. 64. Wilson, J.E. Brain hexokinase, the prototype ambiquitous enzyme. Cute. Top. Cell. Regul. 16: 1-44, 1980. 65. Wokoma, A. and I.A. Johnston. Anaerobic metabolism during activity in the rainbow trout ($almo gairdneri). Experientia 39: 1366-1367, 1983.

Hochachka and Mommsen (eds.), Biochemistry and molecular biology of fishes, vol. 4 9 1995 Elsevier Science B.V. All rights reserved.

CHAPTER 14

Histidine-related dipeptides: distribution, metabolism, and physiological function HIROKI ABE Department of Food Science and Nutrition, Kyoritsu Women's University, 1-710Motohachioji, Hachioji, Tokyo 193, Japan

I. II. Ill. IV. V. VI. VII.

Introduction Distribution of free L-histidine and related dipeptides in fish tissues Effects of physiological conditions on the dipeptide concentrations in fish muscle Metabolicturnover of L-histidine in fish Metabolismand interorgan transport of the dipeptides in fish Physiologicalfunctions of histidine-related dipeptides as proton buffers References

I. Introduction Animal tissue has long been known to contain quite a few different types of L-histidine-related dipeptides (imidazole dipeptides). The dipeptides refer to carnosine (fl-alanyl-L-histidine), anserine (fl-alanyl-Tr-methyl-L-histidine), balenine (/5-alanyl-r-methyl-L-histidine, also called ophidine), and homocarnosine (yaminobutyryl-L-histidine). Of these dipeptides, carnosine, anserine, and balenine may occur in vertebrate skeletal muscle in large amounts depending on the species. Fig. 1 shows the structures of these muscle dipeptides.

+

CO0-

0

1

i

Carnoslne

I

i

Anserine

H,,N.~N~.H

+ CO0t I 3 N ' ~ N H ' ~

0 +

C H3"N'~N'~H

CO0-

0

1 i BalenIne H,,N,~N~cH9

Fig. 1. Chemical structures of histidine-related dipeptides.

310

H. Abe

Camosine was first isolated from meat (came) extract by Gulewitsch and Amiradzibi 27 in 1900 and the structure was established by Baumann and Ingvaldsen 14. Anserine was isolated from goose (Anseriformes) muscle in 1929 by Ackermann and eoworkers n and identified as ~-alanyl-l-methyl-L-histidine. In the ease of balenine, however, confusion has arisen over its structure. The compound was first isolated in 1962 by Pocchiari and colleagues 67 from the muscle of a baleen whale, Balaenoptera sp., hence the name. Long before this discovery, Imamura 3s, in 1939, isolated a dipeptide named ophidine (derived from Ophiophagus sp.) from snake muscle and it was wrongly identified as/5-alanyl-2-methyl-L-histidine43. The correct structure of ophidine was finally established by Nakai and Tsujigado 6~ in 1965 and found to be identical to that of balenine. Thus, the name of the dipeptide was finally unified into balenine sg. However, the confusion about the name goes on unabated today, since some people still use the term ophidine and, not to be outdone, ophidine is identified as ~-alanyl-2-methyl-L-histidine even in Chemical Abstracts. The nomenclature of methyl derivatives of L-histidine has also been somewhat confusing because the numbering system of the imidazole ring of L-histidine was reversed in the chemical and the biochemical literature. IUPAC-IUB 39 finally, in 1974, recommended that the imidazole ring nitrogen close to the a-carbon of Lhistidine should be named as pros (Tr) and that far from the a-carbon as tele (r). Thus, formerly 1-methyl-L-histidine should be designated as ~r-methyl-L-histidine and the 3-methyl derivative as r-methyl-. Since the discovery of these dipeptides, various methods have been developed to determine their concentrations in animal tissues. These preparative and analytical methods were reviewed by Crush 23. Supplanting early methods using paper chromatography and cation exchange chromatography, modified techniques of amino acid analysis for physiological fluid were used in the 1970s2~ Finally, fast and accurate determination methods using ion-exchange and reversed phase highperformance liquid chromatography (HPLC) were adopted in the early 1980s1'9.1s. Fig. 2 shows a typical separation of histidine-related compounds by ion-exchange HPLC. Extensive studies have been undertaken to date on the distribution, metabolism, and physiological functions of these dipeptides in animal tissues. Table 1 represents a brief summary of the distribution of histidine and the dipeptides in the skeletal muscle of various animals. Although the distribution of the dipeptides is restricted in the muscle of invertebrate and most white-fleshed fishes such as percid and pleuronectid fishes, large amounts of these dipeptides are found in the wide variety of vertebrate muscles. Their distribution, however, lacks any phylogenetic basis. Balenine, for instance, which is found only in trace amount in most vertebrate muscles, is abundant in the muscle of snakes and whales. Camosine, on the other hand, is rather common in vertebrate muscles, but some fishes and birds contain much higher amounts of anserine than camosine. In spite of such high concentrations of these dipeptides in animal muscle, their physiological functions have not been satisfactorily elucidated. Free L-histidine which is one of the constituents of camosine is abundant in every tissues of animals because it is a proteinaceous amino acid, although it is

Histidine-related dipeptides: distribution, metabolism, and physiological function

311

m

,.?,

i

r--'I

0

I

III I

I

I

IN

I

10 20 RETENTION TIME (min)

II I am

30

Fig. 2. Separation of authentic histidine-related compounds (2 nmol each) by high-performance liquid chromatography. Conditions: chromatograph, Shimadzu LC-6A; column, Nucleosil 5-SB cationexchanger (diameters: 4.6 x 250 ram, Nagel); mobile phase, 90 mM KI-12PO4 (pH 5.0) containing 10% methanol; temperature, 55"C; flow rate, 1 ml/min; detection, UV 210 nm. Crn = creatinine; His ffi L-histidine; Car - carnosine; ~r-meHis = rr-methyl-L-histidine; ~-meHis = r-methyl-L-histidine; Ans ffi anserine; Bal ffi balenine.

normally below 1 mM in animal muscles. As seen in Table 1, however, several fishes such as tunas and mackerels, salmon, and carp contain large amount of free L-histidine in their white muscle. Physiological function of this large amount of L-histidine has also been obscure. In contrast to these compounds, levels of rr- and r-methyl-histidine and homocarnosine are very low in animal muscles. Therefore, in the following, we focus our attention mainly on free L-histidine, carnosine, anserine, and balenine.

II. Distribution of free L-histidine and related dipeptides in fish tissues Since the early work of Lukton and Olcotd s, there have been many reports on the distribution of these dipeptides in fish muscle. Table 2 shows a typical distribution of these dipeptides in fish and whale muscles containing large amounts of the dipeptides mainly determined during this decade. Marine pelagic high-speed swimmers with myoglobin-rich dark flesh, such as tuna, sardine, mackerel, contain

312

H. Abe TABLE 1 Distribution of histidine-related dipeptides in animal skeletal muscles

Animals Invertebrates Fish Elasmobranchii Teleostei Scombrinae Salmoninae Anguillinae Cyprininae Percinae

Histidine

Carnosine

4-

4-

+

+

20,,- 120 ~10 + 4~ 25 +

~ 10 + 7-,,25 + 4-

Anserine 4-

Balenine 4-

1-,-33

+

~ 120 7~42 + + 4-

44+ 44-

Amphibians Frog

+

2~17

+

4-

Reptiles Snakes Crocodiles, turtles

+ +

l,-d0 +

+ 1-,- 7

2,-,13 4-

Birds Pigeon, chicken

+

,-,13

5,,~43

4-

Mammals Kangaroo Baleen whales Toothed whales Seals Cat, lion Ox, bison Sheep, goat Horse Pig Rabbit Deer Rat Apes Human

+ + + + + + + + + + + + + +

2"-, 4 6-,-13 10~20 9,-,26 4~ 16 10,-,20 2-,- 8 -~34 12~21 ~2 ~-4 4,-, 6 17---23 2~ 8

8,-,16 + 1~ 6 3,-, 5 6~ 18 1-~ 4 5~ 9 + -,,1 14-, 19 -,,14 3,~ 9 + ~2

450-~65 16-,,25 44+ + + ~,2 4~4 444-

Values are given in/zmol per g muscle weight. + = < 1/zmol g-1 muscle; 4- = trace or not determined. Data compiled from references 1-3, 6, 7, 20, 21, 23, 72, 75-79, and 82.

copious amounts of free L-histidine in their white muscle. This is an extraordinary feature absent in other animals thus far examined 44,ts,75. The L-histidine content in the white muscle ranged from 15 to 94 ?tmol/g in dark-fleshed fishes, from 4 to 20/~mol/g in 'intermediate' fishes, and from 0.07 to 1/~mol/g in white-fleshed species 3. Carp muscle is an exception in that it contains rather high concentration of L-histidine 2, though muscle color is white. The level in red muscle of these fishes, however, is much lower than that of white muscle as seen in Table 2. Together with L-histidine, carnosine, anserine, and balenine are also observed in the muscle of various fishes as well as other vertebrate. In contrast to L-histidine, contents of these histidine-related compounds do not depend on the muscle color 3 but are highly species specific3'23. These dipeptides may be widely distributed in fish

Histidine-related dipeptides: distribution, metabolism, and physiological function

313

TABLE 2 Free L-histidine and related dipeptides in fish white and red muscles and whale muscle Classification

Species

n

Muscle

His 39.6 9.64 4.59 0.41 2.57 0.59 1.80 2.38

Car

Ans

Bal

+ +

+ +

+ +

0.56 + + + + +

24.5 16.8 17.2 3.02 5.30 1.59

+ + + + +

+ + +

+ + <0.16

+ + +

18.3 5.16

<0.3 <0.85

Fishes

Clupeioidei

Clupeidae, sardine 3

6

Salmonoidei

Salmonidae, salmon 59,69 (4 species) Rainbow trout (j)2,4 Rainbow trout (C) 4

6 5

Osmeridae, smelt (wild) 75 Osmeridae, smelt (cultured)

7 6

White Red White White White Red White White

Cyprinoidei

C~rinidae, carp 2 Others2(6 species) Cobitidae, pond loach 2

3 3

White White White

10.1 5.10 4.52

Anguilloidei

Anguillidae, Japanese eel 2 Congridae, conger eel 2

10 2

White White

0.37 4.35

Exocoetoidei

Exocoetidae, flyingfish (A) 3

5

Scombroidei

Scombridae, tunas 79,s0 (4 species)

-

Skipjack tuna 0 ) 3

3

Skipjack tuna (H) 6

5

Kawakawa (H) 6

3

Common mackerel 3

6

Histiophoridae, blue marlin (H) 7

5

Carangidae, horse mackerel 3

6

Sphyraenidae, Japanese barracuda 3

5

White Red White Red White Red White Red White Red White Red White Red White Red White

31.1 1.75 62.9 il.0 89.5 17.3 93.5 13.3 75.4 6.51 24.9 9.88 15.9 4.79 11.2 2.35 4.86

Fin whale Sei whale Little piked whale Sperm whale Pilot whale

5 5 3 2 2

Dorsal Dorsal Dorsal Dorsal Dorsal

0.11 0.13 0.24 0.18 0.17

Mugiloidei

+ 8.43 + 1.59 + 27.3 + 7.92 4.52 15.4 0.69 3.99 2.90 51.1 0.56 7.31 1.09 27.7 0.35 4.48 <0.24 <0.05 <0.06 + 2.64 105 + 21.1 + + + + + <0.95

<0.15 <0.3 + + + + + + + + + + + + + + +

Whales 7s

Mystacoceti (Baleen whale) Odontoceti (Toothed whale)

5.74 9.35 5.92 8.89 1.11

0.20 0.47 1.44 4.37 1.59

61.0 72.9 78.0 0.10 23.0

Values are presented as averages in /zmol g-1 muscle. + = trace; - = not determined. His = Lhistidine; Car = carnosine; Ans = anserine; Bal = balenine. Numbers in parentheses are species numbers averaged. Place of capture: (A) = Atlantic Ocean; (J) = Japan; (H) = Hawaii. Fishes without a designation were all caught around Japan.

muscles, a l t h o u g h the distribution of b a l e n i n e may be limited. T h e c o n c e n t r a t i o n s o f b a l e n i n e a r e v e r y l o w in m o s t fish m u s c l e s , b u t q u i t e h i g h in t h e s k e l e t a l m u s c l e of several whales, and no systematic differences are apparent between baleen and toothed whales.

314

H. Abe

In fish red muscle concentrations of histidine-related peptides are generally low, corresponding to one tenth to one quarter of their concentration in white. As far as the dipeptide distribution is concerned, the concentrations in white muscle are closely related to the swimming ability of the fish species. These compounds in fish muscle showed no significant difference among muscle portions of the dorsal or ventral muscle, though dorsal muscle showed slightly higher levels3. Individual difference of the concentration of these compounds is small in a given species, and, as results for histidine in rainbow trout (Oncorhynchus mykiss) and for anserine in skipjack tuna (Katsuwonuspelamis) show, rather substantial differences between regional populations may exist (Table 2). Generally, fishes belonging to the same suborder group, however, reveal quite similar distribution patterns of these compounds irrespective of the family as seen in Salmonidae, Cyprinidae, and Anguillidae2. Other fishes with less pronounced swimming ability contain much smaller amounts of these compounds in their white muscle. In contrast to the large amounts of these compounds in fish skeletal muscle, the levels are extremely low in cardiac muscle as well as smooth muscles, such as stomach and intestine. In the skipjack tuna, only small amounts of the dipeptides are found in non-muscular tissue, such as liver, brain, gill, kidney, spleen, pylorie caecum, and ovary3. Blood levels of these compounds are also very limited and well below concentrations determined in skeletal muscle tissue. Therefore, the distributions of these compounds in fish tissues clearly suggest that these compounds in fish muscle play a role in swimming function and performance. Among all fishes examined to date, skipjack tuna Katsuwonuspelamis and Pacific blue marlin Makaira nigricansexemplify one end of the extremes. Live skipjack tuna (1-2 kg body weight) captured off Hawaii on hook and line showed large amounts of L-histidine and anserine and lesser amount of carnosinr in their white muscle. The level of L-histidine was on average 93.5/~mol/g reaching a maximum of 105 /zmol/g, which is highest value measured for all species thus far examined. Ansefine was similarly abundant, reaching a maximum of about 69.1 ~mol/g, with an average concentration of 51.1/~mol/g (ref. 6). The total content of these compounds thus approaches 150/~mol/g in the white muscle. In contrast to skipjack tuna, Pacific blue marlin (50-100 kg body weight), caught off Hawaii during the Annual Billfish Tournament, showed the highest anserine content of all animals thus far examined, with an average value of 105 ~mol/g, and a maximum of 120/~mol/g (ref. 7). The combined total of histidine and related compounds in marlin white muscle reached above 120/~mol/g. The levels in the red muscle of both species, however, amount to 15-30% of those attained in the white muscle. Fish white muscle is known to be a highly anaerobic muscle and thought to be employed only during the burst swimming. In contrast, the red muscle is used during, generally slower-speed, sustainable swimming33's4. In this connection, tunas arc considered to be the 'ultimate teleosts '33 because of their ability to reach high burst swimming speeds 25,s7 and maintain muscle temperatures as high as 10~ above ambient temperatures 25.71 . The ability to engage in burst swimming of tunas and marlin is sustained by the elevated capacities for anaerobic glycolysis in the white muscle, typically represented

Histidine-related dipeptides: distribution, metabolism, and physiologicalfunction

315

by the activity of lactate dehydrogenase, which in tuna is the highest so far found in any animal 28. During burst swimming, large amounts of lactate, nearly 100 #mol/g muscle, are accumulated as an end product in tuna white muscle 29. Therefore, the highest amounts of L-histidine and related dipeptides in the anaerobic white muscle of tunas and billfishes may indicate that these dipeptides contribute to the extreme ability for anaerobic burst swimming. A critical analysis the data collated in Tables 1 and 2 clearly leads to the conclusion of a positive correlation between the concentration of these dipeptides and the ability of a given species for the anaerobic exercise such as burst swimming, sprint running, flying, and diving.

III. Effects of physiological conditions on the dipeptide concentrations in fish muscle It may be quite difficult to understand the unknown physiological function of a compound in spite of the unequivocal distribution of the compound in tissue. Fishes, however, should be most suitable experimental animals to unveil the physiological role(s) of these dipeptides, because of the wide spectrum of their distribution patterns is found in fish species, fish comprise two functionally and spatially different muscle types (white and red), and fish can adapt and acclimate to various physiological and ecological environments. Thus, in this section I would like to examine the effects of physiological conditions on the concentrations of these dipeptides for obtaining some clues for their physiological roles. A number of independent observations have been reported in the literature and these may lead to a unified interpretation. As seen in Table 2, the content of anserine in the white muscle of cultured smeltfish Plecoglossusaltivelis is lower than that determined for their feral counterparts. Hawaiian skipjack tuna contains much higher concentrations of anserine than the Japanese counterpart. Existing large concentration differences of these dipeptides in different subpopulation of one species, suggest that environmental factors/life style factors may play an important role. It has been reported that carnosine in chinook salmon (Oncorhynchus tshawytscha) decreased when the fish were maintained on a histidine-deficient diet, while anserine did not 47. Similarly, the anserine level was also shown to be kept fairly constant during spawning migration of sockeye salmon (Oncorhynchus nerka), at a time when the white muscle concentration of L-histidine decreased sharply 22,59,92,93. Because of these substantial concentrations and their obvious contribution to intramuscular osmotic pressure, the potential role of these compounds in osmoregulation in fish muscle needs to be examined. As shown in Fig. 3, carnosine concentrations in the muscle of Japanese eels (Anguillajaponica) were largely independent of the external salinity 9,36, though L-histidine was slightly increased in fish adapted to half-strength and full-strength seawater. Along with the increase of external salinity total amino acids in the white muscle of rainbow trout (Oncorhynchus mykiss) increased about two-fold, mainly accounted for by increases in

316

H. Abe w ",

2.0r

o

9

/

r/

= 125

/ I

'/

3

=

I'

~

//Trout-His\

i

i

i ...... =

='|

Eel-Car

'i

15 ir~T Trout,_~Ar~

l, okX..Imx / a -H"

/

,.

/9 s i (~.-9Eel=His 9. n ~ 0l i ' i , , FW SW FW FW SW FW -

(--)

Fig. 3. Changes of L-histidine and dipeptides in the muscle of eel, rainbow trout, and Japanese dace during the acclimation to sea water. Mean and the standard deviation of three fish are shown. FW = freshwater; SW = seawater. See the legend of Fig. 2 for additional abbreviations. Redrawn from ref. 9.

non-imidazole amino acids such as glyeine and alanine 9. Histidine as well as lysine and arginine also increased about 5-fold of the fresh water level in half-strength seawater and seawater (Fig. 3). These increases in histidine, however, correspond to only 4% of the total augmentation of the tissue amino acid pool. Anserine in trout white muscle was maintained at an almost constant level during the seawater acclimation, although it was slightly increased in seawater-adapted specimens. This is also the case for histidine in a cyprinid, Japanese dace Leuciscus haRonennsis. Because eel and trout contain fairly large amounts of carnosine and anserine, respectively, relative to the total amino acid pool, it might be predicted that these dipeptides contribute to the basal tissue osmotic pressure. The contribution of the dipeptides to osmoregulation of the white muscle is, however, likely to be negligible. Fish usually show appreciably high tolerance to prolonged starvation, and an analysis on changes in histidine and dipeptide behavior during starvation may deliver important clues as to the general function of these compounds in teleostean fishes. During the starvation of eel and rainbow trout for 200 and 62 days, respectively, white muscle L-histidine decreased significantly in both species9 as depicted in Fig. 4. This is also the case for Japanese dace (Leuciscus hakonennsis), a species containing large amounts of free L-histidine9. Camosine in eel and anserine in trout, on the Other hand, showed significantly small changes during starvation, with eamosine in eel gradually decreasing after 100 days starvation. The decrease of free L-histidine may be related to this compound serving as a direct energy source for skeletal muscle itself during starvation. As described above, Mommsen et al. 59 showed that free L-histidine in white muscle of sockeye salmon plummeted to one

Histidine-related dipeptides: distribution, metabolism, and physiologicalfunction

2.5

0.5

2.0

0.4 ~

9

1.0

lo .

I:" == I - I-I ir

-

-*

-0.2 "~

.......

I

4-,

"0"5 r '\. 4r~ C: o

$"

u 0 , 25

E15 m

'l

I

I

I

-

.,

/o Trout-

.

":I0

u

0

I

L

~E 0'1~"0E

"~:q

-e

n

l

317

/

0

4"OC5"

~-*"~"*Dace-His

U

0

I

0

I

40

I

I

I

I

80 120 160 200 Days

Fig. 4. Changes of the level of L-histidine and dipeptides in the white muscle of eel, rainbow trout, and Japanese dace during starvation. Mean and the standard deviation for three fish are shown. Redrawn from ref. 9.

tenth of control values during the spawning migration upstream, while the anserine content did not change at all. This may be due to the forced starvation of this species during the prolonged upstream migration. Much more dramatic changes with starvation are seen in skipjack tuna 6, a species living in the fast lane compared with the trout, eel or dace. Table 3 shows L-histidine and related dipeptides in white and red muscles of starved tuna. The well-fed controls just after catch are the same individuals as shown in Table 2. Individual difference in the concentration of each compound was rather small for control tuna, and the combined total pool size in the muscle was quite similar among individuals. Although during starvation rather large individual difference in each compound is found, histidine levels in white muscle decreased significantly on average compared to those in controls, suggesting this amino acid is utilized as an energy source or a glucose source during starvation.

318

H. Abe TABLE 3

Effect of starvation on the levels of L-histidine and related dipeptides in white and red muscle of skipjack tuna (Katsuwonuspelamis) Days after capture Control (n = 5) Tuna no. 1 2 3 4 5 6 7 8

White muscle His Car

Arts

Total

Red muscle His Car

Ans

Total

13.3 4-1.1

7.31 4-1.28

21.3 4-2.1

0

93.5 4-9.2

2.90 4-1.66

51.1 4-10.2

147.4 4-3.5

1 1 2 2 2 3 5 12

79.9 87.7 74.1 69.1 40.3 65.3 31.5 3.46

14.7 5.89 4.94 12.1 7.70 13.9 10.6 24.9

19.1 40.6 47.8 34.3 38.3 36.5 64.0 58.8

113.7 134.3 126.8 111.5 86.3 115.7 106.1 87.2

1.05 6.59 8.67 10.9 7.21 9.71 3.92 3.59

56.4 4-26.9"

11.8 4-5.9'

42.4 4-13.4

110.7 4-16.0"*

6.46 4-3.16"*

Mean 4- SD

0.556 4-0.299 0.439 0.994 0.713 2.66 1.57 3.04 1.96 4.86 2.03 4-1.37"

0.580 5.51 9.12 7.91 8.90 9.00 9.78 13.3

2.07 13.1 18.6 21.5 18.0 21.8 16.2 21.9

8.01 4-3.46

16.7 4-6.2

Average values given in/zmol g-l muscle; * = p < 0.01; ** ffi p < 0.001. From ref. 6.

Carnosine, on the other hand, showed a marked increase, whereas anserine showed no large changes. Tunas 7 and 8, which were starved for 5 and 12 days after capture, respectively, showed an extremely large decrease in histidine and an increase in carnosine, suggesting at least a part of histidine appears to be converted into carnosine and anserine. Anserine concentrations were practically identical to those in the controls, though it decreased at the initial stage of starvation. This tendency was also observed for skipjack tuna red muscle. Kawakawa (Euthynnus affinis), a species closely related to skipjack tuna, also showed similar results, though the degree of change with starvation was somewhat attenuated 6. The total concentration of these compounds was rather similar among individuals even during starvation, decreasing by 30-40% of the control fish. Thus, a metabolic response to starvation appears to be the defence of the total pool size of histidinerelated compounds. This implies that the physiological roles of free L-histidine accumulated specifically in tuna white muscle - as well as other scombroid fishes - is the same as those of the related dipeptides. The drawback of free L-histidine is that, as described above, it is more metabolically reactive than the dipeptides. That is presumably why the conversion of histidine into more metabolically inert forms, such as carnosine and anserine, is necessary. Histidine and its related dipeptides show only minor changes during exhaustion, an observation that holds for tuna muscle 6 as well as for the rat 2~ With increasing body mass, large accumulation of histidine and anserine occur/ed in the early stages of growth of carp and trout, respectively 4. Irrespective of the actual compound, these substances increase with growth of the fish and reach a plateau well before their adult size for both species. Similar data have also been obtained from mammals and birds 26'3~

Histidine-related dipeptides: distribution, metabolism, and physiologicalfunction

319

TABLE 4 Half-lives of NC-histidine in rainbow trout (Oncorhynchus mykiss) and skipjack tuna (Katsuwonus

pelamis) Tissue

Species

Blood

Rainbow trout Skipjack tuna Rainbow trout Skipjack tuna Skipjack tuna

Half-life (h) at 4"C a

White muscle Red muscle

2.0 181

at 14"C b 1.0 1.4 90 132 105

at 24"C a 0.72 66 53

a Measured at the given temperature. b Calculated for 14"C, using an estimated value for Q l0 of 2.0. Compiled from references 6 and 8.

ll:. Metabolic turnover of L-histidine in fish From the above considerations, it is expected that the turnover rate of L-histidine in fish muscle is fast and that of dipeptides is slow. Therefore, the metabolic turnover of 14C-histidine was examined on the blood and muscle of skipjack tuna and rainbow trout 6,8. In Table 4, metabolic half-lives of 14C-histidine are summarized. As the experimental temperatures were 4 and 24"C for trout and tuna, respectively, calculated half-lives at 14"C are also shown in Table 4 after making a simple Q10 correction of 2. After bolus intraarterial injection of 14C-labeled histidine into the aorta of trout and tuna, the label was rapidly removed from blood in both species. The metabolic half-lives were 2.0 4- 0.17 and 0.72 4- 0.124 h for trout and tuna, respectively. This difference between the two species disappears if one makes a simple Q10 correction, in which case the half-life of histidine in tuna is longer than that in trout. These results suggest that the uptake of the amino acid by the tissues is fairly rapid. At the same time, histidine washout from trout white muscle after intramuscular injection and absorption from the peritoneal cavity after intraperitoneal injection is also found to be fairly fast in trout. In free swimming tuna, if the label was administered into white muscle, the decay of the radioactivity from the muscle was slow, whereas the label was taken up by red muscle promptly after injection (Fig. 5). The metabolic half-lives of L-histidine in white and red muscles were 66 and 53 h, respectively, which implies much faster turnover in tuna than in trout muscle (181 h). Again, this difference is temperature dependent and disappears after the above temperature correction to 14"C, resulting in a slower rate for the tuna. Thus, the half-life of L-histidine in muscle may be almost the same irrespective of the level of the amino acid in the muscle. Since skipjack tuna maintains muscle temperatures significantly above ambient temperatures, this fast turnover in tuna muscle suggests that histidine metabolism is probably quite rapid under the normal conditions of this species at sea. These muscle turnovers in fish, on the other hand, are much slower than those in rat gastroenemius muscle, showing a half-life of 3.6 h (ref. 82).

320

H. Abe

I

- "

I

............. I -

I

A

m

B

m

IO

'o

~4 X

D

g

o, , I ....... 0

20

I

i,

i

40

60

80

! O0

0

20

40

60

80

1 O0

TIME (11)

Fig. 5. Total and specific radioactivities in white and red muscles of skipjack tuna after intramuscular injection of L-[U-14C]histidine (30 #Ci/tuna). Each point represents one skipjack tuna. A -- white muscle; B -- red muscle. O - Total activity; O--" specific radioactivity of histidine. From ref. 6.

Metabolic turnovers of the dipeptides have not been examined for fish muscle, but the half-lives of the dipeptides were reported to be of the order of 3 weeks in rat muscle 31,s2 and 4 weeks in chick31, respectively. Judging from the rate of synthesis described later, the turnover of the dipeptides in fish muscle is also expected to be slow.

E~Metabolism and interorgan transport of the dipeptides in fish Metabolism of histidine-related dipeptides has been examined mainly on terrestrial animals and little information has been available on fish. After intramuscular injection of 14C-histidine into trout, the label was gradually incorporated into muscle anserine s as shown in Fig. 6. Fourteen days after injection, the specific radioactivity of anserinc reached the same level of that of histidine. In this case, the incorporation of the label into r was small, but increased after 14C/5-alanine injection intramuscularly. The radioactivity of r was highest one day after 14C-fl-alaninc injection and decreased gradually within eight days, accompanied by the increase in anserine radioactivity. As the conclusion of these experiments, therefore, the biosynthesis of anserine should occur through carnosine as an intermediate step (Fig. 7). Carnosine biosynthesis from histidine and fl-alanine was also confirmed in eel muscle ~. These

Histidine-related dipeptides: distribution, metabolism, and physiological function 1.6

e

~. . . .

i'

"l

e

i

321

I

1.4 A

~1.2 .,,,. -1.~) Zl.0 i-,

0.8

J

I,U

9< 0.6 0.4

"

I,

0

T--

~o.2 , I 0

2

4

6

8 TIME

I

I

i

I

10

12

14

16

18

(days)

Fig. 6. Ratio of specific radioactivity of anserine to that of L-histidine in the white muscle of rainbow trout after intramuscular injection of L-[UJ4C]histidine (2/xCi/trout). Mean and the standard deviation of three trout are shown on each day. From ref. 8.

experiments also revealed that the rate-limiting factor for the biosynthesis of the dipeptides is the #-alanine level in muscle as is the case in mouse 55 and rat 82. Anserine biosynthesis has been known to occur via two or three different pathways in chick and rat. These pathways are: (1) direct condensation of rr-methyl-L-histidine with fl-alanine by a carnosine synthetase-like enzymea1,41,s2,ss; (2) N-methylation of carnosine by carnosine N-methyltransferaseS6; or (3) fl-alanyl transfer from carnosine to Jr-methyl-L-histidine. The biosynthetic pathway of anserine, however, is as yet controversial even in terrestrial animals. At least in trout, the main pathway of anserine biosynthesis may follow pathway (2) as described above. The major organ responsible for the biosynthesis of the dipeptides is confirmed to be skeletal muscle in chick and rat. As mentioned above, carnosine biosynthesis also occurred in the white muscle of eel 86. Similarly, 14C-histidine was incorporated into earnosine and anserine only in white and red muscle of trout and tuna 6,8. At least in skipjack tuna, the rate of incorporation of the label into anserine is higher in white muscle than red. Thus it can be concluded that the biosynthesis of these dipeptides in the skeletal muscle is a generalized feature in vertebrate animals. These dipeptides are known to be catabolized by carnosinase and anserinase (see Fig. 7) and degraded to the corresponding constituents, histidine and methyl-Lhistidine. Anserinase is known to occur in the skeletal muscle of a codling (Gadus

322

H. Abe

Glucose Glutamate CO 2 + H 2 0

Histidlne

Carnosine ....

~:-Methylhistidine

~

Anserine

Fig. 7. Metabolic pathways of L-histidine-related compounds in fish. 1 = carnosine synthetase (EC 6.3.2.11); 2 = carnosinase (EC 3.4.13.3); 3 = carnosine N-methyltransferase (EC 2.1.1.22); 4 = anserinase (EC 3.4.13.5).

callarias) 4~ Lenney and coworkers 46 purified N-acetyl-L-histidine deacetylase, which is now considered to be identical to anserinase, from the brain of skipjack tuna and showed the enzyme also acts on carnosine and anserine. In addition, the activity of this enzyme was found in tuna ocular muscle, but not in the skeletal muscle. In codling, however, weak activity of the enzyme was also located in skeletal muscle. The properties of this enzyme are similar to those of hog kidney carnosinase 45. Trout anserinase activity is highest in brain followed by eye and kidney94. The activity in kidney is reported to be high in cod, pollack, smeltfish, and trout, but undetectable in tuna, flounder, carp, and sardine sl. Also in mammals and birds, the activities of carnosinase and anserinase are fairly high in kidney and olfactory mucosa and low in liver and muscle 53,54,9~ As shown below for the rainbow trout, when large amounts of carnosine or anserine are injected into white muscle, these dipeptides are degraded into L-histidine and ~r-methyl-L-histidine, respectively. In contrast to the synthetic pathway of anserine which involves carnosine as an intermediate, anserine is not catabolized v/a carnosine to L-histidine 5. Major organs responsible for the dipeptide degradation may be the kidney and the liver, even in fishs,6,s. Although methyl-L-histidine produced from anserine and balenine decomposition must be excreted without further reutilization, L-histidine is known to be degraded to glutamic acid v/a urocanic acid 42 and reutilized for energy or glucose sources or for protein synthesis (see Fig. 7). The activity of histidine degrading enzymes is also high in kidney and liver and low or undetectable in muscle 42. In fish, the organs responsible for the biosynthesis and degradation of these dipeptides are different as described above. Thus, these facts give rise to interesting problems concerning the interorgan transports of these dipeptides. As already shown in Fig. 5, labeled histidine administered into tuna white muscle is promptly transported into red muscle. The label was also, as shown above, washed out quickly from trout muscle and intraperitoneal cavity into blood and from blood into tissues.

Histidine-related dipeptides: distribution, metabolism, and physiological function

323

If the levels of these dipeptides which accumulated in large amounts in fish muscle are closely regulated in the muscle, it would be anticipated that after administration of large amounts of these dipeptides into muscle they may be decomposed in situ or washed out into blood, transported to several other organs, and finally decomposed or excreted. To test this hypothesis, large amounts of carnosine and anserine were injected intramuscularly into white muscle of small trout and their respective metabolic fates were traced 5. Fig. 8 gives the results from anserine administration. Almost the same data were obtained from carnosine injection.

60 c ~ r ' ~ r - - ' l ~ ~ / / - r ' e ' n - H - 1 - 1

,

,

,

i ,~,', ,',',

,',',

6

8 24 96 240

RM

~ 20

z

_o

O~

'

'

'

',;," "b"

//"~

~ 1 4 , k " ~ i ' ~ r ~ r ~ r -t,"r-r

An l

Z' ll

I

2

2

4

6

8 24 96 240 2 TIME(h)

4

Fig. 8. Interorgan transport of anserine in rainbow trout and the degradation into 7r-methyl-L-histidine after intramuscular injection of 100 rag/trout of anserine. WMA = anterior white muscle (injection port); WMP - posterior white muscle; RM -- red muscle. Ans -- anserine; 7r-meHis -- 7r-methyl-Lhistidine. Each point represents one trout. Modified from ref. 5.

324

H. Abe

Anserine in the anterior white muscle (injection port) showed a sharp decline 2 min after injection and returned to the control level after 24 h. In the posterior white muscle, however, the anserine level did not change and remained at a slightly higher level than in the control. Red muscle anserine was about twice as high 20 min after injection, but decreased to the control level after 7 h. This fact is consistent with that for tuna mentioned above. These were also the ease for camosine injection. After anserine injection, the levels of eamosine and L-histidine did not change throughout the experimental period in all tissues and blood, suggesting anserine is not degraded via carnosine as mentioned above. However, lr-methyl-L-histidine increased about 10-fold in each muscle. The rate of this increase was rather rapid in red muscle. Just after injection as shown in Fig. 8, a large amount of anserine was washed out into blood and the level sharply declined to the control level after 5 h. The anserine leached out was incorporated into kidney, reaching a maximum 20 rain after injection. The incorporation was less pronounced in liver. The anserine incorporated into kidney was immediately degraded to ~r-methylL-histidine which reached a maximum 5 h after injection and kept this level until at least 24 h after injection. Thus, kidney rather than liver seems to be responsible for the catabolism of the dipeptides. The pattern of increase and decrease of 7r-methylL-histidine was almost identical among liver, red muscle and blood, suggesting the incorporation of the compound produced in kidney via blood. In contrast to breakdown of anserine, carnosine may even be catabolized in situ in muscle because the L-histidine level increased in every muscle prior to the increase in kidney. These data clearly indicate that carnosine and anserine levels must be closely regulated metabolically in fish white and red muscle and there may be a close relationship between white and red muscle of fish. Therefore, large part of L-histidine derived from fish diets may be absorbed from intestine into blood and transported to several tissues and used for protein or energy sources. A small part of the amino acid, however, may be incorporated into dipeptides in muscle and accumulated in situ.

VI. Physiological functions of histidine-related dipeptides as proton

buffers Many efforts have been carried out to date in order to clarify the physiological functions of these dipeptides mainly for terrestrial animals. Earlier work has been reviewed by Crush 23 and Christman 21. Five lines of evidence have been obtained from these efforts: (1) (2) (3) (4) (5)

direct activators of some enzymes in the glycolytic pathway; enhancers for Ca2+-sensitive systems such as myofibriUar Mg2+-ATPase; oxygen or copper transporters by chelating action of these dipeptides; free radical scavengers; buffering components for protons.

Histidine-related dipeptides: distribution, metabolism, and physiological function

325

TABLE 5 Apparent pK values of the histidine imidazole group in various naturally occurring compounds Compound

Temperature

pK

(*c) lmidazole L-Histidine ~r-MethyI-L-histidine r-MethyI-L-histidine Carnosine Anserine Balenine Homoearnosine

20 20 20 20 20 20 20 20

7.23 6.21 6.62 5.98 7.01 7.15 6.93 7.10

"'Bjpical" histidyl-imidazole in proteins Adjacent to acidic ( - ) group Adjacent to basic (+) group

25 25 25

6.5 7-8 5-6

Compiled from references 10 and 35.

Of these functions, we have focussed on the buffering capacity of these compounds accumulated in large amounts in vertebrate skeletal muscle. Maintaining the intracellular pH of skeletal muscle is critically important during the anaerobic burst locomotion of the animals, because otherwise the fall of pH due to proton production as a result of ATP consumption 34 will cause glycolytic enzymes to cease their actions 35. Since the early work of Bate-Smith ~3, the importance of non-bicarbonate buffering of vertebrate muscle has been emphasized by many researchers 7,17.19,24,35,70,74. The intracellular buffering capacity in vertebrate muscle is dominated by the imidazole group of L-histidine, which occurs as histidine residues on protein, as free L-histidine, and as histidine-related dipeptides. One of the two nitrogens of imidazole ring on histidine molecule can be protonated at physiological pH ranges and act as a buffering component for protons produced during the anaerobic muscle exercise. Table 5 represents the apparent pK values determined on histidine-related compounds at 20~ All the p K values fell into the range of pH 6-7.3, indicating the appropriate p K characteristics for buffering constituents in the physiological pH range which is pH 6.8-7.2 at 20~ in the muscle cytosol 7~ Other than the free imidazole compounds, histidine residues on proteins and inorganic phosphate are considered to be good buffers (Table 5). Other candidates are organic phosphate compounds such as nucleotides and sugar phosphates and several organic acids such as citrate and succinate only when they exist in large amounts in muscle cytosol. In aerobic exercise, protons produced along with the ATP consumption are turned over through oxidative phosphorylation and not accumulated in the intracellular fluid 34. In anaerobic metabolism during 02 limitation typically occurred in the fast-twitch white muscle of fishes, however, large quantities of proton would accumulate and lower the muscle pH 34,35. Consequently, all the animals performing anaerobic exercise must evolve the means maintaining the muscle pH as far as

326

H. Abe

possible to enhance the anaerobic capacity. This is the reason why fish white muscle have a higher buffering capacity compared to other tissues such as slow-twitched red muscle. Proton buffering capacity (termed fl value) due to non-bicarbonate buffering compounds was defined as the /~moles of sodium hydroxide required per gram tissue to change the p H by one unit 7,13,19. This unit is termed a 'slyke' based on the original technique of Van Slyke 85. Buffering capacities of several vertebrate muscles are listed in Table 6. Individual variation of the buffering capacity in a species was small compared to the large variations in interspecific comparisons. T h e buffering capacity was typically high in the muscle of marine diving m a m m a l s TABLE 6 Buffering capacity (fl) of skeletal muscles of marine and terrestrial mamals and birds and various fishes Species

n

Buffering capacity (/~mol NaOH/pH. g muscle; pH 6-7)

1 5 1 4 4 3 1 2 1

111 84.1 79.2 76.2 72.1 70.6 70.0 61.2 47.5

1 1 1 1 3

82.8 78.6 63.2 69.0 66.9 50.2

5 5

136 68.8 109 107 105 102 96.4 56.8

Marine mammals and birds

Little piked whale Spotter porpoise Northern fur seal Harbor seal Weddell seal Sea otter adult Adelie penguin California sea lion California gray whale calf Terrestrial mammals and birds

Chicken, pectoralis minor Pig, psoas muscle Pig, biceps femoris Ox, biceps femoris Rabbit Dog Warm-bodied fishes and related species

Skipjack tuna, white muscle Skipjack tuna, red muscle Frigate mackerel, white muscle Albacore, white muscle Yellowfin, white muscle Black skipjack, white muscle Blue marlin, white muscle Blue marlin, red muscle

5 5

Pelagic ~ h e s

Common mackerel, white muscle Common mackerel, red muscle Rainbow trout, white muscle Rainbow trout, red muscle Mean of 11 species, white muscle

5 5 5 5

81.3 51.2 63.3 30.1 63.3

Deep sea fishes

Mean of 9 species, white muscle Data compiled from references 7, 19, and 64.

46.0

Histidine-related dipeptides: distribution, metabolism, and physiological function

327

and warm-bodied tuna fishes. Marine mammals showed a higher average fl value than terrestrial mammals. This may relate to the anaerobic capacity for prolonged breathhold diving. Castellini and Somero 19 clearly revealed that the buffering capacity of mammalian muscle correlated strongly with muscle myoglobin content, which is more abundant in the muscle of marine mammals. The capacity also correlated with muscle lactate dehydrogenase activity 19. Among the fish species listed in Table 6, the white skeletal muscle of warmbodied tuna fishes and related species, which are noted for their extremely high burst swimming abilities, showed the highest buffering capacity. These fishes possess the highest muscle lactate dehydrogenase activities as well as the highest muscle buffering capacities in any animals ever been known 19. Deep sea fish species on average, on the other hand, possess markedly lower buffering capacities and the lactate dehydrogenase activities in their muscle. Pelagic fishes, actively swimming predators and foragers such as mackerel and trout, showed the intermediate value in buffering capacity and lactate dehydrogenase activity between the other two fish groups. Intraspecific difference of buffering capacity is again very small compared to the large interspecific variations. Aerobic slow-twitch red muscle is found to have lower buffering capacities than white muscle from the same species as shown in Table 6 for skipjack tuna, blue marlin, common Japanese mackerel (Scomberjaponicus), and rainbow trout. The capacity in red muscle is almost half of that in white muscle. These data clearly indicated that the non-bicarbonate buffering capacity in animal skeletal muscle is strongly correlated with the anaerobic locomotory ability of the animal species and the anaerobic capacity of the muscle. The buffering capacities and carnosine content in human skeletal muscle were also reported to be significantly higher for anaerobically trained elite athletes (sprinters and rowers) than more aerobically trained marathoners or untrained men 66. Thus, the high buffering capacity in the vertebrate skeletal muscle is considered to be a universal strategy for enhancing the anaerobic exercise capability beyond the species. In view of the correlation between buffeting capacity and anaerobic capability, it is appropriate to know what compounds in the vertebrate skeletal muscle actually participate in the elevation of the muscle buffering capacity. Fig. 9 represents the brief summary of muscle homogenate buffering and the major compounds contributing to the buffering in the pH range of 6.5-7.5. Almost the same tendency was also the case for the pH range 6.0-7.0. Contribution of contractile proteins is as low as several percent to the homogenate buffering. Histidine residues in the muscle soluble proteins, however, contribute from 9 to 38% relative to the homogenate buffering. This contribution is rather high for dark-fleshed fish muscle containing much myoglobin, such as tuna and mackerel. Not all of the histidine residues in the native proteins, however, can contribute to the total muscle buffering 64. Buffering capacities of histidine-related compounds of skipjack tuna and marlin white muscle are as high as 40 and 60%, respectively, relative to their muscle buffering. This is also the case for whale skeletal muscle in which case the contribution was about 25%. The ratio is also high for bovine, porcine, and chicken

328

H. Abe

Fig. 9. Contributions of proteins, inorganic phosphate, and histidine-related compounds to the buffering capacity of muscle homogenate from several vertebrates. Buffering capacity is represented as #mol NaOH required per g of muscle to change the pH by one unit over 6.5-7.5. WM = white muscle; RM = red muscle; SM = skeletal muscle; BF - biceps femoris; PSM = psoas muscle; PM = pectoralis minor. From ref. 64.

muscle, ranging from 12 to 23%, but only 1 to 6% for carp and flounder white muscle containing below 10/zmol/g muscle of histidine-related compounds. For red muscle, however, the ratio is much lower than that for corresponding white muscle, as seen in tuna and martin red muscle. The ratio of inorganic phosphate buffering to total muscle buffeting capacity in tuna and marlin white muscle and whale skeletal muscle is relatively low, while in the white muscle of trout, carp, and flounder which contain only small amounts of histidine-related compounds the ratio is significantly high, ranging from 50 to 80%. This may stem from rather species independent content of inorganic phosphate in muscle. The phosphate buffering, however, would be overestimated because inorganic phosphate is liberated from organic phosphate, such as creatine phosphate or ATP during the postmortem changes of animal muscle. The contribution of total buffering capacities of histidine-related compounds and inorganic phosphate to muscle homogenate buffeting is from 50 to 83%, except for mackerel red muscle and the muscle of whale, ox, and pig in which mammalian case rather high contribution is attributed to unknown compounds. These unknown compounds may comprise some nucleotides, organic acids, and/or taurine 64. As no large difference was observed in the phosphate concentration and buffering capacity among animal muscles thus far examined, inorganic phosphate may contribute to the basic muscle buffering together with the muscle proteins. Thus, the large variation of muscle buffering capacity would mainly be attributable to the

Histidine-related dipeptides: distribution, metabolism, and physiological function

329

levels of histidine-related compounds in animal muscle. These data and the previous d a t a 7,19,24,37 clearly indicate that the accumulation of histidine-related compounds in animal muscle enlarge the muscle buffering capacity and therefore the muscle anaerobic capability. This is realized in some animals such as tunas, billfishes, and marine mammals. Judging from these results, it is apparent that the large amounts of histidinerelated dipeptides in fish muscle enhance the muscle buffering capacity. However, every fish as well as invertebrate contains small amounts of all of these dipeptides in their muscles (unpublished data). Moreover, we cannot find any clear difference among the properties as buffer constituent of these three dipeptides 1~ Thus, we can conclude that these dipeptides have some other physiological roles related to the muscle functions and several animals adapted to the anaerobic muscle exercise have evolved to select and accumulate one or some of them for the muscle buffer components. Several other possible physiological roles mentioned above are worthy of attention from these considerations. These dipeptides may play a role in the regulation of glycolytic enzymes 37 and myofibrillar ATPase 12,65, and in the stimulation of contraction of muscle fibers 15,6a. Other workers also suggested that as a chelator these dipeptides are involved in the intracellular transport of copper for activation of cytochrome oxidase and in the regulation of anaerobic glycolysis 16. More recently, N-acetylated forms of histidine related compounds were found to be distributed together with carnosine and anserine in cardiac and skeletal muscles, brain, and eye lens of several vertebrates 61-63. In vertebrate brain, carnosine has been known to be a neurotransmitter in the olfactory system 5~ In the animal skeletal and cardiac muscle, these dipeptides and their N-acetylated forms are attracting much attention due to their ability to enhance the calcium sensitivity of contractile proteins 32,58, their t~-adrenergic antagonist properties 57, and their scavenging activity for oxygen free-radicals 49. Careful considerations should be given to these attractive hypotheses in the future.

VII. References 1. Abe, H. Determination of L-histidine-related compounds in fish muscles using high-performance liquid chromatography. Bull. Jan. Soc. Sci. Fish. 47: 139, 1981. 2. Abe, H. Distribution of free L-histidine and related dipeptides in the muscle of fresh-water fishes. Comp. Biochem. Physiol. 76B: 35-39, 1983. 3. Abe, H. Distribution of free L-histidine and its related compounds in marine fishes. Bull. Jpn. Soc. Sci. Fish. 49: 1683-1687, 1983. 4. Abe, H. Effect of growth on the concentration of L-histidine and anserine in the white muscle of carp and rainbow trout. Bull. Jpn. Soc. Sci. Fish. 53: 1657-1661, 1987. 5. Abe, H. Interorgan transport and catabolism of carnosine and anserine in rainbow trout. Comp. Biochem. Physiol. 100B: 717-720, 1991. 6. Abe, H., R.W. Brill and P.W. Hochachka. Metabolism of L-histidine, carnosine, and anserine in skipjack tuna. Physiol. Zool. 59: 439-450, 1986. 7. Abe, H., G.P. Dobson, U. Hoeger and W.S. Parkhouse. Role of histidine-related compounds to intraceUular buffering in fish skeletal muscle. Am. J. Physiol. 249: R449-R454, 1985. 8. Abe, H. and P.W. Hochachka. Turnover of 14C-labelled L-histidine and its incorporation into carnosine and anserine in rainbow trout. Bull. Jpn. Soc. Sci. Fish. 53: 1089-1094, 1987.

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9. Abe, H. and S. Ohmama. Effect of starvation and seawater acclimation on the concentration of free L-histidine and related dipeptides in the muscle of eel, rainbow trout and Japanese dace. Comp. Biochem. Physiol. 88B: 507-511, 1987. 10. Abe, H. and E. Okuma. Effect of temperature on the buffering capacities of histidine-related compounds and fish skeletal muscle. Bull. 7p.n. Soc. Sci. Fish. 57: 2101-2107, 1991. 11. Ackermann, D., O. Timpe and K. Poller. Uber das Anserin, einen neuen Bestandteil der Vogelmuskulatur. Z. Physiol. Chem. 183: 1-10, 1929. 12. Avena, R.M. and W.J. Bowen. Effects of carnosine and anserine on muscle adenosine triphos. phatase. 7. BIOL Chen~ 244: 1600-1604, 1969. 13. Bate-Smith, E.C. The buffering of muscle in rigor: protein, phosphate and carnosine. Z. Physiol. 92: 336-343, 1938. 14. Baumann, L. and T. Ingvaldsen. Concerning histidine and carnosine. The synthesis of carnosine. J. Biol. Chem. 35: 263-276, 1918. 15. Boldyrev, A.A. and V.B. Petukhov. Localization of carnosine effect on the fatigued muscle preparation. Ge~ Pharmac. 9: 17-20, 1978. 16. Brown, C.E. Interactions among carnosine, anserine, ophidine and copper in biochemical adaptation. Z. Theor. Biol. 88: 245-256, 1981. 17. Burton, R.E Intracellular buffering. Respir. Physiol. 33: 51-58, 1978. 18. Carnegie, P.R., M.G. Collins and M.Z. llic. Use of histidine dipeptides to estimate the proportion of pig meat in processed meats. Meat ScL 10: 145-154, 1984. 19. CasteUini, M.A. and G.N. Somero. Buffering capacity of vertebrate muscle: correlations with potentials for anaerobic function. 7. Comp. Physiol. 143B: 191-198, 1981. 20. Christman, A.A. Determination of anserine, carnosine, and other histidine compounds in muscle extractives.Anal. Biochem. 39: 181-187, 1971. 21. Christman, A.A. Factors affecting anserine and carnosine levels in skeletal muscles of various animals. Int. J. Biochem. 7: 519-527, 1976. 22. Cowey, C.B., K.W. Daisley and G. Parry. Study of amino acids, free or as components of protein and of some B vitamins in the tissues of the Atlantic salmon, Salmo salar during spawning migration. Comp. Biochem. Physiol. 7: 29-38, 1962. 23. Crush, K.G. Carnosine and related substances in animal tissues. Comp. Biochem. Physiol. 34: 3-30, 1970. 24. Davey, C.L. The significance of carnosine and anserine in striated skeletal muscle. Arch. Biochem. Biophys. 89: 303-308, 1960. 25. Dizon, A.E., R.W. BriU and H.S.H. Yuen. Correlations between environment, physiology, and activity and the effects on thermoregulation in skipjack tuna. In: Physiological Ecoloo of Tunas, edited by G.D. Sharp and A.W. Dizon, New York, Academic Press, pp. 233-259, 1978. 26. Fisher, D.E., J.E Amend and D.H. Strumeyer. Anserine and carnosine in chicks, rat pups, and duckling: comparative ontogenetic observations. Comp. Biochem. Physiol. 56B: 367-370, 1977. 27. Gulewitsch, W. and S. Amiradzibi. Ober das Carnosine, eine neue organische Base des Fleischextraktes. Bet. Dtsch. Chem. Ges. 33: 1902-1908, 1900. 28. Guppy, M. and P.W. Hochachka. Controlling the highest lactate dehydrogenase activity known in nature. Am. J. Physiol. 234: R136-R140, 1978. 29. Guppy, M., W.C. Hulbert and P.W. Hochachka. Metabolic sources of heat and power in tuna muscles. II. Enzyme and metabolite profiles. Y. E~. Biol. 82: 303-320, 1979. 30. Hama, T., N. Tamaki, H. lizumi and M. Kita. Observation on the changes of ~-alanine, anserine and carnosine contents in liver and gastrocnemius of growing rat. Y. Jpn. Soc. Food Nutr. 23: 389-393, 1970. 31. Harms, W.S. and T. Winnick. Further studies of the biosynthesis of carnosine and anserine in vertebrates. Biochim. Biophys. Acta 15: 480-488, 1954. 32. Harrison, S.M., (2. Lamont and D.J. Miller. Carnosine and other natural imidazoles enhance muscle Ca sensitivity and are mimicked by caffeine and ARL 115BS. Y. Physiol. 371: 197P, 1985. 33. Hochachka, P.W. Living Without Oxygen, Cambridge, Harvard University Press, pp. 85-94. 1980. 34. Hochachka, P.W. and T.P. Mommsen. Protons and anaerobiosis. Science 219: 1391-1397, 1983. 35. Hochachka, P.W. and G.N. Somero. Biochemical Adaptation. Princeton, NJ, Princeton University Press, pp. 337-348, 1984. 36. Huggins, A.K. and L. Colley. The changes in the non-protein nitrogenous constituents of muscle during adaptation of the eel (Anguilla anguUla L.) from fresh water to sea water. Comp. Biochem. Physiol. 38B: 537-541, 1971.

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37. Ikeda, T, K. Kimura, T Hama and N. Tamaki. Activation of rabbit muscle fructose 1,6-bisphosphatase by histidine and carnosine. J. Biochem. 87: 179-185, 1980. 38. Imamura, H. Chemie der Schlangen - I. Ober die N-haltigen Extraktivstoffe der Schlangenmuskeln. J. Biochem. 30: 479-490, 1939. 39. IUPAC-IUB. Symbols for amino acid derivatives and peptides (Rules approved 1974). IUPAC Pure AppL Chem. 40: 317-331, 1974. 40. Jones, N.R. The free amino acids of fish: 1-methylhistidine and fl-alanine liberation by skeletal muscle anserinase of codling (Gadus caUarias). Biochem. J. 60: 81-87, 1955. 41. Kalyankar, G.D. and A.J. Meister. Enzymatic synthesis of carnosine and related fl-alanyl and 0-aminobutyryl peptides. J. Biol. Chem. 234: 3210-3218, 1959. 42. Kawai, A. and M. Sakaguchi. Histidine metabolism in fish - II. Formation of urocanic, formiminoglutamic, and glutamic acids from histidine in the livers of carp and mackerel. Bull. Jpn. Soc. Sci. Fish. 34:507-511, 1968. 43. Kendo, K. Ober die Konstitution des Ophidins,. eines N-haltigen Extraktivstoffes der Schlangenmuskeln. J. Biochem. 36: 265-276, 1944. 44. Konosu, S., K. Watanabe and T Shimizu. Distribution of nitrogenous constituents in the muscle extracts of eight species of fish. Bull. Jpn. Soc. Sci. Fish. 40: 909-915, 1974. 45. Lenney, J.E Specificity and distribution of mammalian carnosinase. Biochim. Biophys. Acta 429: 214-219, 1976. 46. Lenney, J.E, M.H. Baslow and G.H. Sugiyama. Similarity of tuna N-acetylhistidine deacetylase and cod fish anserinase. Comp. Biochem. Physiol. 61B: 253-258, 1978. 47. Lukton, A. Effect of diet on imidazole compounds and creatine in chinook salmon. Nature 182: 1019-1020, 1958. 48. Lukton, A. and H.S. Olcott. Content of free imidazole compounds in the muscle tissue of aquatic animals. Food Res. 23: 611-618, 1958. 49. MacFarlane, N., J. McMurray, J.J. O'Dowd, H.J. Datgie and D.J. Miller. Synergism of histidyl dipeptides as antioxidants. J. Mol. Cell Cardiol. 23: 1205-1207, 1991. 50. Margolis, EL. Carnosine in the primary olfactory pathway. Science 184: 909-911, 1974. 51. Margolis, EL. Neurotransmitter biochemistry of the mammalian olfactory bulb. In: Biochemistry of Taste and Olfaction, edited by R.H. Cagan and M.R. Kate, New York, Academic Press, 1981, pp. 369-394. 52. Margolis, EL. and M. GriUo. Catnosine, homocatnosine and anserine in vertebrate retinas. Neurochem. Int. 6: 207-209, 1984. 53. Margolis, EL., M. Grillo, C.E. Brown, T.H. Williams, R.G. Pitcher and G.J. Elgar. Enzymatic and immunological evidence for two forms of carnosinase in the mouse. Biochim. Biophys. Acta 570: 311-323, 1979. 54. Margolis, EL., M. GriUo, N. Grannot-Reisfeld and A.I. Fatbman. Purification, characterization and immunocytochemical localization of mouse kidney catnosinase. Biochim. Biophys. Acta 744: 237-248, 1983. 55. Margolis, EL., M. Grillo, T. Kawano and A.I. Farbman. Carnosine synthesis in olfactory tissue during ontogeny: effect of exogenous/5-alanine. J. Neurochem. 44: 1459-1464, 1985. 56. McManus, I.R. Enzymatic synthesis of anserine in skeletal muscle by N-methylation of catnosine. J. Biol. Chem. 237: 1207-1211, 1962. 57. Melville, C.A., M. Trainor, J.C. McGrath, C. Daly, J.J. O'Dowd and D.J. Miller. Catnosine shows ot-adrenoceptor agonist and antagonist properties in saphenous vein isolated from rabbit. J. Physiol. 427: 29P, 1990. 58. Miller, D.J., J. Campbell, J.J. O'Dowd and D.J. Robins. Novel endogenous imidazoles calciumsensitize chemically skinned rat heart muscle. J. Physiol. 427: 54P, 1990. 59. Mommsen, T.P., C.J. French and P.W. Hochachka. Sites and patterns of protein and amino acid utilization during the spawning migration of salmon. Can. J. Zool. 58: 1785-1799, 1980. 60. Nakai, T. and N. Tsujigado./]-Alanyl dipeptide preparations from whale muscles made by several workers. J. Biochem. 57: 812-814, 1965. 61. O'Dowd, J.J., M.T. Cairns, M. "lTainor, D.J. Robins and D.J. Miller. Analysis of carnosine, homocarnosine and other histidyl derivatives in rat brain. J. Neurochem. 55: 446-452, 1990. 62. O'Dowd, A., J.J. O'Dowd, J.J.M. O'Dowd, N. MacFarlane, H. Abe and D.J. Miller. Analysis of novel imidazoles from isolated perfused rabbit heart by two high-performance liquid chromatographic methods. J. Chromatosz., 577: 347-353, 1992. 63. O'Dowd, J.J., D.J. Robins and D.J. Miller. Detection, characterisation, and quantification of carnosine and other histidyl derivatives in cardiac and skeletal muscle. Biochim. Biophys. Acta 967:

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241-249, 1988. 64. Okuma, E. and H. Abe. Major buffering constituents in animal muscle. Comp. Biocher~ Physiol. 102A: 37-41, 1992. 65. Parker C.J. Jr., and E. Ring. A comparative study of the effect of r on myofibrillar-ATPase activity of vertebrate and invertebrate muscles. Comp. Biochem. Physiol. 37: 413-419, 1970. 66. Parkhouse, W.S., D.C. McKenzie, I'.W. Hochachka, T.P. Mommsen, W.K. Ovalle, S.L. Shinn and E.C. Rhodes. The relationship between carnosine levels, buffering capacity, fiber type and anaerobic capacity in elite athletes. In: Biochemistry of Exercise, edited by H. Kunttgen, J. Vogel and J. Poortmans, Champaign, Human Kinetics Publication, pp. 584-589, 1983. 67. Pocchiari, E, L. Tentori and G. Vivaldi. The presence of the dipeptide fl-alanyl-3-methylhistidine in whale meat extract. Scient. Rep. 1st. Super Sanita 2: 188-194, 1962. 68. Severin, S.E., I.M. Bocharnikova, P.L. Vulfson, A. Grigorovich Yu and G.A. Solovyeva. On the biological role of carnosine. Biokhimiya 28: 510-516, 1963. 69. Shirai, T., S. Fuke, IC Yamaguchi and S. Konosu. Studies on extractive components of salmonids II. Comparison of amino acids and related compounds in the muscle extracts of four species of salmon. Comp. Biochem. Physiol. 7413: 685-689, 1983. 70. Somero, G.N. pH-temperature interactions on proteins: principles of optimal pH and buffer system design. Mar Biol. Lefts. 2: 163-178, 1981. 71. Stevens, E.D., H.M. Lam and J. Kendall. Vascular anatomy of the counter-current heat exchanger of skipjack tuna. J. Exp. Biol. 61: 145-153, 1974. 72. Suyama, M., T. Hirano, N. Okada and T. Shibuya. Quality of wild and cultured Ayu - I. On the proximate composition, free amino acids and related compounds. Bull. Jpn. Soc. Sci. Fish. 43: 535-540, 1977. 73. Suyama, M., T. Hirano and T. Suzuki. Buffeting capacity of free histidine and its related dipeptides in white and dark muscles of yellowfin tuna. Bull. Jpn. Soc. Sci. Fish. 52:2171-2175 1986. 74. Suyama, M., J. Koike and K. Suzuki. Studies on the buffering capacity of fish muscle - IV. Buffering capacity of muscles of some marine animals. Bull Jpn. Soc. Sci. Fish. 24: 281-284, 1958. 75. Suyama, M. and H. Suzuki. Nitrogenous constituents in the muscle extracts of marine elasmobranchs. BulL Jpn. Soc. Sci. Fish. 41: 787-790, 1975. 76. Suyama, M., T. Suzuki, M. Maruyama and IC Saito. Determination of carnosine, anserine, and balenine in the muscle of animal. Bull Jpn. Soc. Sci. Fish. 36: 1048-1,053, 1970. 77. Suyama, M., T. Suzuki and J. Nonaka. Chromatographic determination of imidazole compounds in the whale meat. Bull Jpn. Soc. Sci. Fish. 33: 141-146, 1967. 78. Suyama, M., T Suzuki and A. Yamamoto. Free amino acid and related compounds in whale muscle tissue. J. Tokyo Univ. Fish. 63: 189-196, 1977. 79. Suyama, M. and Y. Yoshizawa. Free amino acid composition of the skeletal muscle of migratory fish. Bull Jpn. Soc. Sci. Fish. 39: 1339-1343, 1973. 80. Suzuki, T., T. Hirano and M. Suyama. Free imidazole compounds in white and dark muscles of migratory marine fish. Comp. Biochem. Physiol. 87B: 615-619, 1987. 81. Suzuki, T, T. Hirano and M. Suyama. Distribution of anserinase in organs of several fish. Bull Jpn. Soc. Sci. Fish. 54: 541, 1988. 82. Tamaki, N., S. Morioka, T. lkeda, M. Harada and T. Hama. Biosynthesis and degradation of carnosine and turnover rate of its constituent amino acids in rats. J. Nutr. Sci. Vitaminol. 26: 127139, 1980. 83. Tamaki, N., M. Nakamura, M. Harada, K. Kimura, H. Kawano and T. Ham& Anserine and carnosine contents in muscular tissue of rat and rabbit. J. Nutr. ScL VitaminoL 23: 319-329, 1977. 84. Tsukamoto, IC Contribution of the red and white muscles to the power output required for swimming by the yellowtail. Bull. Jpn. Soc. Sci. Fish. 50: 2031-2042, 1984. 85. Van Slyke, D.D. On the measurement of buffer values and on the relationship of buffer value to the dissociation constant of the buffer and the concentration and reaction of the buffer solution. J. Biol. Chem. 52: 525-570, 1922. 86. Watanabe, IC and S. Konosu. Incorporation of 14C-histidine into carnosine in eel, AnguUla japonica. Bull Jpn. Soc. Sci. Fish. 45: 1513-1516, 1979. 87. Waters, V. and H.L. Fierstine. Measurement of swimming speeds of yellowfin tuna and wahoo. Nature 202: 208--209, 1964. 88. Winnick, R.E. and T. Winnick. Carnosine-anserine synthetase of muscle. I. Preparation and properties of a soluble enzyme from chick muscle. Biochim. Biophys. Acta 31: 47-55, 1959. 89. Wolff, J., K. Horisaka and H.M. Fales. On the structure of ophidine. Biochemistry 7: 2455-2457, 1968. -

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90. Woios, A., K. Piekarska, J. Glogowski and I. Konieczka. Two molecular forms of swine kidney carnosinase. Int. J. Biochem. 9: 57-62, 1978. 91. Wolos, A. and K. Swidowicz. Kidney and liver carnosinase activity and carnosine level in goose. Comp. Biochem. PhysioL 62B: 515-519, 1979. 92. Wood, J.D. Biochemical studies on sockeye salmon during spawning migration - IV. The nonprotein nitrogenous constituents of the muscle. Can. J. Biochem. PhysioL 36: 833-838, 1958. 93. Wood, J.D., D.W. Duncan and M. Jackson. Biochemical studies on sockeye salmon during spawning migration - IX. The free histidine content of the tissue. J. Fish. Res. Bd. Can. 17: 347-351, 1960. 94. Yamada, S., Y. Tanaka, M. Sameshima and M. Furuichi. Distribution of N-acetylhistidine-deacetylating enzyme in tissues of rainbow trout. Bull. Ypn. Soc. Sci. Fish. 57: 1601, 1991.

Hochachka and Mommsen (eds.), Biochemistryand molecularbiologyoffishes, vol. 4

9 1995ElsevierScienceB.V.All rightsreserved.

CHAPTER 15

The metabolic consequences of body size EDWARD M. GOOLISH National Oceanic and Atmospheric Administration, Southwest Fisheries Science Center, La Jolla, CA 92038, U.S.A. and Scripps Institution of Oceanography, Center for Marine Biotechnology and Biomedicine, University of California, San Diego, La JoUa, CA 92093, U.S.A.

I. II.

Introduction Theoretical explanations for the negative allometry of aerobic metabolism 1. Muscle contraction time 2. The effect of body size on the rate of oxygen delivery III. Symmorphosisand the scaling of individual respiratory traits 1. Blood oxygen carrying capacity 2. Gill surface area 3. Cardiac output 4. Tissue respiration and enzyme activity IV. Tissue-levelexceptions to negative allometry in aerobic metabolism V. The scaling of maximum whole-body aerobic capacity VI. Aerobicallometry: a comparison with hypoxia VII. Effects of body size on anaerobic metabolism 1. Enzymatic evidence 2. Power requirements during burst swimming 3. Constraints on anaerobic scaling VIII. Behavioral and ecological implications of metabolic scaling Acknowledgements IX. References

I. I n t r o d u c t i o n

How should the body size of an organism affect the metabolic biochemistry occurring within individual cells? There are no obvious direct effects of body size on cell metabolism, since the size of individual cells remains relatively unchanged. What does change with increased body size, however, is the interaction between the cells and the environment. With larger body size cellular metabolism is increasingly removed from the environment by distance and, therefore, also by time. In his analysis of the interaction between animal metabolism and the environment, EE.J. Fry 37 defined as limiting factors those identities which govern metabolic rate by restricting the supply or removal of materials in the metabolic chain. In the case of aerobic metabolism this would include such things as oxygen, CO2, and substrate (e.g. glucose). The distancing between the cell and environment which occurs with increased body size will result in a decreased rate of delivery of these limiting factors. The purpose of this review is to discuss how this limitation in the delivery

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of materials can be expected to influence the physiology and cellular metabolism of fish.

II. Theoretical explanations for the negative allometry of aerobic metabolism The scaling of aerobic metabolism has been the subject of extensive theoretical and empirical study 14,11~ It is not my intent here to review this material in detail, however some theoretical overview is necessary to understand the patterns of metabolic allometry in fish and how they are different from other groups of animals. Many issues remain controversial, even including what the actual value is for the scaling exponent b (where 17o2 ffi a Weightb). Peters 11~ analyzed a data set of 146 mammalian species and obtained a mean intraspeeific value for b of 0.74, confirming the 3/4 law or Kleiber's rules3. Heusner 62 examined a similar set of data for mammals and concluded that the actual interspecific value for b is 0.68, in accordance with theories based on surface area. The available information for fish is summarized in Fig. 1 as a frequency distribution of the intraspecific scaring exponent b for standard, i.e. basal, metabolism. The data are taken from early reviews ~,7s,~~176176 together with new values reported over the last two decades as referenced in the legend. In studies where estimates of b were obtained under varied conditions (such as at different temperatures, salinities or rations), each value of b was included individually. The mean of 104 reported values for b is 0.790 with a standard error of 0.011. This mean value for b is significantly higher than

Fig. 1. Frequency distribution of the intraspecific scaling exponent b for the standard metabolic rate (SMR) of the fish, where SMR - a Weight o, b - 0.79, n -- 104. Values taken from refs. 4, 10, 15, 20, 21, 24, 25, 30, 44, 60, 66, 69, 78, 80, 81, 98, 100, 104, 106, 107, 111, 122, 140, 156, 160 and 161.

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both 0.67 (p <0.001) and 0.75 (p <0.01). For now let us just recognize that aerobic metabolism displays negative allometry in fish and consider the mechanisms which have been proposed to explain this relationship. Theoretical explanations for the decrease in metabolism with size can be divided into two categories; those based on the rate of energy use by animals, and those based on the rate of energy production (i.e. the delivery of oxygen and other metabolites). Included in the first group are mechanisms such as changes in tissue composition with size, the energetic costs of homeothermy, and the energy demand by muscles during locomotion 26,s3,Ss,11~ The arguments made by this group of explanations are for the most part scientifically valid, however, their lack of generality suggests that they are not fundamental mechanisms for the negative allometry of metabolism. In this regard fish provide a useful comparison and test of these theories. For example, the locomotion model proposed by McMahon 94 is based on terrestrial animals, laws of elastic criteria, and the observation that larger animals are progressively stockier. Most fish are neutrally buoyant and do not experience the types of structural stresses during locomotion on which this model is based; and yet their metabolism also shows negative allometry. Similarly, most fish are not endothermic, and therefore are not subjected to the decrease in relative homeothermic costs with increased size. The universality of aerobic allometry suggests that it is not the result of changes in energy demand, which will be different for different groups of organisms, but rather the result of decreased energy production (i.e. a 'supply-side' limitation). 1. Muscle contraction time

One area of theory which has been developed to account for the negative allometry in metabolic rate is based on the maximum rate of energy use by the skeletal muscles during locomotion 94,128. A particular line of reasoning among these 'demand-side' theories notes that the rate of muscle contraction decreases with increased body size, which suggests a concomitant decrease in muscle metabolism. And, since muscle mass is such a large proportion of whole-body mass, overall metabolic rate will also presumably decline. Theoretical-estimates of the maximum velocity of shortening by mammalian muscle fibers do indicate negative allometry 63,86,95 with a recent empirical study reporting a scaling exponent of -0.18 for slow fibers 123. In the cod (Gadus morhua) 3, muscle mass, myotome cross-sectional area, and fiber length scale geometrically, whereas twitch contraction time is proportional to L ~ L ~ Furthermore, as individuals of this species increase in size, the cycle frequency for maximum power output decreases from 12.5 to 5 Hz, or as L -~ (~, W -0.17, ref. 2). Wardle 15~ also observed that the contraction time of white lateral muscle more than doubled with increased body size for a variety of teleosts ranging in size from 10 to 75 cm. Assuming that contraction time was reflected in the frequency of tail-beats during swimming, Wardle used this relationship to account for the scaling of maximum swimming speed among fish. Although all of these observations appear to be valid, they are weakened as explanations for metabolic allometry for two reasons. Firstly, the universality of metabolic allometry, even among sessile

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species with little or no muscle tissue, suggests that the scaling is not solely the result of locomotor costs. Secondly, even with muscle respiration rate decreasing with increased size, one would expect that this aerobic capacity would be exploited by the fish for other fitness-related processes such as growth or reproduction.

2. The effectof body sizeon the rate of oxygen delivery The limitation to material delivery imposed by increased size is illustrated here using oxygen. The same principles hold true, however, for all other materials whose rate of delivery or removal will affect metabolism; e.g. CO2, NH3, and organic substrates. It is tempting to consider oxygen as the most limiting factor on the basis that some other components can be stored within in the body. For example, glucose can be stored either in the liver or locally as glycogen. However, similar adaptations also exist for oxygen in the form of cellular myoglobin and the splenic storage of erythrocytes. Ultimately, all of the materials involved in metabolism are under similar limitations of delivery rate for sustained aerobic energy production. The movement of oxygen from the environment to mitochondria in fish can be divided into three general steps; uptake by diffusion at the gills, convective transport in the blood, and finally diffusion again into the cell (Fig. 2). We should ask, to begin with, how an increase in body size would affect each of these individual steps, and secondly how much potential capacity exists at each step to compensate for the effects of body size. The uptake of oxygen at the gills by diffusion is a function of the surface area available, the diffusion distance, the diffusion coefficient, and the concentration difference across the respiratory surface. Assuming structural similarity, among these factors only surface area would be expected to change systematically and appreciably with increased size; scaling as Weight2/3. This decrease, however, could be compensated for by increasing the proportion of tissue allocated for respiration or by increasing the structural

Fig. 2. Summary of respiratory steps involved in the delivery of oxygen from the environment to the cells of fish. The proportion of total energy demand supplied by either aerobic or anaerobic energy production will vary with tissue type and body size.

The metabolic consequences of body size

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complexity of the respiratory surface. Respiratory surface area can and does display isometric scaling for mammals 154, amphibians 143, and insects s2, as well as for some fish 67'71'125. The area available for oxygen uptake, therefore, does not appear to be the major limitation for aerobic metabolic scaling. The convective transport of oxygen in the circulatory system is determined in general by the oxygen carrying capacity of the blood, the proportion of oxygen unloaded at the tissues, and the volume of blood per gram of tissue which can be delivered in a given unit of time. There is no underlying reason to expect either oxygen carrying capacity or the percent of unloading to be greatly affected by body size. However, the volume of blood which can be delivered to a gram of tissue over a given time interval will be dramatically affected by body size. Total blood volume displays isometric scaling for animals in general 121 and for fish in particular 67, which means that a gram of tissue in a large fish is supplied by the same volume of blood (approximately 50/zl) as in a very small fish. Therefore, as has been eloquently noted by Coulson and colleagues 18,19, the rate of oxygen delivery to a unit of tissue will be inversely related to blood circulation time, which can vary by several orders of magnitude. For mammals 136, circulation time increases as body weight raised to a power of 0.79. Consider, for example, the situation in a 100-cm fish and a 2-cm fish. If blood velocity is 1-cm/s and independent of size 58, during a 100-s interval an average gram of tissue in the large fish will be supplied with a single unloading of blood oxygen (i.e. 2 x 50 cm). During the same time interval in the small fish, an average gram of tissue will be supplied with the oxygen from 50 unloadings. Since orders of magnitude increases in blood velocity are improbable, this physical distancing and its effect on oxygen and metabolite delivery appears to be the most direct and inescapable consequence of increased body size. The final step in the movement of oxygen from the environment to the mitochondria involves diffusion from the capillaries into the cell. Changes in body size would not be expected to directly affect processes at this level since cell size and capillary structure are relatively unchanged. Of the three general steps in oxygen transfer (uptake by diffusion, convective transport and diffusion into the cell), it is the convective transport by the circulatory system that appears most clearly limiting. Increased capacity for non-limiting steps, e.g. diffusion into the cell, would not increase the overall rate of metabolism; nor would increased capacity for the use of oxygen such as mitoehondrial density. However, compensation for decreased oxygen delivery capacity with increased size might be expected to occur for components of the limiting step, convective transport.

III. Symmorphosis and the scaling of individual respiratory traits If the overall rate of oxygen delivery and use decreases with increased body size, how would this be expected to affect the scaling of the individual steps involved in respiration? The concept of symmorphosis provides a framework for addressing

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this question. As originally proposed by Taylor and Weibe1141 the hypothesis of symmorphosis states that 'the formation of structural elements is regulated to satisfy but not exceed the requirements of the functional system'. When applied to allometrie variation among mammals ls4, the hypothesis of symmorphosis appears to be acceptable for all internal compartments of the respiratory system (blood, heart, muscle capillaries, and mitoehondria). However, syrnmorphosis does not seem to be supported by data on the lung, which forms the interface to the environment. It makes intuitive sense that selection would operate towards a system where individual steps in a multi-step process such as respiration would be matched. It is not appropriate, however, to test the concept of symmorphosis on single structural parameters4~ the entire structural system must be considered in context. Consider for example the Antarctic ieefish (Chaeniehthyidae) whose blood lacks hemoglobin 67. The decrease in oxygen carrying capacity which results should not necessarily be expected to be accompanied by a decrease in the capacity of other components of oxygen transfer, such as cardiac output. In fact, relative heart size and cardiac output in these fish is extraordinarily high61, as a probable compensation for the absence of hemoglobin. The proper question to ask when applying symmorphosis is whether capacity at one functional level, e.g. circulatory transport, is matched to the capacity of other functional levels, such as the aerobic capacity of the tissues. 1. Blood oxygen carrying capacity Whichever process limits oxygen delivery and use, symmorphosis predicts that all other steps will decrease in capacity with increased body size to match this rate. However, to the extent that it is physically possible, positive eompenstaion may be expected for components of that process which is limiting. If, as discussed above, convective transport and increased blood circulation time is the limiting factor in aerobic respiration, then one would not expect to see a decrease but rather a compensatory increase in its various components. This appears to be the situation for fish. Fig. 3 summarizes data on the sealing of hematoerit values for fish, and the pattern that emerges is that larger fish have higher concentrations of circulating erythrocytes. The associated increase in hemoglobin concentration, and higher blood oxygen carrying capacity which would result from this, may mitigate some of the effects of increased blood circulation time. The hematoerit values presented in Fig. 3 were not obtained from eannulated fish but from fish which would have experienced some degree of physical activity during sampling. Stress in general and physical activity in particular are known to cause an increase in the hematoerit values of fish 132. It is not dear then, if the reported positive allometry in hematoerit is a general pattern existing in resting fish or if it is the result of activity during sampling and the release of erythroeytes from the spleen or other organs. The scaling of spleen weight in fish has, in fact, been reported to display positive allometry 67, with a mean sealing exponent for five species of 1.20. A study of splenic contraction in tilapia (Oreochromis niloticus) 16z further suggests that the allometry in hematocrit occurs as the result of handling and the energy

The metabolic consequences of body size

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Length (cm) Fig. 3. Effects of body size on the hematocrit values reported for fish. Intraspecifically, larger fish appear to have higher concentrations of circulating erythroeytes and also, presumably, increased oxygencarrying capacity. Data are for tilapia 124 (Sarotherodon mossambica; O, now Oreochromis mossambicus), largemouth bass 17 (Microptems salmoides; ~ ) , American plaice 130 (Hippoglossoides platessoides; - - -), North Sea plaice 120 (Pleuronectes platessa; 0), cutthroat trout 85 (Salmo (Oncorhynchus) clarki; 0), rainbow trout 38 (Oncorhynchus mykiss; ..... ), European sea bass 38 (Dicentrarchus labrax; ...), tilapia 119 (+), and Ophiocephalus sp.119(O). Length-weight transformations for rainbow trout and European sea bass were made using the data from refs. 153 and 138, respectively.

demands of physical activity. Relative spleen weight and total spleen hemoglobin content were determined for a wide size-range of fish before and after 3 minutes of forced exercise (Fig. 4). The amount of hemoglobin (i.e. erythrocytes) released into the blood increased dramatically with increased body size, presumably resulting in increased oxygen carrying capacity. It appears, in fact, that small fish (less than 6 g) were able to accommodate the increased energy demands of swimming activity without any release of splenic erythrocytes at all. This pattern, where the energetic costs of swimming seem relatively minor to small fish, is one that occurs at many levels as we will see later in the context of anaerobic scaling. A further way that fish could compensate for changes in oxygen delivery capacity with increased size is by altering blood oxygen affinity. Half-saturation pressures (P s0) decrease with increased body size for birds 88, lizards 113 and mammals 88. The decrease in blood oxygen affinity with decreased body size that this represents is thought to favor unloading of oxygen in small individuals with a high aerobic demand. The relationship for snakes appears to be the opposite, with an increase in oxygen affinity with decreasing body size 112. This unique allometry has been

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attributed to differences in pulmonary gas concentrations which are produced by the specialized lung morphology and breathing movements of snakes. No such multi-species data set on the scaling of blood oxygen affinity exists for fish. A recent study of Dover sole (Microstomuspacificus) captured off the Coast of California did report a pattern of increased blood oxygen affinity with increased size, with P50 values decreasing from 4 torr at 100 g to approximately 2 tort at 1500 g (ref. 148). However, this species makes an ontogenetic vertical migration from shallow water into the Oxygen Minimum Zone where oxygen concentrations fall to as low as 0.30 mg/l (ref. 73). It is not yet clear, therefore, if this allometric relationship can be generalized to all fish. Multiple hemoglobins are common among fish11s, and changes in the frequency of particular forms with increased size and age have been reported for many species including herring (Clupea harengus )159, salmonlSS, Seorpaeniehthyes9~ and skate 91. Whether these shifts in hemoglobin have any functional significance with regard specifically to body size is not known. Compensation by larger fish for decreased oxygen delivery capacity could also occur through an increase in tissue myoglobin concentration. This, like the apparent increase in splenic storage, would provide the tissue of larger fish with a greater reservoir of oxygen to use during periods of high energy demand. Increased concentrations of myoglobin in larger species have been reported for the heart and skeletal muscle of mammals22, with a sealing exponent of 1.31 calculated for whole-body content t. A similar pattern is seen for the aerobic tissue of fish, with cardiac myoglobin content displaying positive allometry for both salmonids 32 and tuna ~16. No information exists for the skeletal muscle of fish, however it is known that for some species the proportion of myoglobin-rieh red muscle to total muscle increases as fish become larger46'54.

The metabolic consequences of body size

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2. Gill surface area Respiratory surface area can and does scale isometrically, or nearly so, for many animals. Interspecific data are most complete for mammals where scaling exponents of 0.95 and 1.19 have been reported for mammals in general and wild species, respectively 41. A recent analysis of total pulmonary diffusing capacity for animals ranging in size from a mouse to cow indicates a scaling exponent of 1.084 (ref. 154). Studies of fish have also found that gill surface area can scale isometrically, at least within a species. Such results have been reported for the mackerel (Trachurus trachurus71; b = 1.17), the carp (Cyprinus carpio99; b ~ 1.0), the dragonet (Callionymus/yra71; b = 1.52), an erythrinid (Hoplias malabaricus35; b = 1.18), and the icefish (Chaenocephalus aceratus67; b = 1.09). Taken together these results suggest that relative respiratory surface area need not necessarily decline with increased body size, and that it may not be a limiting factor in oxygen delivery. In general, however, intraspecific scaling exponents for the gill surface area of fish are mostly between 0.8 and 0.9 (refs. 99, 107), with actual values of b ranging widely from as low as 0.60 to the examples cited above which display positive allometry. The fact that most species display negative allometry seems unusual since information for other groups of animals, and some fish, suggests that there is no physical constraint to respiratory surface area increasing in proportion to body size. A decrease in relative gill surface area could be explained, however, if the concept of symmorphosis is correct in its assertion that the various steps involved in respiration are matched. The decreased capacity for oxygen delivery and use with increased body size may require less respiratory surface area for oxygen uptake. Pauley 1~ has proposed that low values of b for gill surface area, near 0.6, are observed for small species of fish but that values of b approach unity for species with increased maximum body size. His arguments are based in general on the yon Bertalantiy growth equation, and on the allometry of oxygen requirements for growth. However, no clear explanation is given for why the value of b should differ between small and large species. The evidence supporting a relationship between the value of b for gill surface area and maximum body size is equivocal 1~ and the relationship might be dismissed were it not for a similar pattern which appears to exist between the value of b for metabolic rate and maximum body size. A definitive analysis of this latter relationship is precluded by the indeterminate nature of fish growth, and the difficulty in objectively defining the maximum size of a species. Also, most studies only include individuals over a portion of the species' size range, and relatively few small or large species have been studied. Furthermore, temperature appears to influence the value of b (ref. 44), and the studies cited in Fig. I have been conducted at a variety of ambient temperatures. If we focus, however, on those studies which have examined only small species of fish (less than about 5 g), we find that all report very low scaling exponents for metabolic rate compared to the overall mean value for fish of 0.79 (Fig. 1). The earliest of these, b = 0.71, was derived by combining data for three species of Cyprinodontiformes all less than 1.4 g (ref. 160). Even lower values have been reported for the pupfish (Cyprinodon macularius81; b = 0.63), the mosquitofish

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(Gambusia affin/$97; b = 0.64), a Cyprinid minnow (Phoxinus phoxintt$2~ b = 0.55), and the sand goby (Gobius minutust~ b = 0.62). While the value of b for metabolism varies widely for the majority of fish studied (i.e. species of intermediate size), the consistently low values for small species does suggest that a single value of b may not be representative of all fish and that the actual value of b may increase with the maximum size of the species. Supporting such a relationship are values of b for gill surface area, such as that for one of the word's smallest species, the Philippine goby (Mistichthys luzonensisl~ b = 0.60). Recent studies which have re-analyzed the mammalian literature have independently come to the same conclusion, that the actual value of b for a species may depend on its maximum size. Jurgens 7s re-examined the data of Bartels 5 and found that the value of b was significantly lower in small mammals (0.60) than in large mammals (0.77). A re-analysis 7s of the data of Hayssen and Lacy59 revealed values of b for small and large mammals of 0.65 and 0.86, respectively. Finally, the metabolic data of Elgar and Harvey29 indicated a scaling exponent of 0.65 for small mammals and one of 0.86 for large species. Why metabolic rate should display greater allometry over the life-span of a small species compared to a larger one is not dear, however with regard to fish two possible explanations are most apparent. The first concerns the relatively large influence of growth rate on the metabolism of fish and other ectotherms. For although the basal metabolism of fish is approximately an order of magnitude lower than mammals, organismal-level rates of growth in these groups can be similar. Hence, among fish and other ectotherms ingestion and growth rates will largely determine an individual's overall rate of metabolism 4s's2'l~176 If we assume that, for species of all size, initial larval rates of growth are at some similar maximum, and that at that their respective adult size growth rates are also the same (i.e. zero), then the same decrease in growth rate (and therefore metabolism) must occur over a much more narrow size range for smaller species. This would result in more severe negative allometry for small species and a lower value for the metabolic scaling exponent b. A second interpretation of higher scaling exponents in larger species involves the energetic cost of swimming and how it is affected by body size. The hydrodynamics of locomotion in water are such that the power required to overcome drag increases dramatically with larger size lsl. Scaling exponents of approximately 1.17 and 1.34 have been calculated for sustained and sprint swimming, respectively 152. In larger species, such as salmon, higher swimming costs in larger individuals are indicated by scaling exponents for oxygen consumption during aerobic activity which are 1.0 or higher ~~ This increase in aerobic scope, and the resulting size-independence of active metabolism, may also be reflected in relative isometry for standard metabolism in large species. Small species would not require this increased aerobic scope, and would therefore display greater negative allometry. 3. Cardiac output The cardiac output of fish is determined by contraction frequency and by stroke volume, the latter of which is presumably limited by heart size. As discussed earlier

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for skeletal muscle, increases in body size would be expected to result in increased muscle contraction times and, therefore, decreased contraction frequencies. While such changes in the skeletal muscle may not limit overall metabolism, decreases in cardiac frequency with increased size would directly influence the whole-body rate of oxygen delivery by its effect on cardiac output. Data for mammals, birds, and lizards do indicate negative allometry for cardiac frequency 11~ with most relationships showing the rate decreasing as approximately W -~ Limited data for the heart of the skate, Raja erinacea, also shows a decrease in frequency with increased body size 1~ The relative size of the heart is not under any such constraint, and scales according to an exponent near 1.0 for most groups of animals including fish 13'33'53'72. The scaling of total cardiac output has not been examined systematically for fish, but it almost certainly displays negative allometry as is the case among mammals 57 (b ~ 0.75). Compensation for decreased frequency by increasing relative heart size is limited, of course, since the heart itself will have increasingly higher oxygen requirements. This is largely the same conclusion reached by Jones 74, who analyzed the metabolic costs of the cardiac and branchial pumps in fish and their possible role in limiting maximum oxygen consumption. The effect of negative allometry in cardiac output on oxygen delivery (let us assume it is near W~ would be in addition to the decrease in oxygen transport in large fish due to increased circulation time. By simple dimensional analysis, the effect of increased circulation time on the mean rate of oxygen delivery is equivalent to W 0"67. The scaling exponents for cardiac output and increased circulation time, when combined, would suggest that the overall rate of oxygen delivery should increase according to W~176 It is perhaps surprising then, that the decrease in metabolic rate with increased body size is as small as it is (scaling as W~ and even more remarkable that the maximum metabolic rate during activity can remain unaffected by size 11, scaling with a b-value near 1.0. At least two explanations could account for this discrepancy. Firstly, larger fish may compensate for the effects of size on cardiac output and circulation time by increasing capacity at other steps in the delivery of oxygen. Some evidence for this possibility has already been discussed, in that larger fish appear to have greater hematocrit values and higher myoglobin concentrations. Gill surface area may also scale with higher b-values in larger species. However, the potential for such compensation seems limited, and it is not likely that increasing capacity at a non-limiting step (such as oxygen uptake at the gills) would have any effect on the overall rate of delivery. A second explanation is that small fish have the capacity to deliver oxygen at a rate in excess of what is needed by the tissues. While this is in contradiction to the concept of symmorphosis, it is not unreasonable to expect that, ontogenetically, small fish are under the developmental constraint to possess the respiratory structure which will be required as large adults when physical limitations will decrease the potential for oxygen delivery. Small species would be less affected by these considerations and, therefore, might be expected to display lower scaling exponents for metabolic rate. As discussed above, this appears to be the case. The resolution of these issues would be benefited by applying the concept of symmorphosis to the study of fish, by testing whether individual respiratory

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processes are matched, and by identifying limiting steps in oxygen delivery. The plasticity afforded by fish would be an advantage in such studies. For example, drastic growth retardation (i.e. stunting) is possible for fish, where by maintaining fish at cold temperatures and with little food they can be raised to adult age but maintained at juvenile size. Scaling studies on such populations may be able to partly separate ontogenetic and actual size effects.

4. Tissue respiration and enzyme activity There are two ways that an individual fish could accommodate a decrease in wholebody metabolic rate with increased size. One way is to shift tissue proportions away from those with high aerobic capacities to those with low aerobic capacities, and this is a pattern which is observed for intraspecific (or ontogenetic) allometry. Essentially all of the tissues of the common carp (C. carpio) display negative aUometry in weight except for the tissue with the lowest weight-specific metabolic capacity, the muscle tissue 53,1~176 From 0.10 g to adult size muscle mass increases from approximately 30% to over 60% of total body mass. This shift in tissue proportions is in accord with energy requirements during ontogeny, i.e. high food processing and growth capacity when small, and high swimming costs at larger sizes. Although these changes do not provide a mechanistic explanation of metabolic aUomctry, they can partly account for the observed whole-body decrease in aerobic metabolism within a species. In the case of interspecific aUometry only adult fish are being considered, all having a growth rate near zero. Any shift of energy use away from the viscera, therefore, would be expected to be less severe or to not occur at all. In the absence of such shifts in tissue proportions, increased body size must be accompanied by decreases in weight-specific aerobic capacity for individual tissues. Unfortunately, almost no information exists for fish on the scaling of tissue aerobic capacity among species of different adult size. One study of succinate dehydrogenase in the brain tissue of 13 species of marine fish96 reported activity decreasing as W -~ However, it was not clear that the individual fish examined represented the maximum size for each species. The remaining data which exist on the scaling of aerobic enzyme activity, represents intraspecific studies, and most of this information is for muscle tissue. This literature, and data on in vitro rates of oxygen consumption by tissues, indicates a general pattern of negative allometry with most scaling exponents for weightspecific activity ( = b - 1) ranging from -0.15 to -0.30. A summary of data for the aerobic enzyme citrate synthase (CS) in white epaxial muscle indicated a mean scaling exponent of -0.23 for 18 species 16. Variability in the value of b was large, however, ranging from near isometry (-0.06) to allometry as severe as -0.68. In the skeletal muscle of the toadfish (Opsanus beta) 149 CS activity scales with exponents of -0.24 and -0.18 for males and females, respectively. A large data set has recently been collected for the white muscle CS activity of a flatfish, the California halibut (Paralichthys californicus) 1~ CS activity declines with a scaling exponent of -0.15 for individuals ranging in size from 0.2 to 1073 g (Fig. 5A).

The metabolic consequences of body size

347

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Body weight (g) Fig. 5. The scaling of citrate synthase (CS) and lactate dehydrogenase (LDH) activity in the white muscle tissue of a flatfish, the California halibut (Pamlichthys califomicus). Scaling exponents are -0.15 and 0.46 for CS and LDH, respectively. From ref. 147.

An analysis of total muscle, i.e. red and white, cytochrome c oxidase (CCO) activity in the common carp showed no decrease in weight-specific activity with increased size 53. This result, and a similar one for in vitro oxygen consumption 146, could be explained by an increase in the proportion of red muscle tissue with increased size which has been demonstrated for fish46. This increase in red muscle tissue, if found to be general among fish, may be an important compensation for decreased weight-specific metabolism with increased size. Negative allometry is also the most common scaling pattern for the aerobic metabolism of other tissues, but again with some exceptions and with widely ranging scaling exponents. The CCO activity of the carp brain 53 and the pooled CS activity of brain tissue for two Paralabrax species 133 both decrease in larger fish, scaling as -0.27 and -0.09, respectively. However, no effect of body size was reported for the brain CS activity of the Dover sole 134. Weight-specific CCO activity in the intestine of carp 53 decreases as W -~ and in vitro rates of oxygen consumption were also reported to decrease for the gills (b = -0.14 ) and kidney (b = -0.15) of the cutthroat trout (Salmo clarki, now Oncorhynchus clarki) 68. Similarly, liver CCO activity in the American plaice (Hippoglossoides platessoides) TM was found to scale as W -0"14. One factor which may be influencing these slopes is the relative cost of growth in each tissue and how that is affected by body size. Fractional rates of

348

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protein synthesis in tissues of the rainbow trout (Oncorhynchus mykiss, previously Salmo gairdneri) display negative aHometry7~ with scaling exponents ranging from -0.14 in the ventricle to -0.49 in white muscle tissue. Both the oxygen consumption rate and CCO activity of the heart of the sea raven (Hemitripterus americanus) decrease with increased size33, displaying scaling exponents of-0.51 and -0.13, respectively. However, five other cardiac enzymes, both aerobic and anaerobic, were found to be size independent. More species need to be examined before the general scaling pattern for heart tissue is clear, but there may be reasons to expect its response to be unique from other tissues. Highly aerobic tissue such as this under continuous energy demand, and with luminal peffusion, may be maintained at a more constant level of aerobic capacity. Compensation for changes in cardiac output demand in fish appears to be made more by increasing the relative size of the heart, and therefore presumably stroke volume. The activity of cardiac enzymes in the almost mammalian-like tuna are no higher than in inactive species12; compensation occurs by increasing heart size. Similarly, compensation by fish for cold temperature occurs by increasing heart size45, and the increased energy demands of feeding and growth are accompanied by increases in heart size but not by changes in weight-specific enzyme activity52. Hochachka et al.65 have proposed that the oxidative capacity of muscle tissue may be limited by mitochondria volume densities, and this principle may be operating in fish cardiac muscle. Finally, enzyme activities were recently obtained for whole-body homogenates of larval Atlantic menhaden (Brevoortia tyrannus) ranging in size from approximately 1 to 10 mg 117. Weight-specific CS and malate dehydrogenase (MDH) activities displayed sharp negative allometry, scaring with exponents of -0.785 and -0.805, respectively. However, this is a period of substantial ontogenetic development, and these extreme exponents may reflect changes in tissue proportion as well as actual scaling effects.

II4. Tissue-level exceptions to negative allometry in aerobic metabolism The only requirement concerning metabolism, given the organismal limitations on oxygen delivery, is that the sum of aerobic capacity for all tissues must decline with increased body size. However, there is no physical reason why the metabolism of all tissues must decline, or that the metabolism of each tissue must decline at the same rate. Specialized patterns of tissue-specific scaling will occur as is beneficial to the species depending on its unique physiological, behavioral and ecological requirements. Hence, there are numerous exceptions to the general pattern of allometry (i.e. W~ and nearly all possible values for the intraspecific scaring exponent b have been reported for various tissues. In larval fish, even positive allometry has been observed for muscle CS activity, with scaling exponents of 0.21 and 0.24 reported for northern anchovy (Engraulis mordax) and Pacific sardine (Sardinops sago.x), respectively76. These increases in aerobic capacity were attributed to the increased energetic costs of swimming

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during that period, and presumably prior to the level when delivery constraints would limit oxidative metabolism. Subsequent studies are suggestive of an inflection point at approximately 100 mg for the northern anchovy77. Much variability would be expected for the scaling of liver metabolism because of its role in energy storage, and this appears to be the case. During the period of smoltification in Atlantic salmon (Salmo salar), for example, liver CCO activity actually increases with larger body size 8. Patterns such as this most likely reflect periods of energy mobilization rather than scaling effects. Perhaps one of the most interesting exceptions to the general relationship of negative allometry in aerobic metabolism is the sex-related differences in scaling for enzymes in the sonic muscle of the toadfish, O. beta 149. This muscle, in association with the swimbladder, is used by the males to generate a mating call during certain seasons of the year. In females sonic muscle CS activity declines with increased body size as expected, however over the same size range in males sonic muscle CS activity increases several-fold (Fig. 6A). The relationships are linear and do not fit the usual logarithmic transformation well, but when expressed this way for comparative purposes indicate scaling exponents of -0.13 and 0.43 for females and males, respectively. Here is a clear demonstration that fish can overcome the constraints of increased body size by altering enzyme levels, and presumably also perfusion rates, to maintain and even increase aerobic energy production when there is a strong selective advantage to do so. An expected consequence of this increase, of course, is that some other tissue and physiological process must show greater than normal negative allometry for whole-body allometry to be maintained. An additional notable exception to the general scaling paradigm concerns the tissue with the highest known aerobic capacity among vertebrates, which occurs not in a small mammal but among large species of fish. This tissue is in the heater organ of the endothermic scombroid fishes, and in some species it displays higher CS activity than even that reported for hummingbird flight muscle 9. High aerobic enzyme activity in this tissue is associated with the highest known mitochondrial volume (63-70%), made possible in part by the absence of contractile filaments. This observation further supports the proposal that mitochondrial volume may be the limiting factor in cellular oxidative capacity. Scaling information is limited, and confounded by other factors, but it appears that among species which possess this heater organ tissue aerobic capacity for adults displays the usual negative allometry. The effects of body size within a species are more complex. Based on the scaling of heat loss and thermal inertia one would expect that the requirements for heat production would be greater in smaller individuals. However, because it is not practical to maintain elevated temperatures when small, young individuals of endothermic species do not even possess well-developed heater organs. This is also reflected in the fact that the heat-generating red muscle of endothermic scombroids displays positive allometry 54. Therefore, it is probable that if the entire size range of individuals within a species is considered, heater tissue aerobic capacity would display positive allometry at least over some initial size range. This would be a pattern similar to that which seems to occur during the ontogeny of locomotor muscle tissue in larval fish 77. The heater organ system of scombroids further

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Fig. 6. Sex-related differences in metabolic scaling for the sonic muscle tissue of the toadfish, Opsanus beta. A: effect of body size on muscle citrate synthase activity for female (e, b ffi -0.13) and male (o, b ffi 0.43) toadfish. B: effect of body size on muscle lactate dehydrogenase activity for female (O, b ffi -0.09) and male (m, b ffi - 0.24) toadfish. From ref. 149 with permission.

demonstrates how it is possible for fish to overcome, if only locally, the restrictions imposed on tissue metabolism by body size.

V. The scaling of maximum whole-body aerobic capacity We began this discussion by considering the allometry of standard metabolism in fish, which appears to scale as W~ followed by an analysis of what factors may limit aerobic capacity in larger individuals. If the mechanisms presented are

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correct, however, then the limitations they impose should be reflected even more directly in the scaling of maximum whole-body aerobic metabolism. This appears to be the case for mammals where both basal and maximum aerobic metabolism scale approximately as W~ (ref. 142). Maximum aerobic metabolism in fish, as with mammals, has been determined during maximum sustainable locomotor activity (= active metabolism). Among fish, however, maximum metabolic rate has been reported to scale isometrically, or even more remarkably with positive allometry 11,14~ It seems inconsistent, therefore, to develop scaling theory to explain negative metabolic allometry when maximum aerobic capacity, i.e. oxygen consumption, is unaffected by size or actually increases per gram of tissue in larger individuals. Considering the universality of negative aerobic allometry, the most likely resolution to this paradox is that the active metabolism of fish does not represent the maximum metabolic rate for all sizes of fish. That this should be the case is suggested, firstly, by aerobic locomotor muscle proportions. Unlike mammals where locomotor muscle comprises nearly half oftotal body mass 1~ fish typically possess only about 3% of body mass as aerobic locomotor muscle - and many species have none. The relative amount of red muscle in fish also appears to decrease in smaller individuals 46,s4. A second indication is that theoretical estimates of the energetic costs of swimming in fish increase dramatically with body size. Even accounting for a length-specific decrease in performance, the power requirements to overcome drag at maximum sustainable speeds have been predicted to increase as W 1"17 (ref. 152). Furthermore, since we are discussing mostly intraspecific comparisons here, we must also consider the scaling of feeding and growth which would be expected to be a larger component of energy allocation among small (i.e. young) fish. One approach to this issue would be a complete partitioning of aerobic capacity to individual tissues and, by inference, to physiological function. This has been done for the common carp using CCO activity as a measure of tissue-specific aerobic metabolism 4a,53. The proportion of whole-body CCO activity contributed by each of the white muscle, the red muscle, and the viscera, are summarized in ~ig. 7 for male fish ranging in size from approximately 2 to 2200 g. The first thing to note is that most of the aerobic capacity in these fish (40-65%) occurs in the characteristically anaerobic white muscle tissue. This is aerobic capacity which is not even called upon during the sustained swimming activity used to measure maximum metabolic rate. It is employed, however, following exhaustive anaerobic activity when energy is required for recovery processes. This large aerobic capacity in the white muscle can account for the observation that oxygen consumption rates following exhaustive exercise can exceed that during aerobic swimming in adult fish 135. The next largest component of whole-body CCO activity in male carp is the summed activity for the viscera, i.e. those tissues involved in food processing and growth. In the smallest fish the viscera contributes almost 40% to whole-body CCO activity, with this proportion declining with size to equal the contribution of red muscle in 55 cm fish at approximately 15%. This pattern suggests that the rate of oxygen consumption following a meal (Specific Dynamic Action, SDA) may exceed that for active metabolism, a function of red muscle, in small individuals but not for

352

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Fig. 7. Tissue-specific partitioning of aerobic capacity in male carp, Cyprinus carpio, as measured by cytochrome c oxidase (CCO) activity. In small fish the summed aerobic capacity of the viscera, used in food processing, is far greater than that which occurs in the red aerobic locomotor muscle. From refs. 48 and 53.

larger ones. The only data available for the scaling of SDA supports this prediction. The maximum postprandial rate of metabolism in the largemouth bass (Micropterus salmoides) is higher than that during sustained aerobic swimming for individuals up to 100 g, after which active metabolism far exceeds maximum S D A 139. It appears then that fish differ from mammals in that a variety of physiological activities can be responsible for maximum aerobic metabolism among individuals of different size. Unique characteristics which contribute to this include an extremely small minimum size (mg), a buoyant medium which permits large anaerobic muscle mass, no maintenance of elevated body temperature in small individuals, and, having a lower metabolism, their energy allocation is influenced to a larger degree by the costs of growth. Active metabolism as it has been determined is an accurate measure of the energetic costs of swimming, but it does not seem to represent the scaling of maximum aerobic metabolism. When the aerobic contribution of other physiological activities are included, such as the elevated metabolism of postprandial young fish, it is likely that maximum aerobic capacity will conform to the expected relationship of negative aUometry.

I/7. Aerobic allometry: a comparison with hypoxia Nearly all of the mechanisms which have been proposed to account for the negative allometry in aerobic metabolism, such as respiratory surface area or circulation time, are based on the decreased rate of oxygen delivery to the tissue. From the cells

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perspective, then, the effects of increased body size are little different than those imposed by environmental hypoxia. For this reason, it is interesting to compare a few of the metabolic adjustments made by fish to these two respiratory challenges. Beginning at the gills, compensation for hypoxic conditions occurs largely by increasing the amplitude of ventilation, and ventilation volume 43. This indicates that under hypoxia oxygen delivery is more diffusion-limited than perfusion-limited, as is the case under normoxie conditions 89,126. That no systematic pattern similar to this is observed with increased body size suggests that oxygen uptake at the gill, e.g. surface area, is not the limiting factor for scaling. During hypoxic exposure blood oxygen carrying capacity is increased by higher hematocrit levels and higher oxygen affinity (a lower P50). Hematocrit levels also seem to increase with body size (Fig. 3), however, in contrast to hypoxia this appears to be more of an episodic increase in larger fish as energy demands require. Whether there are general patterns for the scaling of Pso among fish is still not clear, but any effects of size if they exist are likely to be small and not of the kind which could overcome the influence of increased circulation time. Predicting changes in cardiac function is more complicated, because although higher cardiac output may increase the rate of oxygen delivery, it will also increase metabolic costs. Hypoxic bradycardia during acute exposure is often fully compensated for by an increase in stroke volume to maintain cardiac output 34, however long-term studies at clearly limiting low oxygen concentrations are rare. A recent study of Dover sole maintained at 0.50 mg/1 for 8 weeks suggests that when metabolic rate is permanently depressed, relative heart size decreases 51. The decrease in cardiac output which would result may facilitate oxygen uptake at the gills or, alternatively, the reduction in cardiac tissue may represent a meaningful energy savings. No such changes in relative heart size occur as a result of scaling, however an almost certain decrease in heart rate would similarly lower total cardiac output. Increased cardiac myoglobin concentrations appear to be associated both with hypoxia tolerance among species 23, and increased body size 32'116. A study of long-term acclimation to hypoxia by the killifish (Fundulus heteroclitus) reported only selected and transient changes in liver enzyme activity56. However, other respiratory adjustments such as hematocrit and oxygen affinity were observed which may have provided nearly complete compensation. A more severe and longer exposure to hypoxia is encountered by the Dover sole during its ontogenetic migration into the Oxygen Minimum Zone. At a size of approximately 300 g it encounters the lowest oxygen concentrations (0.3-0.5 mg/l), and it is in the muscle of these fish that sharp decreases are observed in both aerobic and anaerobic enzyme activity (Fig. 8) 147. This response is very different than the general scaling pattern of enzyme activity, where with increased size decreases occur in aerobic enzymes but increases are seen for anaerobic metabolism. The limiting influence of oxygen delivery during hypoxia does not appear to allow anaerobic energy production by the muscle during locomotor activity. In contrast, the positive allometry for anaerobic metabolism suggests that, although oxygen delivery may be limiting metabolism in larger individuals, these fish retain the necessary unused aerobic capacity to repay the oxygen debt following an anaerobic episode.

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Fig. 8. White muscle citrate synthase (CS) and lactate dehydrogenase (LDH) activities from Dover sole, Microstomus paciIfcus, ranging in size from 2 to 2000 g. Shallow-water fish (<200 m), in water of higher oxygen content, are indicated with open circles. CS and LDH activities for these fish scale with exponents of-0.36 and 0.14, respectively. From reL 147.

VII. Effects of body size on anaerobic metabolism 1. Enzymaticevidence Anaerobic metabolism is not, at least initially, constrained by the delivery rate of oxygen and other materials as is true for aerobic metabolism. We would, therefore, expect it to be influenced by body size differently that aerobic metabolism and this is indeed true for certain tissues. Anaerobic energy production in fish generally occurs through one of two pathways: (1) the high-power, low efficiency lactate pathway used primarily by muscle tissue during extreme activity; and (2) the low-power, higher efficiency pathway leading to ethanol which is employed by some cyprinids

The metabolic consequences of body size

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during environmental anoxia. The scaling of this second pathway has not been investigated, however it would not be surprising to find that it scales with the negative allometry of aerobic metabolism. This is expected because it is usually activated during episodic exposure to low oxygen, and would therefore have to supply the higher whole-body maintenance requirements of smaller fish. The lactate pathway is called upon to produce energy at rates greater than that possible aerobically during periods when high swimming performance is critical 49. The first evidence that this form of anaerobic metabolism does not decrease but in fact usually increases with body size came from the scaling of weight-specific white muscle glycolytic enzyme activities. Mean intraspecific scaling exponents of 0.35 and 0.21 were reported for lactate dehydrogenase (LDH) and pyruvate kinase (PK), respectively, among 13 species of marine fish 133. A later study of LDH in four Antartic mesopelagic fish 144 found their activity to scale with exponents of 0.23, 0.76, 0.95 and 1.05; while the macrourid, Coryphaenoides armatus 129, displayed a value of 0.66. Similar positive allometry has been observed interspecifically for the locomotor muscle LDH activity of mammals 31. In contrast to muscle tissue, brain LDH activity in fish is not higher in larger fish 133,134.This suggests that the changes seen in muscle are the result of muscle function and the costs of locomotion, and not simply reflecting whole-body changes in metabolism. How the scaling of these anaerobic enzyme activities are regulated is largely unknown, and this issue remains as a challenge for molecular biologists in the next decade. Positive allometry in muscle anaerobic potential is believed to be the result of an increase in the relative power required by larger fish to swim at burst or sprint speeds. This has led to a comparison of the scaling exponents for LDH among two active pelagic species, the kelp bass (Paralabrax clathratus) and rainbow trout (O. mykiss), and a benthic flatfish, the Dover sole 134. The scaling exponents for bass and trout were both positive (b = 0.41 and 0.40, respectively), while the weak-swimming Dover sole displayed a negative value of -0.44. However, the decrease in muscle LDH seen for larger Dover sole appears to be the result of factors other than just body size. As discussed earlier, the Dover sole is one of a group of species which make an ontogenetic vertical migration into the Oxygen Minimum Zone off the coast of California. A more complete data set has recently been collected for this species which indicates that once large fish enter the Oxygen Minimum Zone there is a dramatic environmentally-induced depression in metabolic rate as demonstrated by sharp decreases in muscle CS and LDH activities (Fig. 8) 147. If only shallow-water fish are considered (<200 m), occurring in water of higher oxygen, a significant increase in LDH activity is observed in larger individuals (Fig. 8A, b = 0.14), as is typical for most other species. Furthermore, recent data for another flatfish, the halibut (P. califomicus), indicates clear positive allometry in white muscle LDH activity with a scaling exponent of 0.46 (Fig. 5B) 147. The halibut is a extremely strong swimmer, which suggests that the scaling of anaerobic potential is determined more specifically by muscle function and swimming performance than by whether a species is pelagic or benthic. The increase in activity of anaerobic enzymes in the white muscle of larger fish would be expected to result in faster production of and/or higher concentrations

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(cm)

Fig. 9. Comparison of scaling relationships for white muscle lactate dehydrogenase (LDH) activity (- - -) and the concentration of lactate (O) in the white muscle tissue of rainbow trout, Oncorhynchus myk/ss, following 6 min of maximal burst activity. LDH activity is from ref. 134, and muscle lactate from ref. 46.

of lactate following maximal burst swimming. A review of the literature 4s indicates such a pattern of higher white muscle lactate concentrations after exhaustive activity in larger species and, for salmonids, in those studies examining larger individuals. This question has also been addressed directly in a study of rainbow trout~s, a species in which positive allometry in white muscle LDH activity has also been demonstrated 134 (b = 0.40, Fig. 9). Larger rainbow trout have higher muscle lactate concentrations when anesthetized (approximately 30 s of stress), and they also produce lactate at a faster rate when chased to maximal activity~. White muscle lactate concentrations following 6 min of maximal activity are significantly higher in larger rainbow trout, scaling as W~ (Fig. 9). From this one might expect that larger fish would display positive allometry for white muscle buffeting capacity, and this has also been observed for kelp bass 134 (b = 0.064). Evidence of increased anaerobic potential during activity has also been reported among a variety of other ectotherms 7,39,114, us. 2. Power requirements during burst swimming

The increase in anaerobic potential of the muscle of larger fish is believed to be the result of the relative increase with size in the power which is required to overcome drag at burst swimming speeds. Estimates of the scaling exponent for this power requirement range from 1.22 to 1.53, depending on which model is used and whether flow is considered laminar or turbulent 133. In these models length-specific burst performance is assumed to vary little with size. These estimates

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of the scaling exponent for burst power requirements are near those exponents representing the scaling of total muscle LDH activity 133 (~ = 1.55), supporting the original hypothesis. Further analysis has also considered the aerobic contribution of the red muscle to burst power requirements, and compared these two in absolute terms 48. In this analysis weight-specific power output by red muscle is taken to be 4.25 W/kg, and the proportion of red muscle tissue to whole-body mass is 3%. The scaling function which results for total red-muscle power output is shown in Fig. 10, together with the power required to swim at burst speeds as estimated from a simple Newtonian equation for drag. The difference between these two curves, which represents the anaerobic scope, appears to be near zero for small fish (1-3 cm) after which it increases with larger body size. That there may be a minimum size threshold for the use of anaerobic metabolism during swimming is also suggested by the length-specific scaling of sustained (aerobic) and burst swimming performance 48. Burst swimming appears to be little affected by size, with a velocity of 10 body lengths/s being representative of most sizes. Aerobic swimming performance, limited by oxygen delivery, decreases with size from over 12 body lengths/s in small fish to less than 3 body lengths/s in fish greater than 40 cm. These functions intersect for small fish, again at approximately 1-3 cm, which provides further evidence that very small fish can swim at the maximum speeds possible from muscle mechanics without the need for anaerobic energy production. Large and preferential increases in the anaerobic enzyme activity of muscle are also observed during this period 27,28,36,64,77. Limited data are also available for the scaling of anaerobic enzyme activity in other fish muscle tissues. LDH activity in the sea raven heart (H. americanus) increases as W ~ while the aerobic enzyme CCO declines 33 (b = -0.13). The sea raven heart differed from the muscle of this species, however, by displaying negative allometry in pyruvate kinase (PK) activity. From these results the authors propose that the hearts of larger fish may be more effective at utilizing exogenously produced lactate. New data for the heart tissue of Dover sole indicates a similar pattern 148, with scaling exponents of 0.22 and -0.17 for LDH and CS activities, respectively. In contrast to this, LDH activity in the sonic muscle of the toadfish displays negative allometry 149 for both males (b = -0.24) and females (b = -0.09). The scaling patterns for LDH activity in heart tissue and for sonic muscle tissue provide an important comparison to white epaxial muscle. They suggest that the hydrodynamic explanation proposed for locomotor muscle is only one of many mechanisms involved in anaerobic scaling, and that like aerobic metabolism anaerobic metabolism is very tissue-specific. Information on the scaling of LDH in other tissues would be useful, particularly for those like the swimbladder where its anaerobic role is central to tissue function.

3. Constraints on anaerobic scaling The anaerobic potential of fish muscle appears to increase with body size for most active species. Is there an upper limit to this source of energy production, and

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Fig. 10. Theoretical scaling functions for the power requirements during burst swimming (Pb) and the maximum power output from the red muscle tissue of a typical fish (Pro). In small fish power requirements at burst speeds appear to be met by the aerobic power output of red muscle, without any anaerobic contribution. From ref. 48.

will that limit influence the swimming performance of large fish? This issue has been addressed for mammals65, where the upper limit was considered to be set by a compromise between myofilament volume densities, and the combined volume densities of glycogen, intracellular buffering components and glycolytic enzymes. In fish, muscle glycogen concentrations are relatively constant with regard to size at about 1% of muscle weight sT. Total anaerobic capacity among species will, as a result, scale approximately as muscle mass, i.e. as W1"~ We have already seen, however, that the estimated power requirements to swim at burst speeds increase as W 1"5 ( ~ L 4"4, Fig. 10). Therefore, once a fish reaches the size when all of its anaerobic reserves are required, it will not be able to increase its rate of anaerobic metabolism (scope) without suffering a decline in swimming velocity or stamina. This analysis has been applied to the rainbow trout 46, a species for Which the scaring of total anaerobic capacity has been determined, and it suggests a marked decrease in length-specific swimming performance even over the size range of 5-50 cm. Glycogen concentrations much higher than 1% are seen in liver tissue, which has led to the proposal that muscle glycogen is maintained low as a 'brake' to glycolytic activity, and the subsequent acidification and osmotic perturbation 65. A second apparent limitation to the positive allometry of anaerobic metabolism is the decrease in aerobic capacity of larger fish which is required to repay the 'oxygen debt'. Following exhaustive activity energy is required for, among other things, the metabolizing of lactate and the resynthesis of glycogen. The rates of these processes are dependent on aerobic capacity and therefore also o n size 47,84'145. No data exist for very large fish, however attempts have been made to estimate what these rates

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would be for large animals based on a proportionality with metabolic rate. Assuming an aerobic scaling exponent of 0.75, for example, the time required for complete restoration of muscle glycogen in an 800 cm whale shark has been estimated at over 10 days 4s. This suggests that, from an ecological view, reliance on anaerobically powered burst swimming is not a practical option for very large fish. This is in agreement with the observed swimming behavior of these animals which is slow and periodic, i.e steady. Because of the limitation imposed by the scaling of muscle glycogen, and the further limitation of aerobic recovery, the scaling of anaerobic metabolism in very large fish might not show continued positive allometry. There appears to be an intermediate body size which is optimal for the useful exploitation of anaerobic energy production during activity.

VIII. Behavioral and ecological implications of metabolic scaling The scaling of aerobic and anaerobic metabolism may be a consequence of lowerlevel processes, but they will in themselves also have an effect on organismallevel characteristics such as behavior and ecology. Specifically regarding muscle metabolism, these scaling relationships suggest that very small fish (i.e. larvae) are able to swim at their maximum speeds with little or no anaerobic contribution. Such fish would have unlimited stamina. However, because the relative costs of swimming decrease with smaller body size, the aerobic scope of these small fish due to activity is very narrow. This is reflected in the fact that standard metabolism displays negative allometry (W~ while active metabolism is unaffected by size or for some species may actually increase with size. As a result of these relationships, and the fact that anaerobic metabolism is negligible, the entire range of power exerted by small fish during activity has been measured at only 4- to 6-fold in salmonids 156, and just 2- to 3-fold for larval cyprinids 157. Aerobic scope during activity increases with body size, but more significant is the increased contribution of anaerobic metabolism. When converted to units of oxygen consumption 6, the anaerobic energy production during the first 30 s of sprint activity can be greater than 7000 mg O2/kg/h in large salmonids 137. Therefore, considering also the decline in standard metabolism, the range in activity power in large fish is not 2- to 3-fold as seen for small fish but over 150-fold. This dramatic scaling of the range and potential variance in the rate of energy use during activity would be expected to influence behavior, particularly foraging behavior. If we consider the sit-and-wait through active continuum, the continual aerobic swimming of small fish over a narrow power range characterizes them as active foragers. With larger size, however, behavior consists more and more of periods of low metabolism broken by intermittent episodes of very high power output (anaerobic metabolism). This activity pattern suggests that the mode of foraging by fish will become increasingly sit-and-wait with increased body size. Such a change in foraging will, in turn, affect other ecological characteristics including habitat choice and prey choiceS~ and as such be a factor determining ontogenetic niche shifts 155. This predicted shift in foraging behavior does appear to occur

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among large generalist species. With increased body size foraging switches from zooplankton, to littoral invertebrates, and finally to active prey of large relative size, fish 42'79'92'93. As discussed earlier, however, beyond some optimal size anaerobic metabolism during activity may become less important, and the animals would again display sustained aerobic foraging behavior with low variance in power output. This does characterize the behavior of some of the largest fish, and also whales, which filter prey from the water at a nearly uniform speed.

Acknowledgments. The studies discussed here were made possible by a grant from the National Science Foundation (BSR-8700153), and support for writing was provided by the National Research Council of the National Academy of Sciences. I would also like to thank Dr. Russ Vetter and Eric Lynn (N.O.A.A., Southwest Fisheries Science Center) for contributing unpublished data on the enzymatic sealing of Dover sole and halibut. IX. References 1. Adolph, E Quantitative relations in the physiological constitutions of mammals. Science 109: 579585, 1949. 2. Altringham, J.D. and I.A. Johnston. Scaling effects on muscle function: power output of isolated fish muscle fibres performing oscillatory work. J. Exp. B/o/. 151: 453--467, 1990. 3. Archer, S.D., J.D. Altringham and I.A. Johnston. Scaling effects on the neuromuscular system, twitch kinetics and morphometrics of the cod Gadus morhua. Mar. Behav. Physiol. 17: 137-146, 1990. 4. Barlow, G.W. Intra- and interspecific differences in rate of oxygen consumption in gobiid fishes of the genus Gillichthys. Biol. Bull. (Woods Hole) 121: 209-229, 1961. 5. Barrels, H. Metabolic rate of mammals equals the 0.75 power of their body weight. F_.xp.BioL Med. 7: 1-11, 1982. 6. Bennett, A.E and P. Licht. Anaerobic metabolism during activity in lizards. J. Comp. Physio/, 81: 277-288, 1972. 7. Bennett, A.E, R.S. Seymour, D.E Bradford and G.J.W. Webb. Mass-dependence of anaerobic metabolism and acid-base disturbance during activity in the salt-water crocodile, Crocodylus porosus.J. F_~. Biol. 118: 161-171, 1985. 8. Blake, R.L., EL. Roberts and R.L. Saunders. Parr-smolt transformation of Atlantic salmon (Salmo salar): activities of two respiratory enzymes and concentrations of mitochondria in the liver. Can. J. Fish. Aquatic Sci. 41: 199-203, 1984. 9. Block, B.A. Endothermy in fish: thermogenesis, ecology and evolution. In: Molecular Biology and Biochemistry of Fish, Vol. 1, edited by P.W. Hochachka and T.P. Mommsen, New York, Elsevier, 1991. 10. Brett, J.R. The relation of size to rate of oxygen consumption and sustained swimming speed of sockeye salmon (Onchorhynchus nerka). J. Fish. Res. Bd. Can. 22: 1491-1501, 1965. 11. Brett, J.R. and N.R. Glass. Metabolic rates and critical swimming speeds of sockeye salmon (Oncorhynchus nerka). J. Fish. Res. Bd. Can. 30: 379-387, 1973. 12. Brill, R.W. and P.G. Bushnell. Metabolic and cardiac scope of high energy demand teleosts, the tunas. Can. J. Zool. 69: 2002-2009, 1991. 13. Brody, S. Bioenergetics and Growth, Baltimore, MD, Reinhold, 1945. 14. Calder, W.A. Size, Function and Life History, Cambridge, MA, Harvard University Press, 1984. 15. Caulton, M.S. The effect of temperature and mass on routine metabolism in Sarotherodon (Tilapia) mossambicus (Peters). J. Fish Bio/, 13: 195-201, 1978. 16. Childress, J.J. and G.N. Somero. Metabolic scaling: a new perspective based on the scaling of glycolytic enzyme activities. Am. ZooL 30: 161-173, 1990. 17. Clark, S., D.H. Whitmore, Jr. and R.E McMahon. Considerations of blood parameters of largemouth bass, Micropterus salmoides. J. Fish BioL 14: 147-158, 1979.

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103. Oikawa, S. and Y. ltazawa. Allometric relationship between tissue respiration and body mass in the carp. Comp. Biochem. Physiol. 77A: 415-418, 1984. 104. Paloheimo, J.E. and L.M. Dickie. Food and growth of fishes. II. Effects of food and temperature on the relation between metabolism and body size. J. Fish. Res. Bd. Can. 23: 869-908, 1966. 105. Parry, G.D. The influence of the cost of growth on ectotherm metabolism. J. Theor. Biol. 101: 453-477, 1983. 106. Paul, A.J. Respiration of juvenile pollack, Theragra chalcogramma (Pallas), relative to body size and temperature..l. Exp. Mar. BioL Ecol. 97: 287-293, 1986. 107. Pauly, D. The relationships between gill surface area and growth performance in fish: a generalization of yon Bertalanffy's theory of growth. MeeresforscK 28: 251-282, 1981. 108. Pauly, D. Further evidence of a limiting effect of gill size on the growth of fish: the case of the Philippine goby, Mistichthys luzonensis. Philipp. J. Biol. 11: 379-383, 1982. 109. Pelster, B. and W.E. Bemis. Ontogeny of heart function in little skate Raja erinacea..l. Exp. Biol. 156: 387-398, 1991. 110. Peters, R.H. The Ecological Implications of Body Size. New York, NY, Cambridge University Press, 1983. 111. Pierce, R.J., "I.E. Wissing and B.A. Megrey. Respiratory metabolism of gizzard shad. Trans. Am. Fish. Soc. 110: 51-55, 1981. 112. Pough, EH. The relationship between body size and blood oxygen affinity in snakes. Physiol. Zool. 50: 77-87, 1977. 113. Pough, EH. The relationship of blood oxygen affinity to body size in lizards. Comp. Biochem. Physiol. 57A: 435-441, 1977. 114. Pough, EH. Ontogenetic change in blood oxygen capacity and maximum activity in garter snakes (Thamnophis sirtalis).J. Comp. Physiol. l16B: 337-345, 1977. 115. Pough, EH. Ontogenetic changes in endurance in water snakes (Natrix sipedon): Physiological correlates and ecological consequences. Copeia 69-75, 1978(1). 116. Poupa, O., L. Lindstrom, A. Mareska and B. Tota. Cardiac growth, myoglobin, proteins and DNA in developing tuna (Thunnus thynnus thynnus L.). Comp. Biochem. Physiol. 70A: 217-222, 1981. 117. Power, J.H. and P.J. Walsh. Metabolic scaling, buoyancy, and growth in larval Atlantic menhaden, Brevoortia tyrannus. Mar. Biol. 112: 17-22, 1992. 118. Powers, D.A. Molecular ecology of teleost fish hemoglobins: strategies for adapting to changing environments.Am. ZooL 20: 139-162, 1980. 119. Pradhan, V. A study of blood of a few Indian fishes. Proc. lndianAcad. Sci. 54: 251-256, 1961. 120. Preston, A. Red blood values in the plaice (Pleuronectes platessa L.) J. Mar. Biol. ASS. U.IL 39: 681-687, 1960. 121. Prothero, J.W. Scaling of blood parameters in mammals. Comp. Biochem. Physiol. 67A: 649-657, 1980. 122. Rao, G.M.M. Oxygen consumption of rainbow trout (Salmo gairdneri) in relation to activity and salinity. Can..l. Zool. 46: 781-786, 1968. 123. Rome, L.C., A.A. Sosnicki and D.O. Goble. Maximum velocity of shortening of three fibre types from horse soleus muscle: implications for scaling with body size. J. Physiol. 431: 173-185, 1990. 124. Ruparelia, S.G., Y. Verma, N.S. Mehta, S.R. Saiyed, P.K. Kulkarni and S.K. Kashyap. A size related haematological study on fresh water teleost, Sarotherodon mossambica (Peters). J. Anita. Morphol. 33: 93-100, 1986. 125. Saunders, R.L. The irrigation of the gills in fishes. II. Efficiency of oxygen uptake in relation to respiratory flow activity and concentrations of oxygen and carbon dioxide. Can. J. Zool. 40: 817-862, 1962. 126. Scheid, P. and J. Piiper. Quantitative functional analysis of branchial gas transfer: theory and application to Scyliorhinus stellaris (Elasmobranchii). In: Respiration of Amphibious Vertebrates, edited by G.M. Hughes, New York, Academic Press, pp. 17-38, 1976. 127. Schmidt-Nielsen, K. Scaling, Why is Animal Size So Important? New York, Cambridge University Press, 1984. 128. Seeherman, H.J., Taylor, C.R., Maloiy, G.M.O. and Armstrong, R.B. Design of the mammalian respiratory system. II. Measuring maximum aerobic capacity. Resp. Physiol. 44: 11-23, 1981. 129. SiebenaUer, J.E, Somero, G.N. and Haedrich, R.I.. Biochemical characteristics of macrourid fishes differing in their depths of distribution. Biol. Bull. (Woods Hole) 163: 240-249, 1982. 130. Smith, J.C. Body weight and the haematology of the American plaice Hippoglossoides platessoides. J. Exp. Biol. 67: 17-28, 1977.

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131. Smith, J.C. and C.K. Chong. Body weight, activities of cytochrome oxidase and electron transport system in the liver of the American plaice Hippoglossoides platessoides. Can these activities serve as indicators of metabolism7 Mar. EcoL Prog. Ser. 9: 171-179, 1982. 132. Soivio, A., K. Nyholm and M. Huhti. Effects of anaesthesia with MS 222, neutralized MS 222 and benzocaine on the blood constituents of rainbow trout, Salmo gairdneri. J. Fish Biol. I0: 91-101, 1977. 133. Somero, G.N. and Childress, J.J. A violation of the metabolism-size scaling paradigm: Activities of glycolytic enzymes increase in larger-size fish. Physiol. ZooL 53: 322-337, 1980. 134. Somero, G.N. and J.J. Childress. Scaling of ATP-supplying enzymes, myofibrillar proteins and buffering capacity in fish muscle: relationship to locomotor habit. J. F.~. Biol. 149: 319-333, 1990. 135. Soofiani, N.M. and Priede, I.G. Aerobic metabolic scope and swimming performance in juvenile cod, Gadus morhua L. J. Fish Biol. 26: 127-138, 1985. 136. Stahl, W.R. Scaling of respiratory variables in mammals. J. Appl. Physiol. 22: 453-460, 1967. 137. Stevens, E.D. and Black, E.C. The effect of intermittent exercise on carbohydrate metabolism in rainbow trout, Salmo gairdneri. J. Fish. Res. Bd. Can. 23: 471-485, 1966. 138. Stirling, H.P. Effects of experimental feeding and starvation on the proximate composition of the European bass Dicentrarchus labrax. Mar. Biol. 34: 85-91, 1976. 139. Tandler, A. and Beamish, EW.H. Apparent specific dynamic action (SDA), fish weight and level of caloric intake in largemouth bass, Micropterus salmoides Lacepede. Aquaculture 23:231-242, 1981. 140. Tarby, M.J. Metabolic expenditure of walleye (Stizostedion vitreum vitreurn) as determined by rate of oxygen consumption. Can. J. Zool. 59: 882-889, 1981. 141. Taylor, C.R. and E.R. Weibel. Design of the mammalian respiratory system. I. Problem and strategy. Resp. Physiol. 44: 1-10, 1981. 142. Taylor, C.R., M.O. Maloiy, E.R. Weibel, V.A. Langman, J.M.Z. Kamau, H.J. Seeherman and N.C. Heglund. Design of the mammalian respiratory system. III. Scaling maximum aerobic capacity to body mass: wild and domestic mammals. Resp. Physiol. 44: 25-37, 1980. 143. Tenny, S.M. and J.B. Tenny. Quantitative morphology of cold-blooded lungs: Amphibia and Reptilia. Resp. Physiol. 9: 197-215, 1970. 144. Tortes, J.J. and G.N. Somero. Metabolism, enzymic activities and cold adaptation in Antarctic mesopelagic fishes. Mar. Biol. 98: 169-180, 1988. 145. Turner, J.D., Wood, C.M. and Clark, D. Lactate and proton dynamics in the rainbow trout (Salmo gairdneri). J. Exp. Biol. 104: 247-268, 1983. 146. Vernberg, EJ. The respiratory metabolism of tissues of marine teleosts in relation to activity and body size. Biol. Bull. (Woods Hole) 106: 360-370, 1954. 147. Vetter, R., E.A. Lynn, M. Garza and A.S. Costa. Depth zonation and metabolic adaptation in Dover sole, Microstomus pacij'icus, and other deep-living flatfishes: Factors that affect the sole. Mar. Biol. 120: 145-159. 148. Vetter, R. N.O.A.A., Southwest Fisheries Science Center; unpublished data. 149. Walsh, P.J., C. Bedolla and T.P. Mommsen. Scaling of sex-related differences in toadfish (Opsanus beta) sonic muscle enzyme activites. Bull. Mar. Sci. 45: 68-75, 1989. 150. Wardle, C.S. Limits of fish swimming speed. Nature 255: 725-727, 1975. 151. Webb, P.W. Hydrodynamics and energetics of fish propulsion. Bull Fish. Res. Bd. Can. 190: 1-158, 1975. 152. Webb, P.W. Effects of size on performance and energetics of fish. In: Scale Effects in Animal Locomotion, edited by T.J. Pedley, New York, Academic Press, pp. 315-331, 1977. 153. Webb, P.W. and C.L. Johnsrude. The effect of size on the mechanical properties of the myotomalskeletal system of rainbow trout (Salmo gairdneri). Fish Physiol. Biochem. 5: 163-171, 1988. 154. Weibel, E.R., C.R. Taylor and H. Hoppeler. The concept of symmorphosis: a testable hypothesis of structure-function relationship. Proc. Natl. Acad. Sci. U.S.A. 88: 10357-10361, 1991. 155. Werner, E.E. and J.E Gilliam. The ontogenetic niche and species interactions in size-structured populations. Annu. Rev. Ecol. System. 15: 133-142, 1984. 156. Wieser, W. Developmental and metabolic constraints of the scope for activity in young rainbow trout (Salmo gairdneri).J. Exp. Biol. 118: 133-142, 1985. 157. Wieser, W. and H. Forstner. Effects of temperature and size on the routine rate of oxygen consumption and on the relative scope for activity in larval cyprinids. J. Comp. Physiol. B 156: 791-796, 1986. 158. Wilkins, N.E Multiple haemoglobins of the Atlantic salmon (Salmo salar). J. Fish. Res. Bd. Can. 25:2651-2653, 1968.

Hochachka and Mommsen (eds.), Biochemistryand molecularbiologyoffishes, vol. 4 9 1995 Elsevier Science B.V. All rights reserved. CHAPTER 16

Exercise metabolism of fish CHRISTOPHER D. MOYES AND TIMOTHY G . WEST *

Department of Biology, Queen's University, Kingston, Ontario, K7L 3N6 Canada and *Department of Zoology, Cambridge University, Downing Street, Cambridge CB2 3EJ, U.K.

I.

Introduction 1. The typical fish! 2. Fuel selection II. Steady-state swimming 1. Exercise metabolism 2. Protein and amino acid utilization 3. Carbohydrate utilization 3.1. Muscle glycogen 3.2. Circulatory glucose 3.3. Lactate 4. Utilization of fat fuels 5. Summary of fuel oxidation III. Burst exercise 1. Exercise metabolism 2. Carbohydrate metabolism 3. Phosphagens 4. Exhaustion: the influence of basal glycogen 5. Recovery metabolism 6. Carbohydrate metabolism in recovery from burst exercise 6.1. Fate of lactate 6.2. Route of glycogen synthesis 7. Fuel for recovery metabolism 8. Energy metabolism 8.1. Phosphocreatine 8.2. Adenylates 9. Why is recovery so slow? 10. Summary of burst exercise and recovery IV. References

I. Introduction The arrangement of fish muscle into separate, homogeneous fiber types has long been recognized as advantageous for studying exercise metabolism (see refs. 9, 24, 46). Red muscle is recruited at steady-state swim speeds whereas white muscle is used in short-term burst exercise. As muscle is a high proportion of whole body mass, its recruitment imposes extraordinary demands on whole body metabolism. This fact alone allows confident estimates of the potential contribution of specific fuels, depots and pathways in exercise. Our first goal is to integrate empirical studies with

368

C.D. Moyes and T.G. West

a tissue-mass based model of fish exercise metabolism which sets theoretical limits on which fuels and pathways can be physiologically relevant in exercise. When comparing the many studies of fish exercise metabolism, markedly different responses to exercise are observed under conditions which would, at least superficially, appear to be very similar. These variations are usually attributed to unquantifiable differences in species, stocks, acclimation conditions or exercise regimes. Our second goal is to extract information from these variable responses to exercise in order to generate a model which takes into account inter-individual and inter-species differences in metabolic status. Finally, this review should clearly point to critical deficiencies in the current understanding of exercise and recovery metabolism.

1. The typical fish! Fish demonstrate body forms and locomotory patterns which defy simple generalizations. We consider a salmoniform to be the 'typical fish' solely as a framework for discussion about relationships between anatomy and metabolic performance. Most studies of fish exercise metabolism deal with salmonids, because of their wide availability to researchers and commercial importance in locations where research is supported 23,49,55,59,60,63,64,75,78,81-84,93,94,101,114. Physiologically, they perform better than most species in both steady state exercise and burst exercise. When fish other than salmonids are used, differences from the salmonid response can be highly informative about the fundamental mechanisms which underlie metabolic control of exercise. Across species, a 20-fold range in aerobic capacity of skeletal muscle is observed~6. The differences in rate of recovery from burst exercise spans two orders of magnitude in those few species which have been examined74. Also, the fundamental differences in the fuels utilized by muscle in osteichthians and chondrichthians 73 have not been explored in relation to exercise.

2. Fuel selection Fuel preference studies are an attempt to quantify the contribution of the various carbon substrates to changes in tissue energy demand. There have been few direct in vivo studies of fuel selection by fish muscle. However, we can gain initial insight into the possible metabolic partitioning of different substrates during sustained exercise, burst exercise and exercise recovery from whole-body sites and levels of the main storage substrates. Representative salmonid tissue masses and metabolite levels, per g organ mass and per kg body mass, are summarized in Table I. These metabolite levels represent the maximum mobilizable fuels available to a nonfeeding salmonid. Our first attempt to assess the relative importance of metabolic fuels and storage depots in the normal post-absorptive animal involves a comparison of the demands imposed by the exercise (Table 2) to the quantities of the putative fuels available (Table 1). Where physiologically significant quantities of fuels exist, rates of substrate supply, competition between pathways and the influence of regulatory factors will determine the pattern of fuel utilization. Unfortunately, few studies address these situations.

Exercise metabolism offish

369 TABLE 1

Tissue masses and glycogen stores of a resting salmonid

g tissue/kg body mass

ECF

Liver

250

15

Adipose tissue

Glycogen (as glucosyl) (/zmol/g tissue) (~mol/kg body mass)

0 0

Glucose (/zmol/g tissue) (/zmol/kg body mass)

2 -5 0.5 -2.0

2 0.03-

Lactate (~mol/g tissue) (/zmol/kg body mass)

7 1.75

3 0.045

Triglyceride (/~mol/g tissue) 0tmol/kg body mass) Fatty acids (/~mol/g tissue) (/zmol/kg body mass)

22 5.5 3 0.75

100 -200 1.5 - 3.0

10

-

0.153 0.045

5 0.075

30 0.45

White muscle

20

600

-

40 24

-

800 16

Red muscle 70 40 2.8

2 1.2

2 0.14

3 1.8

5 0.35

15 9 2 1.2

Protein (as amino acyl) (/zmol/g tissue) (/zmol/kg body mass)

2000 500

1000 15

1000 600

Amino acids (~mol/g tissue) (~mol/kg body mass)

40 10

-

50 30

40 3 2 0.14 500 35 9 0.63

ECF = extracellular fluid. Data were compiled from various sources (references 30, 42, 54, 60, 66, 81, 86, 96, 108, and 117). These values are estimates for total fuels and are not necessarily available metabolically.

These analyses focus on maximal capacities for flux which are different both qualitatively and quantitatively for red and white muscle recruitment. As exercise becomes less intense or prolonged, the less abundant fuels and depots may become increasingly important. Where no direct studies have been done, indirect studies using enzyme analysis and isolated mitochondria can give insights into the capacity and preference for fuels in exercise and recovery.

II. Steady-state swimming 1. Exercise metabolism The differences between red and white muscle morphology, orientation, ultrastructure and innervation that allow recruitment at different swim speeds have been reviewed elsewhere,94690 , . Fish are able to swim in a continuous csteady-state , fashion over a broad range of speeds. A e r o b i c red muscle is recruited almost exclusively

C.D. Moyes and T.G. West

370 TABLE 2

A comparison of metabolite changes expected to occur if skeletal muscle is recruited at maximal work levels in fish red and white muscle . . . .

Red muscle .

.

.

.

.

.... .

.

.

Wllite muscle .

.

.

.

......

.

icon, o tput Maximal work* (roW/g) ATP utilization b (/~mol/min/g)

5 18

- 8 -29

25 - 35 90 -126

Aerobically generated A TP c Oxygen (O2) consumed: (/~mol/min/g muscle mass) (/~mol/min/kg body mass) Glucose utilized: (ttmol/min/g muscle mass) (ttmole/min/kg body mass)

3.0 - 4.8 0.21 - 0.34 0.50-0.81 0.035- 0.057

15 - 21 9 - 12.6 4.22.5-

5.8 3.5

Anaerobically generated A TP d Lactate produced: (~tmol/min/g muscle mass) (/~mol/min/kg body mass)

12.0 -19.4 0 . 8 4 - 1.36

60 - 84 36.0- 50.4

9 Determined in vitro for bullrout (Myoxocephalus scorpius L.). b Assuming -500 kJ/mole 02, 20% mechanical efficiency and 6 ATPIO2. c Assumes red muscle is 7% body mass and white muscle 60% body mass. A yield of 36 tool ATP/mol lucose is assumed. 1.5 ATP/lactate formed implies endogenous glycogen is utilized. If exogenous glucose were the fuel, the values would be 50% greater (1 ATP/lactate formed).

~

in fish swimming up to 80% of fatigue or critical speed (Uc~,), beyond which the glyeolytie white rnusele begins to be recruited as peak levels of whole-body aerobic demand are approached 46's7'9~176 Studies of swimming physiology often have the fish perform at speeds close to 80% of Ucr,, both in recognition of the muscle recruitment pattern and because of the utility of Ucrit a s a standard measure of aerobic performance. We can evaluate the potential role of circulatory fuels during swimming using estimates of red muscle oxidative capacity and circulatory turnover of metabolites. In the absence of such measurements, we can analyze potential rates of depletion of circulatory and intramuscular substrate stores in support of high intensity red muscle recruitment using the values in Tables 1 and 2.

2. Protein and amino acid utilization

In fish, dietary protein levels of 30-50% (w/w) seem optimal for growth2~ With protein providing a potentially considerable portion of the total caloric intake in omnivores, it is not surprising that daffy routine metabolism may be covered in large part by amino acid catabolism. On average, it is estimated that amino acid oxidation accounts for 10-20% of maintenance energy costs~.s3. This value can possibly double after long-term starvation and may be elevated considerably, on a shorter-term basis, immediately after feeding53. With starvation, increased degradation of muscle-protein is most evident after body reserves of fat have been depleted 8. Similarly, amino acid utilization seems important in migratory salmonids

Exercise metabolism offish

371

late in the run when other fuel stores have been nearly exhausted 66. Taken together, these observations suggest that in normal (fed, non-migratory) fish the availability of amino acids for routine oxidative metabolism is influenced by post-feeding absorptive mechanisms moreso than by the actual turnover of body-protein. The effects of activity metabolism on protein or amino acid dynamics in fish have not been examined extensively. Continuous low level swimming increases protein turnover and degradation in feeding rainbow trout (Oncorhynchus mykiss) 41, but the proportion of amino acids utilized for swimming energetics has not been determined directly. In sockeye salmon (Oncorhynchus nerka) starved for 22 days, an indirect assessment of whole-body amino acid utilization (ammonia excretion) indicated that, despite a probable increase in protein degradation, daily increases in routine metabolism were seemingly not supported by amino acids as long as reserves of fat and carbohydrate were available 11,53. Ammonia excretion largely reflects amino acid transdeamination in the liver 194 and will therefore over-estimate rates of amino acid oxidation by the extent that the resulting carbon skeletons are used in gluconeogenesis, which increases during activity and starvation (see ref. 99). In any case, it seems likely that the higher aerobic demands of muscle resulting from a rest to exercise transition, in both normal and food-deprived fish, would rely even more on non-protein related energy reserves since the tissue pools of free amino acids are small (Table 1) and the oxygen cost of ATP production from fat and carbohydrate oxidation is about 30% lower than that of amino acids 2a. In support of this, it has been noted that oxidation of neutral amino acids (serine, glycine, alanine) does not occur at detectable rates in mitochondria isolated from carp (Cyprinus carpio) red and white muscle 72. In addition, one study of in vivo substrate oxidation rates indicated that glutamate, alanine and leucine, the latter classed as a branchchained amino acid which is possibly oxidized at elevated rates in exercising skeletal muscle 51,52, seemed to contribute very little to whole-body oxidative metabolism of rainbow trout (O. mykiss) swimming at 80% of U~rit (ref. 102). Exercising fish may be able to draw on dietary amino acids after feeding as oxidative substrates for swimming. However, confirmation of such use of amino acids awaits more detailed examination of post-feeding fluxes and oxidation rates. In most studies of swimming metabolism the researcher avoids post-feeding energetics by design in order to minimize inter-individual variability in basal oxygen consumption. Direct circulatory infusions of amino acid mixtures (see ref. 100) could be a more useful experimental approach for looking at the effects of amino acid availability on fuel selection in fish. In this way it may be possible to mimic post-feeding levels of plasma amino acids while at the same time eliminate potential influences of gut absorptive rate on whole-body metabolism.

3. Carbohydrate utilization There are potentially three ways to supply carbohydrate to exercising fish red muscle (Fig. 1): (1) mobilization of intramuscular glycogen reserves; (2) uptake of circulatory glucose derived from hepatic glueoneogenesis/glycogenolysis; and (3) oxidation of lactate produced from white muscle glycogen.

372

CD. Moyes and T.G. West

PLASMA

WHITEMUSCLE i azJ,ooGi ', v

G-6-P < |

I

LIVER

" Glucose<

=

~ruvate

,-~ Laetm

I

,

J'aoa A, i

> Lactate

10Pmi~/min

I I I I

GlA se

Glucose<

10umol/m

..

iI iI

G-6-P<

l

8 i

pyn!,,,vate<

~ II

Amh;o (;~c.ro,

Glucosc< 35-56l~nd/min

Acids

Lact=e<

, ~ Lactate

70-112~ n

i tI

!

RED MUSCLE

i !

Fig. 1. Major routes for circulatory carbohydrate delivery (bold arrows) to red muscle in vivo. Glucose is released from the liver, while at high work rates (>80% of Ucrit) circulatory lactate is derived from white muscle glycogen. In a 1 kg fish (70 g of red muscle/kg), maximal rates of circulatory glucose or lactate delivery (from turnover measurements z6,1~ cannot match substrate demand of 56 and 112/zmol/min/kg, respectively (calculated from O2 consumption, Table 2). Combined use of all carbohydrate stores is also of little relevance since in vivo estimates of lactate and glucose contributions to total oxidation are minimal l~

3.1. Muscle glycogen Intramuscular glycogen is not expected to be the primary fuel for steady state exercise since total red muscle glycogen is a fairly small depot (Table 1). if used exclusively, red muscle glycogen would support sustained maximal aerobic swimming for less than 1 h (see Tables 1 and 2). It is likely however, that many fish species deviate somewhat from our 'typical-fish' scenario. In carp (Carassius caras. sius), red muscle glycogen content appears to be 2-8 fold higher than that of white

Exercise metabolism offish

373

muscle 44,47,5~ providing some evidence for a possibly greater reliance on intramuscular carbohydrate for swimming metabolism than is expected in salmonids. Increased mobilization of endogenous red muscle glycogen has been shown in carp during graded levels of swimming intensity48. Glycogen appears to be a minor oxidative fuel for long-term aerobic exercise, although it may augment fuel supply, particularly at higher intensities and in the early phases of a rest to work transition 97.

3.2. Circulatory glucose The capacity for red muscle to utilize glucose is indicated indirectly from activities of hexokinase that generally range from 0.5 to 1.5 ~mol glucose/g tissue/min or total activity of 35-100/zmol/min in a 1 kg fish21,45. From the maximum oxidative demand of the total red muscle mass (Table 1) it can be calculated that similar rates of glucose oxidation are required for maximal red muscle activity (35-60/zmol/ min/kg body mass). As an estimate of fuel use, hexokinase measurements suggest that glucose flux alone could account for the maximal rate of oxygen consumption of fish red muscle. Crabtree and Newsholme 21 have similarly estimated that the glucose utilizing capacity of trout (O. mykiss) red muscle could meet the aerobic fuel demands. Can the delivery of circulatory glucose keep up with the fuel demand during exercise? Glucose release from the liver of fish results from net gluconeogenic flux and glycogenolysis. Both processes are regulated by the action of various hormones and functional peptides 67,68,85,88,99,117, but it is not known if one pathway dominates hepatic glucose production in vivo. The responsiveness of glycogen stores to catecholamines 6,88,117 coupled with the observation that plasma levels of these hormones do not change during submaximal exercise 15,89, suggests that glucose derived from hepatic glycogen may not be different from resting conditions. In any case, liver glycogen levels alone (Table 1) could not support extended periods of maximal aerobic swimming. Regardless of the pathway of hepatic glucose production, the rate of production and release to plasma in vivo is probably within the range of 1-10/zmol/min/kg, estimating from plasma turnover in teleosts under different experimental conditions (reviewed in ref. 31). Unfortunately, reliable determinations of salmonid glucose utilization during exercise are not available. In Fig. 1, we have used the upper end of the range of available data (measured in hypoxic trout 26) as a possible maximum turnover rate for exercising fish to illustrate the likely imbalance between plasma glucose supply and red muscle demand. It is of course expected that glucose in transit though the plasma compartment is distributed to more tissues than just red muscle. However, in using turnover as an initial maximal estimate of red muscle uptake, it is apparent that glucose delivery would have to greatly exceed 10/xmol/min/kg to match the oxidative demand for substrate during high intensity aerobic exercise. Glucose is oxidized at low whole-body rates in swimming trout 1~ but rates of 2-deoxyglucose uptake in vivo verify that glucose utilization is a minimal part of total substrate oxidation in specifically the working red muscle 112. Red muscle glucose utilization at 80% of Ucrit averaged 21 nmol/min/g tissue mass, a rate which accounted for <10% of expected red muscle oxygen consumption.

374

C.D. Moyes and

T.G. West

The capacity of red muscle to use glucose (estimated from hexokinase activity) may mean that red muscle uptake can be expanded beyond the low level observed in exercising trout, perhaps in periods of hypoxia or low lipid availability. However, dependence of red muscle on plasma glucose during maximal aerobic exercise would seem to be always limited by plasma availability since turnover apparently cannot match aerobic demand for substrate. It would be of interest to examine turnover and red muscle utilization of glucose simultaneously during exercise. Since plasma glucose is poorly regulated in fish 31'38'65's~ in vivo uptake of glucose in fish tissues might be expected to correlate with parameters that affect glucose availability (e.g. plasma concentration, turnover or changes in regional blood flow). Increased glucose utilization in trout red muscle during exercise 112 is perhaps related simply to blood flow redistribution to mainly the red fiber masssT. Direct comparisons of turnover and tissue utilization during exercise would further identify major sites of glucose disposal during exercise and could confirm that glucose utilization in red muscle is low, perhaps inhibited, in vivo so that it may be spared for use in other tissues, as suggested from total red muscle uptake in a 1 kg trout (about 1.5/zmol/min/70 g red muscle 112) compared to our best estimate of turnover (10/zmol/min/kg body mass). More investigation of plasma glucose turnover and uptake during exercise, starvation and hypoxia may reveal tissues and conditions in which glucose is quantitatively more important in vivo.

3.3. Lactate Another route for the delivery of exogenous carbohydrate fuel to working oxidative muscle may be via the steady-state release of lactate from white glycolytie muscle 1~ In mammals, the fate of such lactate is predominantly oxidation resulting presumably from a shuttling of lactate from glycolytie to oxidative tissues v/a the circulation 12. It is intriguing to consider that white muscle glycogen stores in fish could supply lactate to red muscle in the same manner. The total white muscle mass represents a substantial glycogen store, about 20-fold greater than liver (Table 1), that could support hours of intense steady-state swimming (again, assuming exclusive delivery of the lactate released to red muscle and a red muscle demand of 70-112 ttmol/min/kg body mass; Fig. 1). If such a pathway operated, lactate must be produced by white muscle at adequate rates, which would be reflected in plasma lactate turnover, assuming a conventional circulatory transfer. Several species are known to recruit white muscle at high steady state swimming speeds but there is no evidence that the white muscle metabolism under these conditions is primarily aerobic. If for example the aerobic demands of high intensity red muscle activity (29/~mol ATP/min/g, Table 2) were imposed on the recruited white muscle its aerobic metabolism could provide <15% of the ATP (e.g. rainbow trout white muscle mitochondria can produce 3.5/zmol ATP/min/g75). (In some species, such as skipjack tuna (Katsuwonus pelamis), the mitoehondrial content and presumably aerobic capacity of white muscle is exceptionally high, 2-fold and 5-fold that of trout 75 and carp (Cyprinus carpio) 76, respectively, even without the higher body temperatures considered.) The possibility that lactate produced by white muscle could serve as fuel for working red muscle might be evident even

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in the initial stages of white muscle recruitment. Hence, studies in which lactate dynamics have been examined in swimming rainbow trout 1~176 have imposed a high level of sustained exercise (85% of Ucrit) with the expectation that any white muscle recruitment would be fueled primarily by anaerobic glycolysis. The problem with relying on circulatory lactate as a red muscle fuel seems to be the same as that stated for exogenous glucose. As reiterated in Fig. 1, turnover simply cannot match the oxidative demand of red muscle. Lactate turnover would have to increase by about 10-fold to cover maximal red muscle metabolic rates calculated for a 1 kg fish (Table 2). Empirical studies indicate that, on average, the lactate turnover rate during sustained exercise at 80% Ucrit doubles over resting fish (to about 10/zmol/min/kg body mass), similar to that in a salmonid recovering from burst exercise63, but this change does not occur consistently in individual fish 1~ Thus, lactate remains of minor importance as an oxidative fuel during an endurance swim 1~ although it may be more important at higher levels of Ucr,. Under these conditions, however, the expected increases in both white muscle recruitment and whole-body oxygen consumption make predictions difficult. Although elevated rates of lactate oxidation are predicted in swimming trout 1~ this does not necessarily reflect red muscle metabolic events. These turnover experiments merely point out the fate of the lactate which appears in, and disappears from, the plasma compartment and are not inconsistent with the limited whole-body dependence on lactate as a fuel. As in mammals, oxidation may in fact be its major fate in vivo since hepatic gluconeogenesis from circulatory lactate is minimal in most instances and in a variety of species63,1~176 Although other fuels seem more important for overall metabolism, tissues that contribute relatively little to the absolute change in metabolic rate with the onset of exercise could still utilize lactate as an oxidizable fuel. Trout myocardium for example, increases its oxygen consumption several-fold at high work intensities, but could rely heavily on circulatory lactate turnover because of its small relative mass and fractional contribution to whole-body oxygen consumption during swimming 58,112. This possibility requires further investigation.

4. Utilization of fat fuels Currently, the major gap in our understanding of fuel utilization during steady-state exercise concerns the relative importance of lipid fuels (Fig. 2). In total, the body stores of triacylglycerol (TAG) represent the largest energy reserve in fish which could, if delivered exclusively to the red muscle, supply enough free fatty acid (FFA) to fuel about 100 h of maximal aerobic red muscle contraction in a 1 kg animal (assumes about 23/,mol O2 utilized per/~mol fatty acid oxidized). However, there are few empirical measurements that help to identify which of major depots are mobilized in vivo during exercise. Such studies are complicated by the fact that TAG is deposited in, and mobilized from, muscle, liver and the viscera96. Quantitative approaches are needed to increase our awareness of the importance of circulating FFA, extramuscular depot fats and of the dynamics of skeletal muscle intracellular and pericellular TAG stores. Presently, any conclusions regarding the

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support 4--5days of continuous maximalred muscleactivity(see text). relative importance of the various lipid stores, and lipid fuels in general, are limited to studies which focus on either changes in lipid content (e.g. observations of whole-body and organ fat depletion during starvation/migration,842 - 44) or on in vitro enzyme measurements and mitochondrial capacities for fatty acid utilization. Such studies support a major role for lipids in steady-state exercise in fish. Although hepatic and skeletal muscle TAG contents are similar (per g tissue), the smaller liver can be viewed as a minor storage organ with respect to steady-state exercise, regardless of the kinetics of hepatic mobilization. On a quantitative basis,

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visceral fat deposits are the richest store of lipid per g tissue, but skeletal muscle, by virtue of its mass possesses similar TAG stores per kg body mass (Table 2). Each of these stores might be drawn upon during aerobic exercise. The degree to which the white muscle and visceral fat may supply FFA to red muscle should be reflected in estimates of circulatory delivery. However, one study of in vivo palmitate oxidation suggests that circulatory fatty acids contribute minimally to the total oxidative fuel required by trout swimming at 85% of tl 1~ Possible delays in the "-"crit" equilibration of labeled circulatory precursor (14C-palmitate) with the intracellular TAG pool at the site of utilization may result in underestimates of circulatory FFA oxidation 36, but it is not known if this is a problem in fish at different levels of activity. Among endothermic species, the likelihood that intramuscular lipid supports aerobic energy demands, at least to some extent, is supported by the observations of increased stores in aerobically trained muscle 4~ and by the proximity and presumed functional association between mitochondria and intracellular lipid droplets in oxidative fibers 33,4~ The possibility that intramuscular TAG serves simultaneously as a sink for circulatory fuels and a source of mitochondrial substrate needs to be evaluated more closely in fish. As with most vertebrate oxidative muscles, red muscle from teleost demonstrates a high capacity to utilize FFA, as indicated by mitochondrial oxidation rates 72'76'77. While this in itself reveals little about the relative importance of fatty acids, comparisons of mitochondrial oxidation rates and carnitine palmitoyl transferase (CPT) activities between species and tissues are more illuminating. As in aerobic training of mammalian muscles, where an increase in the capacity to utilize fatty acids is apparent, a comparison of fish species and muscle types reveals that CPT activity increases with aerobic capacity of the tissue 76. This change is due to both an increase in quantity of mitochondria and CPT activity per unit mitoehondria. Thus, fatty acids may become relatively more important in tissues (white muscle versus red) and species (carp versus tuna) with greater aerobic capacities 76. This agrees fundamentally with conclusions from various comparative studies which have examined the fuel demands in endotherms and concluded that adaptation for increased aerobic capacity is related to an increased dependence on fat fuels 25,11~ One point of interest is that chondrichthian red muscle, unlike that of teleosts, lacks demonstrable capacity to utilize FFA as indicated by low activities of CPT and an inability of isolated mitochondria to oxidize FFA in a variety of forms (short, medium and long chain fatty acids and fatty acyl carnitinesT3). The fuel used in place of fatty acids in these species has not been established, but is thought to be primarily ketone bodies. In mammals, ketone body utilization by skeletal muscle increases with circulating concentrations which rise primarily during starvation (e.g. ref. 91). Teleost skeletal muscle apparently has no capacity to oxidize fl-hydroxybutryate and minimal capacity to utilize acetoacetate 72 ,76,77 . Conversely, chondrichthian red muscle lacks the capacity for FFA oxidation, but does possess an impressive capacity to oxidize ketone bodies. If ketones functionally replace FFA in muscle metabolism, many, as yet undemonstrated, differences in whole-body lipid metabolism are expected in these species 77.

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The expectation that changes in red muscle aerobic demand are dominated by FFA oxidation in fish may mean that FFA inhibits carbohydrate utilization, as seen in heart preparations 92 and in vivo in mammals 29. In this ease FFA availability may spare glucose for use in other tissues. This is supported only indirectly by the possibility that red muscle glucose uptake in a 1 kg trout may be a small proportion of the expected total glucose turnover during exercise (discussed earlier). Ketone body use in ehondriehthians has been suggested to have a similar sparing effect on glucose 69. Investigations which emphasize FFA kinetics in concert with effects on glucose uptake and oxidation (and reciprocal studies of glucose effects on lipolysis and FFA oxidation) in exercise and starvation, or FFA limitation, will help describe the nature of fuel interactions in fishes.

5. Summary of fuel oxidation The utilization of fat fuels in fish during sustained exercise is implied indirectly from enzyme measurements, mitoehondrial flux capacities and an indication that the expansion of aerobic capacity across tissue-types and species seems associated with a greater capacity for mitoehondrial transport of long-chain fatty acids. Fat stores are the richest energy reserve in the whole-animal, but direct quantitative determinations of in vivo lipolysis, fat oxidation and effects of fat availability on fuel use at high intensity steady-state exercise are needed to verify preference for fat in the whole-animal. Low in vivo rates of circulatory glucose and lactate utilization are consistent with potential reliance on fat in oxidative muscle, although a potentially important role for lactate in smaller oxidative tissue masses (e.g. heart) cannot be dismissed. Muscular oxidation of amino acids may be important after feeding, but amino acid availability for continuous exercise seems otherwise limited, except late in migration. Chondriehthians are one group that deviate from the typical fish model presented in that they are possibly reliant on ketone bodies for activity metabolism moreso than fatty acids.

IlL Burst exercise 1. Exercise metabolism White muscle is recruited when the power output needed to achieve relatively high swim speeds exceeds the capacity of red muscle. In the natural world, burst exercise is important iv escape from predators, capture of prey and migration against swift water currents. Experimentally, burst exercise is achieved in a swim tunnel or by manually chasing the fish. Neither of these techniques can be expected to realistically duplicate fully the physiological events occurring in nature 11~. Swimming to exhaustion is typically the endpoint of a burst exercise regime. The need to reach a standard state to reduce experimental variation associated with interindividual differences in motivation is usually thought more important than the fact that this type of exercise would rarely be expected to occur in nature. Although most studies

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report to swim fish to near-exhaustion, comparison of parameters such as tissue lactate suggests that even this end-point may be somewhat subjective. 2. Carbohydrate metabolism Simply stated, the high power outputs of white muscle exceed the capacity of mitochondrial metabolism to meet the demand. However, it should not be interpreted that burst exercise is fuelled anaerobically because white muscle has too few mitochondria (<2% volume density, ref. 46). The metabolic cost of achieving the maximal power outputs of white muscle determined in vitro, 126 ~mol ATP/min/g (Table 2), is well above white muscle aerobic capacity (3.5 /zmol ATP/min/g for trout, ref. 75). As the capacity for a very aerobic muscle such as skipjack tuna heart (42/zmol ATP/min/g, ref. 76) is also well below this ATP demand, it can be argued that even if a comparable mitochondrial volume density (30%) could be achieved in white muscle, it would still be inadequate to meet the demands of burst exercise. In fact, a large proportion of white muscle intracellular space is necessarily devoted to myofibrils precluding the packing of mitochondria to the volume densities observed in more aerobic muscles39,111. Since there is not enough white muscle mitochondrial capacity to meet the demands of burst exercise, questions concerning the adequacy of oxygen delivery are largely irrelevant. Most of the ATP used in burst exercise is obtained from muscle glycogen and glycolysis. It has been suggested that exogenous glucose may be an important fuel during burst exercise 84. This is highly unlikely except, perhaps, at the very lowest intensities for reasons related to the high rate of demand and the large mass recruited. Extramuscular sources of glucose and glycogen are minor compared to the white muscle stores of approximately 24 /~mol glucosyl units/kg body mass (Table 1). The plasma glucose pool size is small, (Table 1) and circulating levels change little in exercise or recovery59. Liver typically has significant glycogen content/g tissue but as total hepatic glycogen represents less than 10% of the intra-muscular depot (Table 1) and it does not change much with exercise59,62, it is unlikely to be an important source of glucose for burst exercise. The minimal role of circulatory glucose in burst exercise has been directly demonstrated in vivo using the glucose analogue 2-deoxyglucose. At high swim speeds glucose uptake rates of 0.3-0.4 nmol/min/g occur 112 compared to lactate production rates of approximately 2 ~mol/min/g 74. It should be noted that this lactate production rate is averaged over an extended exercise regime, consisting of alternating periods of burst and gliding. if the in vitro predictions of ATP demand in burst exercise are valid, instantaneous glucose uptake rates of 63/zmol/min/g would be required (Table 2). 3. Phosphagens An integrated picture of the regulatory control of burst exercise is far from complete. As the rate of utilization of ATP increases, pathways for synthesis of ATP must be activated. Catabolism of immediately available ATP leads to an increase in ADE This shifts the mass action ratio of the creatine phosphokinase (CPK) reaction toward

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ATP production, with concomitant decrease in phosphoereatine and a net release of inorganic phosphate (Pi). When the ATP-buffering role of phosphoereatine is exceeded, breakdown of ATP to IMP (inosine 5'-monophosphate) by AMP deaminase occurs. The net result is a decrease in ATP with a increase in IMP (1 mol/mol ATP), ammonia (1 tool/tool) and Pi (1 tool/tool) 24,64. It has not been shown that Pi released from phosphoereatine breakdown and ATP catabolism is retained within the white muscle. Considering the importance of Pi in metabolic regulation, improved resolution of the changes in Pi occurring in recovery would be most useful. While the changes in adenylates and Pi are important signals for stimulation of glycogenolysis and glyeolysis, they would also be expected to stimulate white muscle respiration during exercise. As mentioned previously, it is unlikely that the ATP produced by mitochondria would form a significant proportion of the total ATP utilized. However, the stimulation of mitochondria during exercise may cause longer lasting effects and have implications for recovery, when mitoehondrial ATP production is critical. It is known, for instance that exercise induces dephosphorylation (activation) of pyruvate dehydrogenase75. If this activation persists, it could have important consequences to regulation of fuel selection in recovery.

4. Exhaustion: the influence of basal glycogen Experimentally, a burst exercise regime usually continues until the fish is exhausted. At this point, the fish is still capable of performing slow, steady state exercise but is unable to burst. Although most studies involve this type of protocol, dramatic differences in the metabolite status at exhaustion are obvious. Several studies employ fish possessing low resting glycogen levels, which may be related to seasonal variations, dietary status, size or age23,64,93. Exercising these fish may actually deplete white muscle glycogen completely in at least some fibers (<1 /zmol/g) and exhaustion apparently occurs when the muscle is starved for the carbon fuel. In studies using fish with higher initial glycogen levels (>20/zmol glueosyl/g) similar exercise protocols result in greater changes in adenylates and lactate 84,94. The actual cause of exhaustion when adequate glycogen occurs may be more related to disturbances of excitation-contraction coupling 113. Not surprisingly, the patterns of recovery in these two groups are different as well. In 'carbon starved' exhaustion, recovery of metabolite status tends to be slower, most obvious with phosphocreatine. As yet, no comprehensive study has directly addressed the influence of basal glycogen on exercise and recovery.

5. Recovery metabolism The exercise-induced imbalances in metabolite levels return to resting levels in minutes to several hours, depending on the metabolite. When a fish is deemed recovered is dependent on which metabolite is examined. Perhaps the most useful parameter on which to base the rate of recovery of carbohydrate status is lactate. The development of techniques to best sample fish is based upon minimization of lactate accumulation in 'resting' fish94,1~ Lactate generally returns to resting

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levels at the slowest rate. It is easily measured with little error arising from tissue extraction (little binding, highly acid-soluble). As resting levels are very low, exercise-induced changes with respect to rest are 10-fold or more. This contrasts to the situation with glycogen, where resting levels are highly variable between individuals, and exercise-induced changes are often less than 30% of the total glycogen level. Changes in lactate, however, offer little insight into the recovery of energy status. The most meaningful energy parameter incorporates some expression of free adenylate concentrations, but these determinations require much more analysis (total ATP, ADP, AMP, pHi, creatine and phosphocreatine) than phosphocreatine, the more commonly determined parameter. Changes in phosphocreatine reflect the adenylate status (free MgATP/MgADP) through CPK, which is assumed to be near equilibrium in vivo 18. It should be emphasized that phosphocreatine determination using freeze-clamped tissue typically underestimates the in vivo level. Using NMR, van Waarde and colleagues 1~176 estimate creatine is 80% phosphorylated in vivo, whereas the best estimates from freeze clamp studies are 62% in salmonids 94, 75% in tuna 2 and 70% with in vitro muscle fibers 7~ While absolute levels are difficult to determine with confidence, the relative changes within a study are probably realistic. Those studies which measure phosphocreatine yet neglect to determine creatine are less useful for assessing changes in energy status and very difficult to compare with other studies. Also, pH/must be considered in the analysis as acidosis will depress phosphocreatine through mass action effects on CPK, even when ATP/ADP is constant. Basing assessment of adenylate status on phosphocreatine changes alone would lead to erroneous conclusions 94.

6. Carbohydrate metabolism in recovery from burst exercise 6.1. Fate of lactate White muscle post-exercise must reduce lactate levels as well as replenish glycogen stores in preparation for the next exercise bout. In mammals, lactate may be oxidized or used as a substrate for hepatic gluconeogenesis or skeletal muscle glycogen synthesis directly. Its fate may be highly dependent on muscle type, species, pH and hormonal conditions 6,7,1~176 In reptiles, lactate produced in white muscle is largely transferred to red muscle for glycogen resynthesis 32. We believe that in fish, with few exceptions, lactate oxidation is minimal. The majority of lactate post-exercise is converted to glycogen and this occurs directly within the white muscle 74. It is clear that lactate disappearance is not due primarily to oxidation in white muscle or any tissue as whole animal respiration would greatly exceed that observed in vivo 93. Lactate may be utilized by tissues such as heart sa, but its oxidation would have little impact on whole body lactate levels. We believe that in recovery lactate disappearance accounts for glycogen reappearance 94, most obvious when single fish are repeatedly sampled 2. This is a critical, and somewhat controversial, conclusion as several studies conclude otherwise based on either of two lines of evidence: (1) lactate disappearance apparently does not parallel glycogen re-appearance with

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the required stoichiometry; and (2) glycogen does not recover to pre-exercise levels. However, the relationship between white muscle lactate and glycogen is very difficult to address where terminal samples constitute the time-course, due primarily to variability in resting glycogen levels. We feel that if the following two criteria are not met in a study it is very diftieult to establish the relationship between lactate and glycogen in recovery. Firstly, as lactate is most certainly derived primarily from muscle glycogen, moles of lactate produced cannot exceed twice the moles of glycogen utilized. Variability in glycogen levels can be so great that, at least in one study84, there was an apparent increase in lactate during exercise that is more than double that possibly arising from glycogen. When the stoiehiometry between glycogen disappearance and lactate appearance is violated during exercise, the relationship in recovery is more difficult to address with confidence. Secondly, glycogen and lactate in unexercised controls at the end of the study must be similar to fish prior to exercise. In studies which employ small fish with relatively low glycogen levels (e.g. ref. 93), there is an apparent depletion of glycogen in both exercised and control fish, suggesting significant utilization of white muscle carbohydrate for basal metabolism. As a consequence of the high and variable glycogen levels typically observed, limited oxidation of lactate (<20%) would be very difficult to detect. Indeed some fuel must be oxidized by white muscle mitochondria to support net glycogen resynthesis in recovery. Given the relatively low capacity for both hepatic glueoneogenesis and white muscle glucose transport (see next section), any metabolic strategy that leads to substantial depletion of white muscle carbohydrate seems maladaptive. As will be discussed in section 7, it is likely that lipid oxidation fuels recovery metabolism despite high levels of lactate 75. Several indirect and direct studies suggest that lactate conversion to glycogen does not involve extramuscular tissues but occurs directly within the white muscle. In mammals, the fiver may be an important site of glueoneogenesis 19 (but see also ref. 12), but the importance of the Coil cycle in fish is dubious due to low hepatic glueoneogenie capacity and limited capacity for glucose uptake by white muscle. The in vitro hepatic glueoneogenie rate of toadfish ~~ and tuna 14 is a small fraction of the observed rate of glycogen replenishment. In vivo, [14C]-laetate injected into the blood remains primarily as lactate in skipjack tuna (/E pelamis) 1~ and coho salmon (Oncorhynchus kisutch) 63, arguing against a significant influence of fiver, or any other gluconeogenic tissue. A recent study concludes that, in channel catfish (Ictalurus punctatus), lactate is primarily released from white muscle 16. Estimation of intraceUular lactate levels, based on eompartmentation analysis, suggests a peak of no greater than 1 /zmol/g (1563 /zmol/kg body mass). As this is far below the lactate level induced in most fish exercise studies, either the white muscle lactate pool was underestimated, and consequently the degree of release into the blood, or this species does not exercise as intensely as others (J.N. Cameron, personal communication). In other species lactate turnover is a small fraction of the rate of lactate disappearance from white muscle (tuna 2'1~ trout63). Even if adequate gluconeogenic potential existed, glucose uptake, as indicated by 2deoxyglucose transport, by white muscle is too low (rainbow trout, 2.5 nmol/min/g,

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T.G. West, unpublished) to account for the observed rate of glycogen repletion (25-50 nmol/min/g) 6~ Thus, both extra-muscular glucose production and white muscle glucose uptake are low compared to the observed rate of glycogen repletion. Since glycogen recovers to control levels, it follows that this pathway occurs within white muscle where lactate is retained. Furthermore, the smaller fraction that is released following exercise may be taken up by white muscle in the latter stages of recovery, relegating the plasma compartment to a short term lactate storage site 2.

6. 2. Route of glycogen resynthesis While it is likely that white muscle glycogen resynthesis is the major fate of lactate, the pathway by which this occurs is not established. At the beginning of the gluconeogenic pathway, key enzymes responsible for net reversal of glycolysis in liver are absent from white muscle. All vertebrate white muscles examined lack pyruvate carboxylase 22. Alternate pathways in mammals and amphibia have been suggested to involve malic enzyme plus phosphoenolpyruvate carboxykinase 7 (PEPCK) and reversal of pyruvate kinase 27 (PK). The situation appears more straightforward in fish because PEPCK does not occur at detectable levels in white muscle 74, except in marlin (Makaira nigricans) 98. Reversal of PK is thought unlikely in liver because mass action ratios suggest it is too far from equilibrium to catalyze a net reversal under physiological conditions. Pyruvate kinase in resting skeletal muscle is much closer to equilibrium than in liver, due to higher free ATP/ADP ratios (liver ~20, ref. 71 versus 200 in rainbow trout white muscle, ref. 94). In recovering trout white muscle, the ATP/ADP ratio increases to approximately 2000 (ref. 94). Increases in pyruvate (6-fold in trout 98, 20-fold in tuna 2) would also favor PK reversal. Changes in phospoenolpyruvate, the product for the reverse direction have not been measured in recovery, but realistic estimates suggest that PK could be close to equilibrium post-exercise 94. No direct evidence for PK reversal as the route of glycogen resynthesis in muscle has been presented in any muscle. Examination of PK activity in those species for which lactate disappearance rates are available reveals an interesting relationship consistent with such a role for PK 74. Fig. 3 compares the rate of disappearance of white muscle lactate following exercise of several fish species against the maximal velocity of PK (forward). A mean of 0.05% of PK maximal velocity would be required for lactate conversion to glycogen at the observed rates. Dyson and coworkers 27 suggest that PK could be driven in its reverse direction at a maximal rate of about 2% of the forward maximal rate, well above that which would be required in fish white muscle. Whatever the route of glycogen production from lactate, mitochondrially produced ATP is required to fuel net glycogen resynthesis.

Z Fuelforrecovery metabolism We have argued that the early stages of recovery may be fuelled by glycolytic ATP production, but it is expected that glycogen resynthesis and most of the recovery costs will be met by mitochondrial ATP production. This stimulation of aerobic metabolism is indicated by the excess post-exercise oxygen consumption 93, although

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this typically exceeds the identifiable costs of recovery in fish93 and mammals 5. The question of fuel oxidized under these conditions has only recently been addressed 5,6,75. It is paradoxical that, under conditions where lactate is present at very high concentrations, fat appears to be the metabolic fuel utilized in recovery metabolism. This has been shown in mammals using arteriovenous metabolite differences and respiratory quotients across exercised muscle groups 3,6. Difficulty in obtaining a viable isolated perfused preparation has prevented a direct assessment of respiratory quotients or metabolite changes across fish white muscle. An early report examining whole animal respiratory exchange ratios, suggested that carbohydrate may be the fuel utilized by fish under similar conditions, but it is unlikely that respiratory gases are in equilibrium at this time 63. Indirect studies using isolated trout white muscle mitochondria suggest that fatty acid oxidation may be preferred fuel in recovery. Fish white muscle mitochondria, as in mammals, oxidize pyruvate based fuels (e.g. lactate) in preference to fatty acids when each is presented singly4,72 but when presented together, fatty acid oxidation inhibits utilization of pyruvate 75. Control of fuel selection at the mitochondrial level in skeletal muscle is primarily through regulation of pyruvate dehydrogenase. Although pyruvate dehydrogenase is subject to both allosteric (stimulation, inhibition) and covalent (activation, inactivation) regulation 35, it is likely that inhibition of pyruvate dehydrogenase is achieved allostericaUy in isolated white muscle mitochondria in vitro and in recovering fish in vivo 75. As white muscle respiration rates in recovering fish are a low proportion of the oxidative capacity, even a modest fatty acid supply would be capable of meeting the needs of recovery metabolism 75.

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8. Energy metabolism During exercise, dramatic changes in the phosphagens occur which allow ATP to be produced at very high rates to meet the demands of burst exercise. Phosphocreatine is depleted as the free ATP/ADP ratio decreases and the tissue becomes acidotic 94. A fraction of the ATP pool is catabolized to IMP via AMP deaminase 64,94. In general the changes in phosphagens are greater than in mammals. In recovery, there is a modest energy cost associated with re-establishing the phosphagen levels 93. Any changes in free adenylates must be integrated with carbohydrate metabolism as they are important regulators of both carbohydrate metabolism and the mitochondrial pathways which must be used to provide ATP for net glycogen resynthesis. 8.1. Phosphocreatine Few studies of fish burst exercise and recovery provide detailed time courses of changes in phosphocreatine and these are limited to even fewer species (skipjack tuna 2, salmonids23, 64,59,84,93,94) . As phosphocreatine resynthesis occurs as a consequence of re-establishment of appropriate free ATP/ADP ratio, with consideration of the intracellular pH, it provides some insight into the changes in adenylate status. Most rainbow trout studies find phosphocreatine levels to recover quickly (<2 h), however, two studies find phosphocreatine recovery to be much slower 23,64. Phosphocreatine resynthesis cannot be regulated separately, so delayed resynthesis does not represent a shift in the intracellular metabolic priorities, but is indicative of a persistent disturbance in either pH or free ATP/ADP. In both of the studies which demonstrate this slow recovery of phosphocreatine, glycogen is severely depleted by the end of exercise (< 1/zmol/g). Also, in studies where time-courses are sufficiently fine and glycogen is not depleted, lactate production is observed to continue after cessation of exercise (e.g. ref. 93). Taken together, these two observations suggest that the earliest phase of recovery metabolism, which includes phosphocreatine resynthesis, is dependent, paradoxically, on anaerobic glycolysis 23. Those individuals which deplete glycogen in exercise, may possess inadequate glycolytic potential to allow a fast recovery of phosphocreatine, indicative of depressed free ATP/ADP. Immediately following exercise the free ATP/ADP ratio is low 94 and stimulatory to both glycolytic and mitochondrial pathways. If post-exercise conditions are such that mitochondrial metabolism is differentially suppressed, glycolytic ATP would be required to meet the non-gluconeogenic demands of recovery. One possible such effector is Pi, which is expected to be high post-exercise and inhibits mitochondrial respiration at expected post-exercise levels 75. Coincident with recovery of phosphocreatine concentration is net utilization of Pi. Thus, recovery of phosphocreatine may be a prerequisite of utilization of mitochondrial pathways for ATP synthesis and in situ glycogen resynthesis. 8.2. Adenylates Investigation of recovery of adenylate status as an indicator of energy metabolism must take into account the metabolite species present. Much of the acid-extractable ADP pool in white muscle is bound to myofibrils. Thus, changes in total ADP

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tell little about energy metabolism. Calculations of unbound adenylate species can be performed assuming CPK and adenylate kinase are near equilibrium and their metabolites are homogeneously distributed within the intraceUular space. When only acid-extractable adenylates are considered, the minor changes observed mask much greater changes occurring in the metabolically-relevant free pool. At present only one study estimates the changes in free adenylates following exercise in fish white muscle 94. Although total ATP is low for many hours following exercise, the energy status (e.g. free ATP/ADP) recovers quickly and is even elevated above rest 94. Both the quick recovery and apparent overshoot have important implications in the integration of the post-exercise recovery pathways. Mitochondrial ATP synthesis is stimulated by a decrease in ATP/ADP ratio, yet in recovery, when an increase in whole animal respiration is known to occur, the ATP/ADP ratio increases 94. If this ratio was the sole parameter to consider, a decrease in white muscle respiration would be expected from the increase in ATP/ADP observed postexercise. It is likely that the increase in Pi stimulates respiration to provide ATP to fuel recovery metabolism 75. Acidosis stimulates mitochondrial respiration when P~ is limiting, possibly by increasing the proportion of diprotonated Pi present. Thus, in less extreme, more physiological exercise, when less Pi is expected to accumulate, cytosolic acidosis would be expected to stimulate mitochondrial ATP production 75. Again, it must be stressed that the changes in P~ expected from net phosphocreatine breakdown and AMP deaminase activity have not been assessed directly. Although energy status recovers quickly, total adenylate concentrations take much longer ~'94. Resynthesis of ATP from IMP requires the re-aminating arm of the purine nucleotide cycle64. During high intensity exercise, ATP is stoichiometrically converted to IMP. As this IMP remains within the cell, there is no requirement for adenosine synthesis for ATP production following exercise. The reaminating arm of the purine nucleotide cycle involves two enzymes and requires input of aspartate as the nitrogen source and GTP as phosphate and energy source 64. 9. Why is recovery so slow?

The earliest studies on recovery metabolism suggested that the rate of recovery from burst exercise in fish was very slow by mammalian standards (e.g. refs. 37 and 56). Recent work using skipjack tuna suggests that this generalization was more related to the choice of species 2. Lactate disappearance from skipjack tuna white muscle following burst exercise occurs at rates within the range observed in mammalian studies but in mammals, much of the lactate is released from the working tissue and oxidized elsewhere 6,13. Thus, when one considers the difference in the fate of lactate, tuna white muscle 2 can convert lactate to glycogen as fast or faster than mammalian skeletal muscle even though the rate of lactate disappearance is greater in mammals 6. When the full range of fish species studied is compared, the rates of lactate disappearance span two orders of magnitude 74. The biochemical basis of the interspecies differences has not been established. The rate of lactate disappearance correlates with the aerobic capacity of the white muscle 74. Resynthesis of glycogen post-exercise demands mitochondrial ATP production

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and white muscle is a mitochondria-poor tissue but the simplest explanation for this relationship, that maximal mitochondrial ATP producing capacity limits the rate of glycogen resynthesis, is unlikely75,94. The high free ATP/ADP observed throughout recovery would actually suppress mitochondrial ATP production to approximately 10-30% of the maximal capacity7s. The high free ATP/ADP ratios, while suppressing mitochondrial respiration, may be required to drive PK in the reverse direction for net glycogen resynthesis94. As was mentioned previously, the activity of PK also correlates with the rate of recovery across these same species (Fig. 3). Direct demonstration that the capacity for PK reversal limits the rate of recovery across species has proven elusive.

10. Summary of burst exercise and recovery Burst exercise is fuelled by glycogen breakdown to lactate. Reliance on circulatory glucose is minimal. Adenylates and phosphocreatine undergo much greater changes than are observed in mammals. Although total ATP remains low for several hours, post-exercise energy status, as indicated by phosphocreatine/creatine or free adenylate ratios, recovers more quickly. In species or stocks with severe glycogen depletion, the time required for recovery of energy status is extended. Reduction of Pi through post-exercise glycolysis and net phosphocreatine synthesis may be a prerequisite for rapid recovery. Resynthesis of glycogen occurs primarily in white muscle and requires much longer to recover than does energy status. Recovery is faster in species possessing: (1) more aerobic white muscle; and (2) more pyruvate kinase. Unlike mammals, oxidation is thought not to be a quantitatively important fate of white muscle lactate post-exercise. Glycogen resynthesis occurs in white muscle, and probably through pyruvate kinase, reversed in vivo by high pyruvate and free ATP/ADP ratios. Direct evidence for this or any route of glycogen resynthesis is presently lacking. Glycogen resynthesis is probably fuelled by mitochondrial oxidation of fatty acids, sparing lactate for glycogen resynthesis.

Note added in proof Recent studies (A. Pagnotta, L. Brooks and L. Milligan. Potential regulatory roles of cortisol in recovery from exhaustive exercise in rainbow trout. Can. J. Zool., in press) have shown that the rate of recovery from burst exercise is profoundly influenced by circulating cortisol levels. If exercised fish are allowed to swim during recovery, cortisol levels remain low and recovery rate increases 2--4 fold.

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58. Milligan, C.L. and A.P. Farrell. Lactate utilization by an in situ perfused trout heart: effects of work load and blockers of lactate transport. J. Exp. Biol. 155: 357-373, 1991. 59. MiUigan, C.L and C.M. Wood. Intracellular and extracellular acid-base status and H + exchange with the environment after exhaustive exercise in the rainbow trout. J. F~. Biol. 123: 93-121, 1986. 60. Milligan, C.L. and C.M. Wood. Tissue intracellular acid-base status and the fate of lactate after exhaustive exercise in the rainbow trout. J. Exp. Biol. 123: 123-144, 1986. 61. Milligan, C.L. and C.M. Wood. Effects of strenuous activity on intracellular and extracellular acid-base status and H + exchange with the environment in the inactive, benthic starry flounder Platichthys stellatus. PhysioL Zool. 60: 37-53, 1987. 62. Milligan, C.L. and C.M. Wood. Muscle and liver intracellular acid-base and metabolite status after strenuous activity in the inactive, benthic starry flounder, Platichthys steUatus. Physiol. Zool. 60: 54-68, 1987. 63. Milligan, C.L. and D.G. McDonald. In vivo lactate kinetics at rest and during recovery from exhaustive exercise in coho salmon (Oncorhynchus kisutch) and starry flounder (Platichthys stellatus). J. Exp. Biol. 135: 119-131, 1988. 64. Mommsen, T.P. and P.W. Hochachka. The purine nucleotide cycle as two temporally separated metabolic units: a study on trout muscle. Metabolism 37: 552-556, 1988. 65. Mommsen, T.P. and E.M. Plisetskaya. Insulin in fishes and agnathans: history, structure and metabolic regulation. Rev. Aquat. Sci. 4: 225-259, 1991. 66. Mommsen, T.P., C.J. French and P.W. Hochachka. Sites and patterns of protein and amino acid utilization during the spawning migration of salmon. Can. J. Zool. 58: 1785-1799, 1980. 67. Mommsen, T.P., P.C. Andrews and E.M. Plisetskaya. Glucagon-like peptides activate hepatic gluconeogenesis. FEBS Letts. 219: 227-232, 1987. 68. Mommsen, T.P., P.J. Walsh, S.E Perry and T.W. Moon. Interactive effects of catecholamines and hypercapnia on glucose production in isolated trout hepatocytes. Gen. Comp. Endocrinol. 70: 63-73, 1988. 69. Moon, T.W. and T.P. Mommsen. Enzymes of intermediary metabolism in tissues of the little skate, Raja erinacea. J. Exp. Zool. 244: 9-15, 1987. 70. Moon, T.W., J.D. Altringham and I.A. Johnston. Energetics and power output of isolated fish fast muscle fibres performing oscillatory work. J. Exp. Biol. 158: 261-273, 1991. 71. Morikofer-Zwez, S. and P. Walter. Binding of ADP to rat liver cytosolic proteins and its influence on the ratio of free ATP/free ADP. Biochem. J. 259: 117-124, 1989. 72. Moyes, C.D., L.T. Buck, P.W. Hochachka and R.K. Suarez. Oxidative properties of carp red and white muscle. J. Erp. BioL 143: 321-331, 1989. 73. Moyes, C.D., L.T. Buck and P.W. Hochachka. Mitochondrial and peroxisomal fatty acid oxidation in elasmobranchs. Am. J. Physiol. 258: R756-R762, 1990. 74. Moyes, C.D., P.M. Schulte and T.G. West. Recovery metabolism in fish white muscle. In: Surviving Hypoxia: Mechanisms of Control and Adaptation, edited by P.W. Hochachka, P.L. Lutz, T. Sick, M. Rosenthal and G. van den Thillart, CRC Press, Boca Raton, 1992. 75. Moyes, C.D., P.M. Schulte and P.W. Hochachka. Recovery metabolism of trout white muscle: the role of the mitochondria. Am. J. Physiol. 262: R295-R304, 1992. 76. Moyes, C.D., O.A. Mathieu-Costello, R.W. Brill and P.W. Hochachka. Mitochondrial metabolism of cardiac and skeletal muscles from a fast (Katsuwonus pelamis) and a slow (Cyprinus carpio) fish. Can. J. Zool. 70: 1246,1256, 1992. 77. Moyes, C.D., R.K. Suarez, P.W. Hochachka and J.S. Ballantyne. A comparison of fuel preferences of mitochondria from vertebrates and invertebrates. Can. J. Zool. 68: 1337-1349, 1990. 78. Neumann, P., G.E Holeton and N. Heisler. Cardiac output and regional blood flow in gills and muscles after exhaustive exercise in rainbow trout (Salmo gairdneri). J. E~. Biol. 105: 1-14, 1983. 79. Pagliassotti, M.J. and C.M. Donovan. Glycogenesis from lactate in rabbit skeletal muscle fiber types. Am. J. Physiol. 258: R903-R911, 1990. 80. Palmer, T.N. and B.E. Ryman. Studies on oral glucose intolerance in fish. J. Fish. Biol. 4: 311-319, 1972. 81. Parkhouse, W.S., G.P. Dobson, A.N. Belcastro and P.W. Hochachka. The role of intermediary metabolism in the maintenance of proton and charge balance during exercise. Mol. Cell. Biochem. 77: 37-47, 1987. 82. Parkhouse, W.S., G.P. Dobson and P.W. Hochachka. Organization of energy provision in rainbow trout during exercise. Am. J. Physiol. 254: R302-R309, 1988.

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83. Parkhouse, W.S., G.P. Dobson and P.W. Hochachka. Control of glycogenolysis in rainbow trout muscle during exercise. Can. J. Zool. 66: 345-351, 1988. 84. Pearson, M.P., L.L. Spriet and E.D. Stevens. Effect of sprint training on swim performance and white muscle metabolism during exercise and recovery in rainbow trout (Salmo gairdneri). J. Exp. Biol. 149: 45-60, 1990. 85. Petersen, TD.P., P.W. Hochachka and R.K. Suarez. Hormonal control of gluconeogenesis in rainbow trout hepatocytes: regulatory role of pyruvate kinase. J. Exp. Zool. 243: 173-180, 1987. 86. Plisetskaya, E. Fatty acid levels in blood of cyclostomes and fish. Env. Biol. Fish 5: 273-290, 1980. 87. Randall, D.J. and C. Daxboeck. Cardiovascular changes in the rainbow trout (Salmo gairdneri Richardson) during exercise. Can. J. Zool. 60: 1135-1140, 1982. 88. Reid, S.D., TW. Moon and S.E Perry. Rainbow trout hepatocyte ~-adrenoreceptors, catecholamine responsiveness, and effects of cortisol. Am. J. Physiol. 262: R794-R799, 1992. 89. Ristori, M.T and P. Laurent. Plasma catecholamines and glucose during moderate exercise in the trout: comparison with bursts of violent activity. Exp. Biol. 44: 247-253, 1985. 90. Rome, L.C., R.P. Funke, R.M. Alexander, G. Lutz, H. Aldridge, E Scott and M. Freadman. Why animals have different muscle fiber types. Nature 335: 824-827, 1988. 91. Ruderman, N.B., C.R.S. Houghton and R. Hems. Evaluation of the isolated rat hindquarter for the study of muscle metabolism. Biochem. J. 124: 639-651, 1981. 92. Saddik, M. and G.D. Lopaschuk. Myocardial triglyceride turnover and contribution to energy substrate utilization in isolated working rat hearts. I. Biol. Chem. 226: 8162-8170, 1991. 93. Scarabello, M., G.J.E Heigenhauser and C.M. Wood. The oxygen debt hypothesis in juvenile rainbow trout after exhaustive exercise. Resp. Physiol. 84: 245-259, 1991. 94. Schulte, P.M., C.D. Moyes and P.W. Hochachka. Integrating metabolic pathways in post-exercise recovery of white muscle. J. Exp. Biol. 166: 181-195, 1992. 95. Schwalme, K. and W.C. McKay. The influence of angling-induced exercise on the carbohydrate metabolism of northern pike (Esox lucius L.). J. Comp. Physiol. 156B: 67-75, 1985. 96. Sheridan, M.A. Lipid dynamics in fish: aspects of absorption, transportation, deposition and mobilization. Comp. Biochem. Physiol. 90B: 679-690, 1988. 97. Spriet, L.L., L. Berardinucci, D.R. Marsh, C.B. Campbell and TE. Graham. Glycogen content has no effect on skeletal muscle glycogenolysis during short-term tetanic stimulation. J. Appl. Physiol. 68: 1883-1888, 1990. 98. Suarez, R.K., M.D. Mallet, C. Daxboek and P.W~Hochachka. Enzymes of energy metabolism and gluconeogenesis in the Pacific blue marlin, Makaira nigricans. Can. J. Zool. 64: 694-697, 1986. 99. Suarez, R.K. and T.P. Mommsen. Gluconeogenesis in teleost fishes. Can. I. Zool. 65: 1869-1882, 1987. 100. Tappy, L., IC Acheson, S. Normand, D. Schneeberger, A. Thelin, C. Pachiaudi, J.-P. Riou and E. Jequier. Effects of infused amino acids on glucose production and utilization in healthy human subjects. Am. J. Physiol. 262: E826-E833, 1992. I01. Tang, Y. and R.(3. Boutilier. White muscle intracellular acid-base and lactate status following exhaustive exercise: a comparison between freshwater- and seawater-adapted rainbow trout. J. Exp. Biol. 156: 153-171, 1991. 102. Van den Thillart, G. Energy metabolism of swimming trout (Salmo gairdneri). J. Comp. Physiol. 156B: 511-520, 1986. 103. Van den ThiUart, (3., A. Van Waarde, H.J. Muller, C. Erkelens, A. Addink and J. Lugtenburg. Fish muscle energy metabolism measured by in vivo 31p-NMR during anoxia and recovery. Am. J. Physiol. 256: R922-R929, 1989. 104. Van Waarde, A. Aerobic and anaerobic ammonia production by fish. Comp. Biochem. Physiol. 74B: 675-684, 1983. 105. Van Waarde, A., G. van den Thillart, H.J. Muller, C. Erklelens, A. Addink and J. Lugtenburg. Functional coupling of glycolysis and phosphocreatine utilization in anoxic fish muscle -an in vivo alp NMR study. J. Biol. Chem. 265: 914-923, 1990. 106. Walsh, P.J. An in vitro model of post-exercise hepatic gluconeogenesis in the gulf toadfish Opsanus beta. J. Exp. Biol. 147: 393-406, 1989. 107. Webb, P.W. The swimming energetics of trout I. Oxygen consumption and swimming efficiency. J. Exp. Biol. 55: 521-540, 1971. 108. Weber, J.-M. Effect of endurance swimming on the lactate kinetics of rainbow trout. J. Exp. Biol. 158: 463-476, 1991. 109. Weber, J.-M., R.W. Brill and P.W. Hochachka. Mammalian metabolite flux rates in teleost: lactate and glucose turnover in tuna. Am. J. Physiol. 250: R452-R458, 1986.

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Hochachka and Mommsen (eds.), Biochemistry and molecular biology of fishes, vol. 4 9 1995 Elsevier Science B.V. All rights reserved. CHAPTER 17

Fasting and starvation ISABEL NAVARRO AND JOAOUIM GUTIl~RREZ

Depanament de Bioqufmica i Fisiologia, Universitat de Barcelona, Unitat de Fisiologia, Avda. Diagonal, 645, E-08071 Barcelona, Spain

I. II.

Introduction Carbohydrate mobilization 1. Liver glycogen 2. Blood glucose 3. Muscle glycogen III. Lipid mobilization 1. Liver 2. Intestinal fat 3. Muscle lipids 4. Lipid classes 4.1. Free fatty acids 5. Influence of starvation on lipolysis and lipogenesis IV. Protein mobilization 1. Liver proteins 2. Muscle proteins 3. Plasma protein and amino acids V. Endocrine regulation 1. Insulin 2. Glucagon and glucagon-like peptide 3. Glucocorticoids 4. Growth hormone 5. Thyroid hormones VI. General conclusions Acknowledgements VII. References

I. Introduction Fish can survive for a long time without food and for many species a fasting period forms part of the natural life cycle. Winter months, spawning migration and/or prespawning stage can all be naturally non-feeding periods. Thus, numerous species can starve for many months and then recover fully after refeeding. Therefore, these species are well adapted to mobilize their metabolic reserves and even body constituents to survive periods of food deprivation. Specific effects of starvation on metabolism are dependent on multiple variables; including the species under consideration, the preferential tissues for metabolic stores, the quantity stored and their availability as well as distinct routes of mobilization. In this sense, the pre-fasting diet may exert substantial influence on the

394

I. Navarro and I. Guti~rrez

metabolic events initiated by fasting75. It is also important to differentiate between experimental and natural fasting, because natural fasting can be accompanied by other, compounding factors, such as gonadal growth, low temperatures, etc. Experimental fasting effects are likely dependent on endogenous and exogenous factors. For instance, the season chosen, temperature and photoperiod, fish age and whether or not the fasting is imposed during a reproductive period will all have substantial bearing on the experimental results. Therefore, all these factors have to be taken into account when comparing fasting responses between species or even within species. Another important aspect when comparing the experimental results of fasting periods in fishes is the way in which the data are expressed. Machado and eoworkers 114, for instance, pointed out that many discrepancies found in the literature can be explained because conclusions are obtained on the basis of changes in the concentrations of tissue metabolites, being more informative than changes in total amounts stored in tissue. Similarly, Black and Love 15 found a great deal of clear information when data were considered in relation to one another. In the same vein, Foster and Moon 55 indicated the advantage of using body weight and tissue DNA content as reference points for metabolic and enzymatic parameters. The aim of this chapter is to give a brief overview of gross metabolic and enzymatic changes observed in fasting and starving fish. We have chosen the storage substrate as the organizing principle, before focusing on various parameters of endocrine regulation during fasting and starvation in fishes.

II. Carbohydrate mobilization 1. Liver glycogen Carbohydrates are stored as glycogen in liver normally amounting to 1-6% of liver weight, although some species, such as carp (Cyprinus carpio), can accumulate values exceeding 10%. When required, hepatic glycogen is enzymatieaUy broken down and transported to the extrahepatie tissues as glucose. On arrival at its target cells, glucose is either metabolized or reconverted into glycogen. Unfortunately, liver glycogen determination has a number of inherent problems which may complicate the interpretation of observed changes in this particular parameter under some experimental conditions. First, variation in the amount of liver glycogen is large between individuals; and second, as the example of the carp shows, concentrations may be exceptionally high. Because liver glycogen is normally determined in terminal samples, it is inherently diftieult to determine (or detect) small changes in liver glycogen and often even large decreases may be missed due to large individual variability. Usually, glycogen depletion is a continuous process from the beginning of fasting 1~ In most species, liver glycogen is mobilized early in experimental fasting in fish; in fact, partially because of the ease of mobilization, liver glycogen is generally the first substrate to be used during fasting. At 5, 8, 15, 20 days

Fasting and starvation

395 TABLE 1

Variation of body parameters and liver and muscle components in fed and fasted trout (Salmo tmtta

fario) Fed (days) 1 15 Body parameters Weight (g) Length (cm) HSI (%) MLI (gcm -1) VI (%)

30

50

Fasted (days) 8 30 135 21.6 0.99 3.2 a

163 22.7 1.24 3.6 8.4

175 22.0 1.09 4.2 9.1

174 24.4 1.03 3.9 6.1

201 25.3 1.08 4.41 9.9

Liver composition Glycogen (%) Protein (%) Lipids (%) Water (%) P-DNA (mg 100 g-l)

4.6 14.3 3.7 75.1 27.8

3.9 15.1 3.3 75.1 35.4 b

3.9 15.3 4,0 74.6 36.2 b

2.5 15.4 4.3 75.3 39.1 b

0.9 a,b

0.9 a,b

0.6 a,b

16.7 a'b 3.7 76.0 a,b 43.0 a,b

16.7 a,b 3.8 75.5 a

16.1 a,b 3.6 a 76.2 a,b 57.3 a,b

Muscle composition Glycogen (%) Protein (%) Lipid (%) Water (%) P-DNA (mg 100 g-l)

0.24 19.7 1.5 77,8 2.5

0.27 19.2 1.6 77.7 2.7

0.27 19.6 1.4 77.5 2.2

0.30 19.3 1.7 76.9 1.9

0.20 18.8 1.5 78.1 3.3

0.10 a,b 18.8 1.1 77.8 2.4

5.0 a'b

134 23.8 0.83 a,b 3.1 a

50

4.5 a'b

51.6 a,b

121 23.7 0.77 a,b 2.9 a,b 3.6 a,b

0.09 a,b 18.2 a,b

1.0 a 78.5 a 2.1

Results are means from 10 fish in each group. a Indicates significantly different (p < 0.05) from fed fish at the same point (Duncan's test). b Indicates significantly different (p < 0.05) from the fed group at day 1 of the experiment (Duncan's test). Abbreviations: HSI ffi Hepatosomatic index (gram liver weight 100 g-1 body weight); MLI = Index of muscle weight (g) over length (era); VI ffi Index of viscera weight (g) without liver per 100 g body weight; P-DNA ffi DNA phosphorous (mg) per 100 g tissue fresh weight. Maximum standard error of individual obervations were below 15%, in most cases below 10% of the mean. Adapted from Navarro et al. TM.

fasting, significant decreases in glycogen have been described in several species of teleostean fishes 65,75,114,115,135,169 o r elasmobranchs 47. Table 1 shows the effects of 15, 30 and 50 days of starvation on liver and muscle glycogen in brown trout (Salmo

trutta fario ). However, several species undergo prolonged periods of fasting in their natural environment with little depletion of liver glycogen. Pacific sockeye salmon (Oncorhynchus nerka) migrate some 1000 km without decrease in this reserve 58. Fasted European eels (Anguilla anguilla) did not show a change in liver glycogen after 95 days fasting 96. As mentioned above, carp (C. carpio) accumulate exceptionally high levels of liver glycogen and after 12 months of starvation still maintain 6% of glycogen in their livers 192. Nagai and Ikeda 133 indicated that 22 days fasting in carp (C. carpio) did not provoke any change in liver glycogen (10.65%) and it was only after 100 days that a clear decrease was observed (1.55%). Such an obvious defence of hepatic glycogen, even after long periods of starvation, may be explained by a compensatory increase in the rate of gluconeogenesis from non-carbohydrate precursors, which has two effects, namely: (1) that hepatocyte glycogen can be spared;

L Navarro and J. Guti~rrez

396 TABLE 2

Variation of body parameters and liver and muscle components in fed and fasted yellow perch (Perca flavescens) Fed

Fasted for 7 weeks

Body parameters Condition factor Hepatosomatic index (%)

1.26 1.99

1.13" 0.85 a

Plasma Glucose (mM/l)

4.27

3.03 a

Liver composition Glycogen (/zMol//~g DNA) Protein (mg//zg DNA) Glucose (/zMol//zg DNA) DNA (~g/g tissue weight)

253 43.9 73.0 2.4

40.8' 28.0 a 98.0 4.5 a

Muscle composition Glycogen (/xMol/ttg DNA) Protein (mg//~g DNA) Glucose (/zMol//zg DNA) DNA (/xg/g tissue weight)

69.4 883 17.4 0.22

43.3 a 700 a 7.6 a 0.25

Values are means of 3 to 12 observations. a Significantly different from fed control fish (~p < 0.05; Student's t-test). Condition factor -- gram body weight per cm ~ body length. Adapted from Foster and Moon 5s.

and (2) that the hepatic production of glucose 6-phosphate can lead to an increased carbon flux into glycogen. Increase in glyconeogenesis was suggested in A. anguilla after 95 days fasting from the increase of the hepatic glutamate-oxaloacetate transaminase (aspartate aminotransferase, AspAT, EC 2.6.1.1), together with the maintenance of glycogen. Liver gluconeogenesis was also enhanced in 6- or 7-week fasted rainbow trout 126 (O. mykiss) or yellow perch 55 (Perca flavescens) (Table 2), respectively. The percentage per wet tissue weight of key gluconeogenie enzymes AspAT, phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32) and lactate dehydrogenase (LDH, EC 1.1.1.27) significantly increased in yellow perch 55. These data are in agreement with the observations of Morata and eoworkers 129 who found increases in liver and kidney gluconeogenesis during the second month of starvation in rainbow trout, and with those of French and coworkers 59,5a who determined relative increases in PEPCK and LDH in starving and exercising rainbow trout and increases in PEPCK and glutamate-pyruvate transaminase (alanine aminotransferase, AlaAT, EC 2.6.1.2) in migrating (starving) sockeye salmon. It is clear that in the face of rapid proteolytie degradation, the activities of key enzymes are somehow defended. However, the mechanism of this defence is not clear until more detailed research, preferably at the molecular level, has been done. For instance, it is not obvious from the descriptive studies mentioned above: (1) whether these enzymes are protected against a generalized proteolytic attack; (2) whether the proteolytic attack, which involves selective activation of eathepsin-like proteases, is general in nature; or (3) whether compensatory increases in enzyme synthesis are responsible for the apparent maintenance of such key enzymes. Thus,

Fasting and starvation

397

this topic would make an ideal area to apply molecular biological techniques to probe mRNA longevity, transcription rates and possible induction of key enzymes. Of the enzymes mentioned, it is known from other vertebrates that transcription of PEPCK is under multifaceted endocrine control, involving crosstalk between different regulatory pathways. The applicability to piscine systems, however, remains to be elucidated. At any rate, the nature of the key enzymes purportedly preserved, more than hints at key metabolic pathways which retain importance and metabolic flux throughout starvation. Applying a teleological view to intermediary metabolism, all key enzymes support the idea of a drastically changed emphasis on amino acid metabolism, possibly with concurrent increases in the importance of gluconeogenesis. The transaminases can be considered feeder enzymes for carbon into oxidative and gluconeogenic pathways, while PEPCK plays a pivotal role in the flux of carbons from C3, C4 and C5 precursors (which can be derived from most amino acids) into glucose. Nevertheless the often times noticed maintenance of LDH poses a bit of a problem to the picture outlined above. At any rate, however, given the decrease in liver glycogen, the overall contribution of glycogen to the total energy expenditure is comparatively small considering the limited weight of the liver (hepatosomatic index ranging normally from 1 to 3%; of. Tables 1 and 2).

2. Blood glucose Although blood glucose in fishes is known to fluctuate widely between species and also within species, within a given, uniform batch of individuals, blood glucose concentrations are maintained at a steady level during long periods of food deprivation. It is thought that this apparent defence of blood glucose against fluctuations or depletion occurs largely at the expense of liver glycogen, at least during the initial stages of fasting. Hochachka and Sinclair77, for instance, found a decrease in liver glycogen without modification of blood glucose in 14-day fasted rainbow trout. Not surprisingly, glycemia profiles during fasting vary depending on the species considered, and this is one area where other physiological factors mentioned above are likely to exert substantial influence. In juvenile sea bass (Dicentrarchus labrax), glycemia is decreased after a 5-day fast, and in juvenile brown trout (Salmo trutta ratio) after a 10-day fast 65'135. Zammit and Newsholme 198, working on adults of the same species, failed to observe changes in glycemia after imposing a 40-day fast. It was only after 100 days of food deprivation that blood glucose levels decreased significantly. The hagfish (Myxine glutinosa) seems to follow a similar pattern as the teleosts, since this species maintained blood glucose throughout a three-week fast 43. An entirely different picture emerges for an elasmobranch fish, exemplified by the response of the common dogfish. In these animals (Scyliorhinus canicula), whose metabolism is even less 'glucosocentric' than in teleostean fishes, plasma glucose decreases rapidly during food deprivation and reaches a low of 0.52 mg/100 ml (0.03 mM) within eight days of food deprivation; however, later during starvation, blood glucose levels recover and reach pre-fasting levels (12.05 mg/100 ml; 0.67 mM) after 67 days of fasting47.

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L Navarro and J. Guti~rrez

In carp and eel, no changes in glycemia were observed after several weeks of fasting 34,131. Starved Clarias lazera (a warm-water catfish) experienced no change in blood glucose after four months, although this parameter decreased to 60% after an additional three months of experimentation 68. Sluggish fish seem to be able to maintain glucose levels for longer periods than more active species, likely reflecting the comparatively smaller demand and hence turnover of glucose in these species. As shown by Weber and coworkers 1~,~89, a strong positive correlation exists between glucose turnover and assumed physical activity of teleostean fishes. In the case of the extremely active skipjack tuna (Katsuwonus pelamis), for instance, glucose turnover rates approach or even surpass those of many mammals (cf. chapter 2, this volume). In carp (C. carpio) an increase in glycemia was observed in early starvation 133. It is possible to distinguish two phases in the maintenance of blood glucose during fasting and starvation in fishes; one is that glucose sequestered from the plasma pool is replenished by liver glyeogenolysis; the other is replenished through the compensatory activation of glueoneogenesis, especially from amino acids mobilized though preferential proteolysis of muscle 114. However, as the studies mentioned above already reveal, a clear temporal or functional relationship between these phases, as exists in many mammals, cannot be delineated. At any rate, a combination of both phases is clearly involved in the stabilization of blood glucose during starvation.

3. Muscle glycogen The amount of glycogen stored in fish muscle falls into the range between 0.2 and 1.3% per wet weight and is thus generally assumed to make only a small potential contribution to total energy expenditure ~14. However, it should be kept in mind, that because of its immediate involvement in muscular activity, muscle glycogen is a most volatile entity and its mobilization is more likely related to an increase in muscular activity than to the fasting process. Not surprisingly, the rate of muscle glycogen mobilization is positively correlated with ambient temperatures. In brown trout (S. trutta fario) significant decreases in muscle glycogen were found at 8 days fasting in summer, but only at 30 days fasting in winter (Table 1) 135. Similarly, in rainbow trout (O. mfldss) muscle glycogen is lowest in summer 61 when high temperature and likely increased activity of the fish combine to drain the muscle glycogen stores. In Atlantic cod (Gadus morhua), at early stages of starvation, both liver and muscle glycogen were mobilized. But red muscle glycogen remained unchanged during most of the depletion 15. However, in several fish species glycogen in muscle is not mobilized and it seems likely that this store is maintained at the expense of blood glucose which is supplied by hepatic processes (gluconeogenesis, glycogenolysis). In juvenile sea bass (Dicentrarchus labrax) fasted 22 clays in summer, muscle glycogen decreased very little 65, while carp fasted 19 or 67 days did not show a depletion in muscle glycogen levels is. Similarly, European eels fasted for 164 days maintained unchanged muscle

Fastingand starvation

399

glycogen concentrations 34. A comparable pattern seems to hold at least for one species of elasmobranch: 82 days of fasting did not provoke a decrease in muscle glycogen in S. canicula 47. In conclusion, although important differences may exist in carbohydrate metabolism among teleostean species, a tendency to maintain stable glucose values during starvation is observed. Concentration of liver glycogen varies between species, but this is the main tissue for glycogen storage, and liver glycogenolysis accounts for maintenance of glycemia during early starvation (weeks). Liver gluconeogenesis from amino acids (AA) and lactate will supply glucose for later (months) stages of starvation. A different picture emerges for the representative (S. canicula) elasmobranch, with lower dependence on glucose and more exaggerated lipid metabolism.

III. Lipid mobilization Apart from the at times obvious changes in carbohydrates, many authors are convinced that lipids are mobilized first. Lipid depletion during starvation has been demonstrated in many species of fish, marine as well as freshwater, and this literature has been comprehensively reviewed by Sargent and coworkers TM. An inverse relationship exists between lipid and water contents and catabolized lipids are replaced by an equal volume of water 1~ Thus, under conditions of moderate starvation and lipid loss, body weight is maintained through the uptake of water. This phenomenon is best exemplified (and exaggerated) in migrating sockeye salmon (Oncorhynchus nerka): these animals use up their entire - substantial storage lipids in the first part of their migration, yet do not change body weight appreciably during this phase 82. Values exceeding a water content of 81% are reached in fish muscle, when only very low levels of liver lipids can be detected 1~ The strategy of unloading body storage lipids is similar in species classified as fatty and those considered non-fatty, in that the bulk of lipid is used first, before proteins are drawn upon. Lipids can be stored in liver, intestinal fat and muscle, with the quantitative importance of each tissue varying between species. Salmonids generally rely on the deposition of visceral fat, while many gadid species accumulate hepatic storage lipid, and clupeiformes contain high contents of lipids in muscle. Independent of the actual site of deposition, lipids will be mobilized from these sites during starvation.

1. Liver As mentioned above, the actual decrease of tissue lipid during fasting varies between species depending on the tissue for lipids storage and also depending on the strategy followed in mobilizing other reserves including carbohydrates. In the Atlantic cod (Gadus morhua), for instance, liver lipids decreased drastically with fasting; in fact this storage depot was exhausted even in the face of an appreciable quantity of the initial glycogen reserve remaining 15. Carp could also be included in this group of species that spare glycogen reserves to a certain degree. In carp (C.

400

L Navarro and Z Guti~rrez

carpio) after 19 days of fasting, an appreciable depletion on liver lipids occurred, while the fish seemed to protect their entire store of hepatic glycogen throughout this period ~7. In longer term experiments, where carp were fasted for 100 days, liver lipid was exhausted, while glycogen level was retained at about 1.6% (ref. 133). On the other hand, the rest of body fat reserves tends to be depleted before liver lipid is mobilized in salmonids. Nevertheless, substantial decreases in hepatic content of storage lipids can be detected in rainbow trout (O. mykiss) 115 or brown trout (Salmo trutta fario) with prolonged fasting (Table 1) 135. In sea bass (Dicentrarchus/abrax), a species which stores lipid predominantly in mesenteric fat, fasting for 22 days 65 or 130 days 27 did not provoke significant decreases in hepatic lipids. In contrast, Stirling 169 did notice decreases in liver lipids from sea bass that were fasted for 26 and 60 days. 2. Intestinal fat The major lipid storage site of many teleostean fishes is mesenteric fat. For instance, lipid accumulated by rainbow trout (O. mykiss) is deposited preferentially in perivisceral adipose tissue 16~ and it is this mesenterie fat that is first mobilized in fasted rainbow trout. In this species, during 48 days fasting, more lipid was mobilized from perivisceral depots than either liver or muscle 9~ Species with sustained accumulation of mesenteric fat usually follow the same strategy of mobilizing this lipid reserve preferentially. In Esox lucius intestinal lipids are mobilized before any change in liver lipid is apparent a6. Similarly, in the sea bass (Dicentrarchus/abrax), mesenteric lipid depots decrease by 41% after 15 days of fasting compared with control fish65, an observation supporting earlier data in the same species 198.

3. Muscle lipids In species storing lipids in muscle, mobilization of intramuscular lipid is initiated as soon as food intake ceases. An excellent case showing this point is the freshwater teleost Rhamdia hilarii. This catfish has no organized or visible adipose tissue in the abdominal cavity and during fasting intramuscular lipid (average of 5% wet weight) plays an important role for the overall energy requirement 114. In this species, after 30 days of fasting, muscle fat was reduced to one-fifth of fed values, which, considering that muscle constitutes 60-70% of fish body weight, represents an important source of energy. In species devoid of intracellular storage lipid, structural muscle lipid is usually retained until later phases of starvation when protein structures begin to be broken down. In Gadus morhua, for instance, a species that has little lipid stored in the skeletal muscle and considerable amounts stored in the liver, this hepatic reserve is used first. Muscle protein is degraded and partial destruction of structural lipids may occur at the final stages of starvation, only when other sources of energy have been nearly consumed. Muscle lipids in sea bass, which preferentially deposits lipids for storage in mesenteric fat, failed to show significant changes during fasting 6s'169, while in starving trout, muscle lipids underwent decreases after a 50-day fast 9~ but

Fasting and starvation

401

experienced no changes during a 27-day fast. Muscle lipids in carp (Cyprinus carpio) are not easily mobilized in fasting, possibly reflecting the sluggish activity pattern of this species. Working on carp, Takeuchi and colleagues 17s failed to observe variations in muscle lipids as a consequence of fasting. Identical results have been reported for fasting plaice (Pleuronectes platessa) 91. However, it should not be forgotten that the metabolic requirements of fasting species may be adjusted to the amount and availability of storage substances accumulated around the body. Fasting yellow perch (Perca flavescens), for instance, were found to be able to enter into a stage of hypometabolism, thus decreasing metabolic output and sparing storage substances 5s. Unfortunately, the potential for such metabolic 'down-regulation' has not been widely appreciated in fishes and is likely overlooked unless stringent controls are incorporated into the experimental protocols. Of course the possibility of behavioral hypometabolic regulation in natural settings should not be overlooked, and likely deserves more attention than it has garnered at this point. For instance, lower rates of oxygen consumption and activities of energy metabolism enzymes in deep-living fishes may reflect a combination of factors: reduced abundance of food; low temperature and water oxygen concentration; and deprived light intensity, which may account for reduced locomotory activity19s. At any rate, the potential for hypometabolic regulation is well described for other vertebrates (hibernators, estivators) and this is certainly an area of research where fish could add another useful model to the analytical arsenal. In fishes, red muscle contains more lipids than white muscle; for example, in Atlantic salmon, white muscle has a lipid content of 2%, while red muscle has about 15% (ref. 108). However, dark muscle lipid is not easily mobilized, which may be related to the special properties of these types of tissues. Red muscle is used for sustained swimming and for activities such as maintaining body position against currents, whereas white muscle is used intermittently, for sudden movements in case of pursuit or escape. Some diminution in locomotory capacity during food deprivation may be tolerable 195. Although generally the muscle of elasmobranchs tends to contain more lipid than that of teleostean fishes, the specific composition may indicate that the bulk of this lipid represents structural rather than storage material and therefore is not easily mobilized. In agreement with this hypothesis, 82 days of starvation did not provoke significant changes on muscle lipids of S. canicula 47.

4. Lipid classes During starvation-induced breakdown of lipids, different phases can be distinguished. The most accessible lipid store appears to be triacylglycerols (TAG, triglycerides), well exemplified in the European eel (Anguilla anguilla). In this species, triglycerides were the main energetic substrate during 95 days fasting96. Other (structural) lipids, in contrast, are generally entirely spared or used only during the later phases of food deprivation. As a result of focused breakdown of triglycerides, the specific fatty acid composition changes in the course of starvation. Since myristate (14:0), palmitoeate (16: 1), and oleate (18: 1) are mobilized prefer-

402

L Navarro and 1. Guti~rrez

entially, their abundance in the remaining triglyceride declines almost continuously in the liver of A. anguilla in the course of starvation 35. As a rule, triglycerides are mobilized before phospholipids during starvation 161, reflecting the duality of storage versus structural lipid. As delineated for the European eel liver above, there also is an apparent selectivity in the fatty acids mobilized. In rainbow trout, saturated fatty acids were mobilized in preference to other types of fatty acids from perivisceral fat deposits. Similarly, the bulk of fatty acids depleted from liver and muscle lipids were 16:1, 18:1 and 20:1 (ref. 90). A marked decrease in the 18: 1 content of body and liver of starved rainbow trout was reported 161. In salmon, spawning depletion resulted in utilization of long-chain mono-unsaturated acids such as 20:1 and 22:1, while those of shorter chains and polyunsaturated acids where consumed later s9. Levels of 20" 5 and 22' 6 were maintained or proportionally increased in Fugu vermicularis porphyreus during early fasting72. In stock fish (Merluccius capensis) a relative increase in the degree of unsaturation is noted during starvation 19~ It should be kept in mind that the proportion of unsaturated fatty acids as well as the degree of unsaturation exert pronounced effects on membrane fluidity. Therefore, it is likely that the retention of certain fatty acids at the expense of others is guided not only by the necessity to requisition carbons for oxidation but also by the need to maintain structural integrity and fluidity of the membrane. As a result it can be expected that the mobilization of unsaturated fatty acids from lipids will differ in warm-water- and cold-water-adapted species. Obviously, there is a natural limit to the amount of structural lipids that must be retained. Below this threshold, essential membrane functions will be compromised and the survival of the fish is in question 1~ In Perca flavescens, this limit seems to be reached when only 2.2% of overall lipid is left. Below this critical value the animals die 137.

4.1. Freefatty acids The fasting-induced pattern of changes in plasma free fatty acids (FFAs) varies between species, but clearly does not lead to the rapid and marked increase of FFA familiar in starving mammals 13. Researchers noted decreases of FFA in 30and 90-day starved oyster toadfish (Opsanus tau) 176while rainbow trout experienced increases 166 or no change under the same conditions. In the European eel (A. anguiUa) no changes in plasma FFA were noticed during the initial 95 days of fast, followed by a marked rise thereafter 96. A noticeable increase with 3 or 5 days fasting is found in Limanda limanda 49. In sea bass (D. labrax) 40 days of starvation caused an increase in plasma FFA concentration of about 65% along with a 3- to 7-fold increase in plasma glycerol concentration 198 . In fasting catfish (R. hilarii) plasma FFA increased two-fold during the first 30 days 114. However, initially FFA levels do not increase, and authors suggested that all FFA produced was first utilized locally, and it is not until later stages of starvation that a spillover of fatty acids into the plasma compartment is noticed. It is well known that in fasted teleosts, in stark contrast to mammals, the production and utilization of ketone bodies does not play an important role 15'147'198.

Fasting and starvation

403

In fact, it is under debate whether ketone body metabolism forms an integral part of fish intermediary metabolism at any time. First, some studies showed that 3-hydroxybutyrate dehydrogenase (EC 1.1.1.28), a key enzyme in ketone body production, was lacking in the livers of teleost fish 9'197'198. Nevertheless, LeBlaneh and Ballantyne ~~ have demonstrated the presence of that enzyme in tissues of some freshwater species such as goldfish (Carassius auratus), brown bullhead (Ictalurus nebulosus), pike (Esox lucius) and crappie (Pomoxis nigromaculatus). 3-Hydroxybutyrate dehydrogenase activity has also been found in marine teleost species: alewife (Alosa pseudoharengus), smelt (Osmerus mordax), and mummichog (Fundulus heteroclitus). The levels of the enzyme in freshwater fishes are highest in the liver and kidney, tissues known to be ketogenic in other vertebrates 9, which could suggest a similar role of ketone bodies in fishes. But the levels of this key enzyme in ketone body formation in the marine species studied did not display the same tissue pattern, since the highest levels in the alewife were found in the brain, while the heart of the smelt had the highest activity. Second, under normal physiological conditions the concentrations of ketone bodies in tissues or plasma and the rate of utilization are very low48 suggesting the idea of a minor role in teleost metabolism. No data exist showing that ketone bodies make a substantial contribution to the energetic requirements of teleost fishes during starvation. On the other hand, the presence of the enzyme 3-hydroxybutyrate dehydrogenase in elasmobranchs was observed in earlier studies 12s,198. The levels of this enzyme and plasma levels of ketone bodies are both higher than in teleosts 47,198 and isolated hepatocytes will readily use and convert added ketone bodies 119. These results indirectly support an earlier contention that plasma transport of fatty acids may be limited in elasmobranchs and other organisms lacking carder proteins such albumin. Thus, ketone bodies may provide a more soluble lipid transport form 4,5,1~ Nevertheless, the role of ketone bodies in energy production and thus their contribution to energy supply during fasting is doubtful. Contrasting with increments of plasma ketone bodies observed in fasted mammals, plasma levels of these metabolites were not significantly altered in starved skate (Raja clavata) 197, but a large increase was observed at 36 days fasting in dogfish (Scyliorhinus canicula)47.

5. Influence of starvation on lipolysis and lipogenesis Generally, information on lipid mobilization in lower vertebrates is scant. Lipolytic activity has been found in salmonid adipose tissue, liver and red (dark) muscle 164. In trout adipose tissue, the triglyceride lipase is subject to covalent modification through phosphorylation, with activity increasing as the enzyme is phosphorylated (K. Michelsen, J. Harmon and M. Sheridan, unpublished results). Activity of an acid lipase of lysosomal origin was found in rainbow trout dark muscle 14 and adipose tissue 16s. Bilinski and coworkers 14 suggested that lysosomal lipase from rainbow trout lateral line muscle serves mainly in the mobilization of intracellular lipids for internal use. Neutral lipase from rainbow trout adipose tissue was characterized 16s and it is this enzyme which seems to play an important role in lipid mobilization in adipose tissue and thus to supplying fatty acids to peripheral tissues 164.

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L Navarro and J. Gutir

At the same time, as the lipolytic machinery is activated during starvation, the activity of the lipogenic pathway seems to be curtailed, at least during the later stages of food deprivation. In coho salmon (O. kisutch), for instance, the activities of several lipogenic enzymes in liver remain unchanged for the first two days of starvation, while significant decreases were noticeable after 23 days 1~ Usually, cytosolic enzymes supplying reducing power in the form of NADPH are considered 'lipogenic'. These include two enzymes of the pentose phosphate shunt, namely glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, as well as malic enzyme (malate dehydrogenase-decarboxylating) and isocitrate dehydrogenase. The total NADPH available for FA synthesis in liver is lower in American eels (Anguilla rostrata) fasted 4-6 months than in fed or freshly caught eels 2. In rainbow trout liver the activities of the NADP-linked dehydrogenases were substantially depressed after four weeks of food deprivation s . As other experiments show, the activities of these NADP-generating enzymes, albeit not always all of them, are governed by the dietary status of fishes 161. Abundance of fatty material, as in the case of elevated concentrations of dietary lipid, decreases the activity of G6PDH and ME in rainbow trout liver 1~ Conversely, feeding a diet high in carbohydrates to channel catfish (lctalurus punctatus) results in increased hepatic titers of all four NADPH-generating enzymes. A more direct measure of lipogenic potential would be the analysis of ATPcitrate lyase and particularly of acetyl-coenzyme A carboxylase, the committed step in the synthesis of fatty acids. In the case of ATP-citrate lyase, activity of the enzyme decreases significantly in carp (Cyprinus carpio) starved for two months (H. Segner and R. B6hm, unpublished results). Unfortunately, the carboxylase is not well described in piscine systems, and none of the existing studies 6~ analyzed carboxylase activity and phosphorylation status during different physiological stages of the experimental animals. In the rat liver, the enzyme is under stringent control though three different mechanism, all of which are known to be influenced by diet and starvation: (1) allosteric effectors such as citrate; (2) covalent modification through phosphorylation and dephosphorylation; and (3) enzyme concentration. Liver slices from European eels fasted for 7-39 weeks incorporated 14C-acetate into FA at lower rates (6- to 20-fold) than those found in fed eels 1. After 95 weeks starvation, eel liver was still able of incorporate ~4C-acetate into FA, albeit at a lower rate 1. Esox lucius starved for 2 months, converted ~4C-acetate preferentially into unesterified fatty acids and phospholipids 94. It appears that during starvation, emphasis is on the synthesis of structural lipids, while carbon will be preferentially funnelled into storage lipids (triglycerides) after re-feeding. In conclusion, lipids are stored as triglycerides in different tissues. But independently of the tissue location, lipid mobilization occurs during fasting, simultaneously or after carbohydrate mobilization, but always before protein degradation. In teleosts, FFA are the main products of lipid mobilization, while in elasmobranchs ketone bodies take their position. In comparison with carbohydrates, lipids represent an important source of energy due to the high depots accumulated and their high caloric value (Table 3).

Fasting and starvation

405 TABLE 3

Initial energy content in total liver and muscle, relative change and rate of energy loss between different periods of fasting in carp

Relative change (%) Day 1-8

Day 8-19

1.12 0.84 0.47 2.42

-57.0 - 18.6 -12.4 - 35.2

- 18.1 - 13.8 -29.8 - 19.2

0.73 21.7 2.0 24.4

+ 1.7 -19.9 + 23.4 - 15.8

-6.5 -22.4 - 17.2 - 21.2

Initial values (kcal) Liver Glycogen Protein

Lipids Total Muscle Glycogen Protein Lipids Total

(Cyprinus carpio)

Rate of energy loss Liver (cal/h) Muscle (cal/h)

5.07 22.9

1.1 16.5

Data are given as means for 8 fish in each group. From Blasco et al. 17.

IV. Protein mobilization The sequence of mobilization of the different sources of energy during the course of starvation seems to be very similar, in general terms, in the different species of fish. Usually, protein reserves are spared at the beginning of fast, thus proteolysis occurs only when more readily available energy reserves have been widely consumed such as liver glycogen and lipid stores as it does in higher vertebrates. Then, as protein is utilized, water moves in to take its place. Fish present special adaptations for protein mobilization: high level of proteolytic enzymes in muscle, coupled with the generalized ability to excrete excess nitrogen as ammonia or ammonium ion. Only very few species go through the metabolic expense of synthesizing - at appreciable metabolic cost - and excreting urea 123. The actual contribution protein makes to meeting the overall energy requirements during fasting depends largely on the species 187. It is trivial to note that the impact of starvation is felt sooner in active than in sluggish fish. The activity of catheptic enzymes is greater and more rapid autolysis is noticed in the muscle of such species as mackerel (Scomberscombrus) as compared with carp (Cyprinuscarpio) or cod (Gadus morhua). Different organs deplete endogenous protein to differing extents during starvation. As detailed below, fish tend to mobilize more protein from white muscle than from dark muscle. While increases in proteolytic activity with starvation have been found in liver, kidney, spleen, and red muscle, not surprisingly, it is the white muscle that experiences the largest increases in proteolytic activity. In the initial phases of starvation in carp (Cyprinus carpio), proteolytic capacity is lowest in muscle, and increasingly larger in spleen, liver, kidney and highest in the intestine. This order in the series is most likely a reflection of normal tissue

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turnover and synthetic demand of the tissue. As the period of food deprivation lengthens, this order is almost reversed. Now intestine has the lowest proteolytic activity while activity increases in the order: liver, kidney, spleen and white muscle. Rates of protein turnover must be matched to the energy demands; allowing for the dietary constraints that mark starvation, protein synthesis rates are adjusted to the new requirements. Again, the white muscle appears to be the fish tissue that is most sensitive to fasting and this tissue responds with a reduction in the rate of protein synthesis after food deprivation 7~'1~ Protein synthesis rates of other tissues such as the liver and gills have been found to be little affected by starvation 81,1s4,1~.

1. Liverproteins In general, fasting exerts only limited effects on hepatic protein suggesting that the bulk of this protein has vital functions 31,126. Observed increases in percentage of liver protein is usually correlated with decreases in liver weight due to the mobilization of other reserves in the course of starvation. These increases are observed in carp 17, eel (,4. rostrata) 124 or brown trout (Salmo trutta fario) 13s after different periods of fasting. Absolute values of the total quantity of the store in the entire organ is a more meaningful physiological measurement, and analysis of data in percentage can lead to erroneous interpretations. Thus, actually, the total quantity of liver protein declines with fasting 17,13s. Fasting exerts little influence on protein synthesis rate in liver, but degradation increases under fasting conditions. Nevertheless, selective destruction of proteins may occur. Effects of starvation on tissue enzyme activities are variable 3~ The activity of gluconeogenic enzymes increases in numerous fasted teleost species 171. Increases in the liver activities of alanine and aspartate aminotransferases has been reported in many teleost species suggesting a stimulation of gluconeogenic flux from amino acids 59.171. These changes are accompanied by increased activities of liver gluconeogenic enzymes including phosphoenolpyruvate carboxykinase (PEPCK) 124,127,171. Another analytical aspect that has to be taken into account is, again, the manner in which the data are expressed and interpreted. Enzyme activity changes expressed in terms of tissue weight are not always adequate, because the liver mass relative to body size declines with fasting in many fish species, the total potential of the liver for some metabolic pathways would be reduced. This is the case in the perch (Perca flavescens) which shows a decrease in overall metabolism including the liver gluconeogenic pathway. It is clear that, particularly with respect to the liver, the use of tissue DNA content as a denominator for various parameters can result in a different interpretation of the fasting response ss.

2. Muscle proteins Fish are in general well adapted to withstand long periods of depletion and this is probably because the muscle is rich in proteolytic enzymes which can mobilize the body tissues for fuel when required 1~

Fasting and starvation

407

The main effects of prolonged starvation are increases in muscle proteolysis activity and mobilization of amino acids derived from muscle proteins for utilization by more 'vital tissues'. During periods of extended starvation, catabolism of muscle protein is initiated and the resultant amino acids are the major sources of energy for the fish 1~ Muscle protein mobilization is produced after glycogen mobilization when glycemia and lactate are in decline. In some species, including Gadus morhua, muscle protein breakdown increases in the early stages of starvation and maximum values were found 3-4 weeks into a fast. Protein content starts to decline after liver lipid has fallen below a critical value. Muscle protein is the most important body energy store, being used mostly during prolonged starvation 31,187. During spawning migration of sockeye and chum salmon, catabolism of muscle protein is preceded by 3- to 7-fold increases in the muscular activities of the proteolytic enzymes (cathepsin D and carboxypeptidase A) 118. In common with mammals, when fish muscle protein is degraded, contractile and soluble proteins are removed preferentially, while connective tissue proteins are used to a lesser extent. The consequence is that the connective fibers rich in collagen are almost entirely spared from degradation. Thus, the proportions of glycine, proline and hydroxyproline (the key amino acids in collagen) in muscle protein increases during fasting. The changes in protein concentration and composition during depletion may be distinctly different in red and white locomotory muscles 1~ For example, in the flounder (Pleuronectes platessa) white muscle loses a significant portion of myofibrillar protein during starvation, but this part of muscle protein was conserved in red muscle. Only mitochondrial and sarcoplasmic proteins were mobilized from red muscle in this species. The overall balance between synthesis and degradation is altered with fasting. Some regulatory adjustments in protein metabolism are needed to bring about these changes in muscle protein concentration and composition. First, rate of protein turnover needs to be adapted to the new energy demands. Studies on protein synthesis in rainbow trout 1~ and barred sand bass ~~ have shown that white muscle protein synthesis rate declines to a plateau after 10-14 days without food. Second, and very important, the turnover of specific proteins in each muscle type must be regulated closely to ensure conservation of physiological capacity as we have also mentioned for liver protein. In this context, studies with barred sand bass (Paralabrax nebulifer) by Lowery and Somero 1~ are illustrative. Also, white muscle myofibrillar protein synthesis rate was more affected by starvation than was the sarcoplasmic protein synthesis rate. Actin, a major myofibrillar component (approximately 10% of total white muscle protein in fish) was very sensitive to fasting (Table 4). That leaves the question of how the synthesis of vital enzymes or other proteins is differentially regulated? For instance, the relative amounts of the different glycolytic enzymes are relatively well conserved during periods of starvation, despite very large decreases in the total activities of the enzymes 11s. The conservation of relative activities could be achieved by different mechanisms. It seems that, at least in barred sand bass, differential effects on synthetic rates may exist. Muscle enzymes present at high concentra-

408

I. Navarro and :I. Guti~rrez

TABLE 4 Changes in protein and actin concentrations during fasting in Pamlabrax nebulifer Days fasting rotein 0 5 10 16 23 Actin 0 5 10 16 23

White muscle . protein (mg/g) 192 174 177 135 130

4- 4 4- 9 4- 24 4. 5" 4. 3"

18.5 4. 15.24. 15.6 4. 12.84. 13.0 4.

1.0 0.5" 0.3" 0.2' 0.4"

.

.

. . % of fed

Red muscle protein (my/g)

% of fed

100 91 92 71 68

247 264 263 222 209

100 107 107 90 85

100 82 84 69 70

4- 12 4- 12 4. 12 4- 17 4. 11"

13.3 4. 12.9 4. 13.5 4. 12.9 4. 12.7 4.

0.3 0.4 0.7 0.5 0.4

100 97 102 97 95

Values are means 4. SEM of four observations. "Significantly different from fed values (p < 0.025). Adapted from Lowery and Somero 1~

tions, low specific activities and possessing long half-fives, show a diminution in synthesis rate with fasting much more pronounced than that found in enzymes present in lower concentrations and higher specific activities. In this way, the protein turnover may be regulated such that the relative proportions of specific proteins or enzymes are conserved. Unfortunately, methods of determining specific protein degradation rates present many problems, the most serious of which is the re-incorporation of radiolabeled amino acids 196. The half-life of a protein, consequence of both degradation and synthesis rates, is likely changed during starvation. 3. Plasma protein and amino acids

There is a clear tendency for plasma proteins to decrease in fasting fish. In both brown trout (S. trutta fario) and carp (C. carpio) the electrophoretic pattern of plasma proteins is substantially altered compared with control fish after 30 days or 6 months fasting, respectively 1~ Plasma protein decreased in fasted mature carp after five days of fasting tT. In addition, the overall concentration of plasma protein is curtailed. In carp, for instance, a 6-month fast decreases plasma concentration from 3.9 to 2.8% (ref. 106). Apparently, albumins are the first plasma proteins to undergo reduction in concentration, followed by the alpha and beta globulins. Gamma globulins, in contrast, were not utilized; in fact, since the overall protein concentration decreases, the percentage of plasma proteins contributed by gamma globulins is increased. A similar picture was observed in Gadus morhua during starvation, while an extreme situation is noticed in sockeye salmon during the spawning migration. The anorexic females of this species build up impressive levels of vitellogenin in their plasma in the course of migration, but at the same time

Fastingand starvation

409

deplete their plasma store of albumin in its entirety. Some authors have suggested using albumin: globulin ratio as a nutritional indicator because of this sharp decline in albumin during starvation 1~ But in any case, the plasma concentration of different free amino acids is of greater interest than plasma protein levels since it reflects results of whole body protein turnover. The composition of the free circulating amino acid (AA) pool changes markedly during fasting in all the species studied. In rainbow trout (O. mykiss) even a relatively short (24 or 48 h) fasting period lead to a decrease in plasma essential amino acids (EAA) but not in the non-essential AA ls6. The significant positive correlation between the levels of plasma EAA and their respective concentrations in the diet, as well as the lack of such a relationship for NEAA appears to be a general phenomenon 16,32,148. The decline in EAA observed in plasma of carp or trout at the onset of fasting may respond to the uptake of these AA by tissues without a new contribution from diet, while NEAA levels are metabolized and altered to a greater extent and can undergo extensive interconversions. Of all the presumed essential AA, histidine dropped most precipitously in fasted migrating salmon 118. It appears that histidine is utilized by the fish at high rates in the early phases of migration, but the exact metabolic role of this amino acid is still not known. In carp (Cyprinus carpio), branched-chain AAs (leucine, isoleucine and valine) were affected most, decreasing by 44% after five days without food (Fig. 1) 16. This decline suggests an increase of oxidation of these amino acids during fasting 16,199. Nevertheless, after 19 days fasting a compensatory increase in plasma AA was noticed in this species. This increase is largely due to increases in branched-chain AAs. Of the non-essential AAs, alanine, glutamate and glutamine levels are augmented with the two five-carbon amino acids experiencing the largest 2500

2000

~L "" 1500 4o 4r I000 o u

500

O

I 2

I 5

1 8

9 19

9 50

I R

I

Day

Fig. 1. Variations in plasma essential amino acids (EAA) and non-essential amino acids (NEAA) levels at different periods of starvation and refeeding (]2 days) in Cyprinus carpio. Control (filled circles), starved (open circles), R = refed (for 12 days). Differences were determined by Duncan's multiple r a n g e test. * Different from control (p <0.05); ** different from control (p <0.01); *** different from preceding point (p <0.05); **** different from preceding point (p <0.01). From ref. 16, with permission.

410

L Navarro and J. Guti~rrez

increases (two-fold over control). The increase in plasma amino acids observed in fasting carp after 19 days is a consequence of muscle proteolysis and thus, it may be expected that generalized proteolysis of muscle proteins will provide a balanced cross-section of all the AA. However, this is clearly not the case. In starving dogfish (Squalus acanthias) alanine was selectively released into the plasma draining the white muscle t~ Similarly, Mommsen and coworkers tls noticed that alanine was the major AA released from white muscle of migrating sockeye salmon for transport of amino acid derived carbon to other tissues. It therefore appears that the main fate of AAs is transdeamination in situ to supply carbon precursors for gluconeogenesis mainly as alanine and possibly also as glutamate (which represent 46% of NEAA) 16. Both alanine and glutamate are excellent gluconeogenic substrates in most teleostean fishes 129,157,171. While this has been shown directly in isolated fish hepatocytes numerous times, this notion is further supported by the marked depletion of the glutamate/glutamine pool at 50 days fasting in carp, coinciding with tight maintenance of glycemia at this time t6. Furthermore, other in vitro studies have demonstrated that fasting induced an increase in uptake of L-alanine by trout (Salmo trutta) hepatocytes that showed an inverse relationship with L-alanine plasma levels2~ Thus, all the above studies support the metabolic model that glutamate and mainly alanine form fundamental precursors for gluconeogenic pathway in fish tissues under fasting conditions. In summary, during starvation, proteins are mobilized at the final stage. Different tissues can contribute to the available pool of AAs, however, skeletal muscle represents the largest store of protein and, as consequence of the percent of muscle weight (with respect to body weight) it constitutes the main energy source during prolonged starvation (Table 3). Blood AA from tissue proteolysis will be transported to gluconeogenic tissues to maintain the necessary glycemic level.

V. Endocrine regulation 1. Insulin Although the role of insulin in mammals under fasting conditions has been widely analyzed, comparative studies on the regulation of fish metabolism by insulin during food deprivation are relatively sparse. Nevertheless, of all the pancreatic hormones, insulin has received the most attention in fish studies t+9. Initial studies on the role of insulin in fish have concentrated on the direct effects of exogenous insulin on blood metabolites and tissue energy reserves. The regulatory function of insulin in fish during different nutritional conditions has been more clearly elucidated when measurements of circulating levels became available. Initial data on plasma insulin levels were successfully obtained by some authors in different teleost species 141.176. These difficulties in measuring fish insulin levels can be explained by the fact that the mammalian radioimmunoassay (RIA) components proved to be useless tools in measuring fish insulins except in a few isolated cases. Cross-reactivities between many fish insulins and antibodies to mammalian insulins are very weak, in spite of

Fasting and starvation

411

the fact that piscine insulins are structurally remarkably close to other vertebrate insulins 42. Thus, the development of RIAs with fish components or homologous RIAs has been an essential step towards understanding the unique role of insulin during fasting in fish. Antibodies raised against insulins isolated from species such as scorpion fish, bonito, cod and anglerfish, together with their respective radiolabeled equivalents, can be successfully used in RIAs to accurately determine insulin levels of other fish species 63,141,178. Nevertheless, the development of a homologous RIA for coho salmon (Oncorhynchus Idsutch) has been described lsl. Clearly such homologous systems are to be favored, since they are less prone to erroneous data and require less effort to verify concentration estimates. The general actions of insulin in fish seem to be similar to other vertebrates, in that insulin is a hypoglycemie hormone, lipogenie and promotes protein synthesis 122,132. Just as in mammals, the circulating levels of insulin diminish during fasting in fish. Table 5 shows the changes in plasma insulin levels in starved elasmobranch and different teleost species. An interesting phenomenon has been described for the eyclostomata. Working on the Atlantic hagfish (Myxine glutinosa), Emdin 41 found that plasma insulin levels declined within 1 month of starvation from 12 to 6 ng/ml. Concomitantly, a decline of blood glucose and an almost complete exhaustion of glycogen reserves in liver and skeletal muscle were observed. Surprisingly, in this species, insulin lacked metabolic effects on the liver whereas the hormone had the expected~ but rather small, effect in muscle, where it stimulates the synthesis of glycogen, protein and lipids. It appears that the physiological role of insulin regulation of skeletal muscle metabolism is similar to that in mammals while its effects in liver are weak or absent. In fact, Emdin 41 considered liver of little significance in fasting or other catabolic situations in this cyclostome. In contrast, more recently, studies on hepatocytes isolated from this species of hagfish s7 reveal unusual effects of insulin in liver carbohydrate metabolism, such as increases in gluconeogenesis and total glucose production. Since it seems that glucagon is absent in the hagfish islet 42 an ancient catabolic function of this hormone may still be present in this primitive group of Agnathans. Similar contradictory insulin effects have also been described in two species of teleosts 53,s4. Thus, it may be necessary to design appropriate fasting studies since agnathan tissue metabolism and its regulation have been poorly studied, and important evolutionary aspects could be elucidated from these studies. Little work has been done on selachian physiology. The studies on the effects of insulin in the dogfish shark (Squalus acanthias) suggest that the dogfish uses ketone bodies as a primary fuel at rest. Glucose would apparently be synthesized by gluconeogenesis to maintain muscle glycogen reserves. These carbohydrate stores would be used for anaerobic glycolysis in situations where vigorous swimming is needed. This hypothesis agrees with the opportunistic food habits of this carnivorous fish altering between consumption of large amounts of food and relatively long periods (about two weeks) of fasting 159. Artificial food deprivation in an elasmobranch (Scyliorhinus canicula) leads to plasma insulin decreases similarly to what is described in the more evolved teleosts. Plasma insulin decrease progressively after 15 and 36 days of food deprivation

412

L Navarroand I. Guti~rrez

with a maintenance of low levels with prolonged fasting62. Plasma glucose levels decreased initially, and recovered during the period of declining insulin titers. It is interesting that the glucose decrease was paralleled by changes in plasma amino acid levels at 36 days of fasting. A similar decrease in total plasma amino acid levels has been also seen in other species of chondrichthyes such as Squalus acanthias 1~ suggesting an increase of gluconeogenic processes would be favored by the diminished plasma insulin levels. More studies are needed to identify the tissue destination and metabolic pathway of the supposed de novo synthesized glucose and possible regulation by insulin and other hormones. Comparatively more studies have been performed in teleosts (Table 5). The general picture emerging from these studies is a clear diminution of insulin plasma levels in fish after just a few days of fasting. Plasma insulin decreased during fasting in goldfish (Carassius auratus) 141. Serum insulin levels of fed animals were nearly twice those found in 5-day fasted goldfish using codfish insulin as standard and guinea pig anti-cod insulin serum and 12sI-labeled cod insulin as other components. The same authors measured insulin secretion in islet incubations of oyster toadfish (Opsanus tau) obtaining values very similar to those determined earlier in the same species by Tashima and Cahil1176 using scombroid (bonito) insulin standards and guinea pig anti-bovine insulin antibody. In fact, these were the first studies describing a decline of insulin levels caused by fasting in teleosts. Subsequently, Thorpe and Ince 177 observed a very strong decline in plasma insulin in the rainbow trout fasted for 7 days, a diminution of 76% in relation to fed animals, and in only 4 days fasted cod a decline of 61% was observed. Furthermore, upon refeeding, the animals showed a clear recovery of the insulin levels after one week of food administration. Plisetskaya and coworkers m developed a homologous radioimmunoassay to determine plasma insulin in salmonids. These authors measured significant decreases (in excess of 50%) in insulin levels in blood of coho salmon (O. kisutch) after one or two weeks of fasting. The observed decreases are entirely reversed after refeeding. Long-term experiments of food deprivation in salmonids always induce a decline in insulin plasma levels. In rainbow trout, a 6-week fast produced a decline in insulin plasma levels. This fact, together with the relative changes in other pancreatic hormones such glucagon and GLP, enhanced the gluconeogenic pathways in the liver, that are activated after this period of fasting 126. This fact is also in concordance with the observations that, after injection of insulin in starved rainbow trout, glucose synthesis from alanine was drastically depressed, when compared with control fish28. The role of insulin in the regulation of gluconeogenic processes is corroborated in salmonids by in vitro experiments. In isolated rainbow trout hepatocytes, insulin curtailed the rate of gluconeogenesis from lactate 146. This gluconeogenic inhibition by insulin is accompanied by activation of pyruvate ldnase activity. However, this pattern does not seem to be universal since in isolated hepatocytes of sea raven (Hemitripterus americanus) porcine and fish insulins, contrary to expectations, increased the flux rate of amino acids into glucose s2. More recent studies show that this insulin-stimulated increase in gluconeogenesis in sea raven may be at least partially related to an inhibition of the fructose-6-phosphatase activity ratio of 6-

Fasting and starvation

413 TABLE 5

Plasma insulin levels of fed or fasted elasmobranch and teleostean fishes State

Insulin (ng/mi 4- SE)

Scyliorhinus canicula 62

Fasted Fasted Fasted Fasted

1d 15 d 36 d 82 d

Carassius auratus 140

Fed Fasted

5d

Cyprinus carpio 17

Fasted Fasted Fasted Refed

1d 5d 50 d 12 d

Cyprinus carpio 18

Fasted Fasted

1d 16 d

10.25 4- 0.71 8.28 4- 0.96

Gadus morhua 176

Fed Fasted

4d

8.43 4- 0.90 3.28 4- 0.20

Gadus morhua 73

Fed Fasted Fasted Fed Fasted Fasted

7d 21 d

Dicentrarchus labrax

Fasted Fasted

I d 22 d

Oncorhynchus mykiss 176 (Salmo gairdneri) Oncorhynchus mykiss 1-/6 (Salmo gairdneri) Oncorhynchus myk/ss 12s

Fed Fasted Fed Fasted Fed Fasted Fed Fasted

Gadus morhua -/3

Oncorhynchus kisutch 150 Oncorhynchus kisutch 150 Salmo trutta (summer) TM

Salmo trutta (winter) 134

7d 21 d

7d 7d 42 d 42 d

0.77 0.64 0.25 0.30

3.50 4- 0.30 1.90 4- 0.50 5.21 2.27 2.23 5.93

3.30 1.60 0.20 4.90 1.90 0.50

4- 0.68 4- 0.27 + 0.29 4- 0.26

4- 0.73 4- 0.44 4- 0.13 4- 0.82 4- 0.82 4- 0.12

10.91 + 0.25 8.68 4- 0.22 5.65 2.20 6.80 1.60 12.10 2.00 13.20 3.00

4- 0.30 4- 0.30 4- 0.60 4- 0.30 4- 1.10 4- 0.10 4- 1.50 4- 0.10 4- 0.80 4- 0.20 4- 0.90 4- 0.10 4- 0.27 4- 0.21 4- 0.11 4- 0.14 4- 0.32 4- 0.20 4- 0.18 4- 0.23 4- 0.23 4- 0.23

Fed Fasted Fed Fasted

21 d

4.50 1.40 4.30 0.90

Fasted Fasted Fasted Fasted Refed Fasted Fasted Fasted Fasted Refed

1d 3d 30 d 50 d 8d 1d 8d 30 d 50 d 8d

4.85 3.85 3.12 1.62 3.69 5.48 2.53 1.29 1.17 4.94

7d

4- 0.04 4- 0.04 4- 0.03 4- 0.04

414

L Navarro and J. Gutic~rrez

phosphofructo-l-kinase (PFK-1 EC 2.7.1.11). Phosphoenol-pyruvate carboxykinase and pyruvate kinase (PK, EC 2.7.1.40) were not affected by insulin. PK is the most probable point for critical hormonal regulation in the gluconeogenic process in mammals. But caution must be used in interpreting these results as the relative importance of the various enzymes in the regulation of these pathways in fish is not yet clearly understood. A variable pattern is revealed in American eel (Anguilla rostrata) hepatocytes, where insulin induced either increases or decreases in alanine gluconeogenesis depending on the time of the year s3. Other fasting experiments, with more dynamic measurements of insulin levels, have been performed in teleosts. In juveniles of Pyrenean brown trout, plasma insulin levels showed a progressive decrease (between 20 and 30%) after short-term fasting (3, 8 or 15 days) or after a more prolonged starvation (a decrease of 67% after 50 days). The decline of insulin titers was more rapid in summer than in winter 135. This diminution of insulin levels was correlated with the mobilization of tissue energy reserves. Thus low levels of insulin could favor a degradation of liver glycogen during short-term fasting, together with the action of other hormones. Knowing the anabolic function of insulin in protein and lipid metabolism in fish 116.1~, a decline in plasma insulin may contribute to the decrease in fat and protein reserves under fasting conditions in this species. A progressive decline of insulin plasma levels was also associated with the mobilization of energy reserves in sea bass, although, in that case, the role of glucagon seems to be more relevant 65. Reduced plasma insulin levels following fasting are common in other species of teleosts, including cod 173'177 and carp 17. In carp, insulin plasma levels always decrease with fasting experiments, although the response of glucagon to fasting is not so uniform in this species of teleost. Blasco and coworkers 17 found a two-fold decrease in insulin with respect to control values, after 8 days of fasting. This rapid decrease correlated nicely with the early mobilization of protein observed in that study. In carp, as well as in trout fasting experiments, the levels of insulin returned to control values after short periods (about 10 days) of refeeding. This fact suggests a rapid and high adaptability of fish to the recovery from fasting periods 76,135. Unfortunately, the above-mentioned changes in insulin concentration and their correlation with metabolic output of fishes, are even less clear-cut under natural, rather than experimentally imposed, periods of food deprivation. For example, during the (anorexic) spawning migration of the pink salmon (Oncorhynchus gorbusha), plasma levels of insulin remained stable or, if any changes were noted, plasma levels were elevated. The elevation of plasma insulin, opposed to the expected decline, in this unique situation may preserve the energy stores instead of an early mobilization, thus enabling the fish to preserve sufficient metabolic reserves for final maturation of gonadal products and the exhausting spawning upon arrival at the spawning grounds ~2e. Another explanation for the elevated insulin titers in pre-spawning non-feeding salmon or lamprey is a possible role of insulin as a gonadotropic hormone similar to the one described for mammals 155. To ultimately make the connection between changing insulin titers in plasma during starvation and the altered metabolic status of fishes, it has become important to examine the interactions of insulin with its receptor in fish tissues. While the

Fastingand starvation

415

first studies on insulin receptor in fishes elegantly, if not conclusively, dealt with evolutionary aspects of both the peptide and its receptor 1~176 more recent studies have focused on functional receptor characterization and the varied influence of different physiological states on insulin receptors in fish tissues. Specific insulin binding to fish liver or muscle 66,67 appears to be lower than that reported in mammals and birds 92,13~ In rainbow trout, a fast of 40 days caused a decline in plasma insulin levels and an increase in the binding capacity (specific number sites) in liver membranes, a situation reminiscent of increasing binding capacity for insulin in mammals and birds after a short fast 3'167. However, at the same time, a decrease in the binding affinity in relation to control fish was noted, and as a result, specific insulin binding remained unchanged. This could be explained as a way to reduce the anabolic fluxes in the liver. Refeeding of fasted fish for 15 days restored plasma insulin levels and increased binding affinity with the presumed receptors attaining a higher specific activity than in control fish. Nevertheless during short-term fasting experiments (a few days) the number of binding sites seems to be a major regulating factor: after 3-6 days of fasting no changes in insulin titers were observed, but specific binding of insulin to the liver plasma membranes increased from 5.0 to 9.3% (ref. 66). We conclude that at the onset of fasting, insulin binding increases in fish liver, possibly as a compensatory mechanism, until all the metabolic fluxes are shifted towards catabolic and gluconeogenic processes, while a prolonged fast leads to a stabilization of the binding of insulin to its receptors. Since hepatic metabolism is strictly linked to the ratio of insulin to glucagon, the analysis of binding of other hormones such glucagon to the liver will be a prerequisite to understand metabolic regulation in the liver. Insulin binding to muscle in fish and its regulation in response to fasting seem to vary between species 14~ It is interesting that omnivorous species such as carp (C. carpio) appear to have more numerous insulin receptors, with higher tyrosine kinase activity than carnivorous species (trout (Salmo trutta), sea bream (Sparus aurata)). The most striking difference was observed between carp and trout. In fed fish, the number of insulin receptors was significantly higher in carp than in trout (Fig. 2). Fifteen days of fasting resulted in a decrease to 50% of the respective control values in insulin binding to semipurified receptors of carp and trout muscle. This observation is supported by the concomitant decreased rate of anabolic processes and utilization of glucose by this tissue during fasting. After 30 days of fasting this tendency is a special feature in carp 17,18. In summary, a number of strategies are available to fish tissues to respond to changes in insulin secretion rate and changes in plasma insulin titers. At least in liver and in white skeletal muscle, the number of hormone binding sites or their affinity can be adjusted, at times concurrently, with different nutritional states, thus resulting in the observed metabolic alterations.

2. Glucagon and glucagon-like peptides It was not until quite recently that attempts have been made to elucidate the role of glucagon in fish under fasting conditions. Unlike insulin, glucagon levels in fish

416

I. Navarro and I. Guti~rrez

Fig. 2. Specific binding of insulin (inset) and binding capacity of muscle insulin receptors (main panel) of starved fed trout (Salmo trutta fario) and carp (Cyprinus carpio). Bars with identical letters a r e n o t significantly different at the 0.05 level. From ref. 140, with permission.

plasma can be measured successfully with a mammalian RIA ~, in spite of the fact that an appreciable variability exists in fish glucagons42. What helps matters is that key regions of the peptide are highly conserved26 and show many structural similarities to the mammalian hormone. Nevertheless, some authors have used homologous RIA systems to assay glucagon titers in teleostean fishes ~52. In mammals, pancreatic insulin and glucagon act together in response to fasting. After a short-term fasting, insulin titers decrease while glucagon levels increase substantially resulting in a high molar ratio of glucagon/insulin. After a more prolonged fast, glucagon levels remain high and plasma insulin concentrations continue to decrease. Thus, the glucagon/insulin (G/I) molar ratio appears to be a key regulatory parameter providing a link to the nutritional state of the organism 179. In most mammals, a normal G/I molar ratio after an overnight fast would be approximately 0.30. Undemutrition or a low carbohydrate meal will drive this molar ratio up towards unity and in total starvation the G/I value will likely approach 1.5-2.0. When the animal is in a catabolic situation, glucagon is instrumental in mediating the retrieval of stored nutrients at a time of need. On the other hand, in an anabolir situation, such as after a meal, the excess of nutrients must be stored, and in mammals G/I ratios may decrease to 0.1-0.05 or even lower if the diet carbohydrate content is high. In fasting experiments in fish, plasma hormone levels follow a somewhat different pattern. In fasted juvenile sea bass (D. /abr~) a steep increase in plasma glucagon was found after 4 days of fasting (Table 6) (similar to that occurring in higher vertebrates). This was accompanied by a stabilization of glucose levels. In contrast to mammals, this increased hormone level is not maintained and glucagon

Fasting and starvation

417 TABLE 6 Plasma glucagon levels in fed or fasted teleosts State

Glucagon (ng/ml 4- SE)

Dicentrarchus labrax 65

Fasted Fasted Fasted Fasted Fasted

1d 3d 8d 15 d 22 d

0.72 1.31 0.54 0.37 0.32

4- 0.14 4- 0.30 4- 0.11 4- 0.05 4- 0.05

Salmo trutta (summer) 135

Fasted Fasted Fasted Fasted Fasted Fasted Refed

1d 3d 8d 15 d 30 d 50 d 8d

0.65 0.91 0.51 0.46 0.25 0.25 0.52

4. 0.09 4- 0.03 4- 0.03 4- 0.04 4- 0.03 4- 0.03 4- 0.06

Salmo trutta (winter) 135

Fasted Fasted Fasted Fasted Fasted Fasted Refed

1d 3d 5d 8d 30 d 50 d 8d

0.44 0.46 0.55 0.45 0.32 0.23 0.43

4-0.04 4- 0.05 4- 0.09 4- 0.03 4- 0.03 4- 0.03 4- 0.04

Cyprinus carpio 17

Fasted Fasted Fasted Refed

1d 5d 50 d 12 d

0.42 0.28 0.14 0.54

4- 0.05 4- 0.04 4- 0.01 4- 0.05

Cyprinus carpio 18

Fasted Fasted Fasted

1d 50 d 65 d

0.37 4- 0.04 0.64 4- 0.04 0.75 4- 0.12

Gadus morhua 7a

Fed Fasted Fasted

7d 21 d

1.10 4- 0.32 0.30 4- 0.09 0.30 4- 0.03

Fed Fasted Fasted

7d 21 d

2.00 4- 0.73 0.30 4- 0.06 0.40 4- 0.13

Gadus morhua 73

concentrations declined again after 8 days. An increase of glucagon levels has also been observed in brown trout after 3 days of fasting 135, although this response was dampened compared to sea bass. It is possible that species differences can account for this difference in response, but surprisingly, no such increase was detected following a two- to four-day fast in the rainbow trout, another salmonid with a very similar lifestyle and diet as the brown t r o u t 152,173. The changes in glucagon and insulin in this short-term fasting lead to a rise in G/I molar ratio. Contrary to mammals, this ratio remains usually below 0.5. Increases of G/I values from 0.1 to 0.2 after a four-day fast in sea bass or from 0.2 to 0.4 in three-day fasted brown trout have been observed 65,135. Immediately following a meal in fish, G/I molar ratios do not decrease as clearly as in mammals.

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High levels of insulin a few hours after ingestion in salmonids 76,134 are compensated by subsequent elevation of glueagon levels which may be related to the high protein content of the diet. Sea bass (Dicentrarchus labrax) fed a natural diet shows a similar pattern of post-feeding hormonal changes 133. In fasted sea bass and trout a correlation exists between high glueagon levels and decreased fiver glycogen reserves. The glycogenolytie action of glueagon has been demonstrated in vivo and in vitro in numerous species of teleosts, although the degree of glyeogenolytie action can vary depending on the season. Glueagon injections induced a decrease in liver glycogen in eel (,4. japonica) 22, coho salmon (O. kisutch) or chinook salmon (O. tshawytscha) 152. Similar potent glyeogenolytie actions of glueagon have been seen in isolated hepatoeytes from sea raven (Hemitripterus americanus) 52, eoho salmon (O. kisutch) 156 or catfish (Ictalurus melas) 139. Thus it can be envisaged that increasing plasma levels of glueagon at the beginning of fasting may induce the mobilization of glycogen reserves. In contrast to mammals, long-term fasting in fish is accompanied by a drop in plasma glueagon (Table 6) and in glueagon-like peptide levels (Table 7) 126. The relative changes in insulin and glueagon family peptides increased the G/I ratio from 0.11 in fed fish to 0.16 in fasted fish and GLP/I molar ratio from 0.08 to 0.25 after 6 weeks of fasting in eoho salmon. Moon and coworkers 126 concluded that the relative hormonal changes are responsible for the observed activation of the glueoneogenic pathway during the later stages of food deprivation. Fasting-dependent increases in liver glueoneogenie capacity - as assessed by maximum enzyme activities of key enzymes - have been described in different species of fish (reviewed in ref. 125). In vitro experiments demonstrated that glucagon and especially GLP appear to be key peptides for regulating hepatic gluconeogenesis in fish 17,12~ It is interesting that this potent gluconeogenic effect of GLP has been found only in fish and not in mammals, where only insulinotropie and intestinal-related functions of GLP have been described 126. In fishes, a high GLP/I ratio appears to enhance gluconeogenie processes and mobilization of carbohydrate reserves. TABLE 7 Plasma glucagon-like peptide (GLP) levels in fed or fasted teleosts "GLP (ng/mi 4-SE)

State Oncorhynchus mykiss 126 Oncorhynchus mykiss 15~ Gadus morhua 73

Gadus morhua 73

Fed Fasted

42 d

0.6 4- 0.10 0.3 • 0.04

Fed Fasted

42 d

1.9 4- 0.40 0.4 4- 0.02

Fed Fasted Fasted

7d 21 d

0.2 4- 0.06 0.2 4- 0.03 0.2 4- 0.03

Fed Fasted Fasted

7d 21 d

0.3 4- 0.06 0.2 4- 0.06 0.2 4- 0.06

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A high G/I ratio associated with long-term fasting has also been described in 50-day starved brown trout (increase from 0.20 to 0.48) 135 but only in the summer season and not in the winter. Immature carp food-deprived for 50 or 65 days demonstrated an unusual increase of plasma glueagon levels 18. Nevertheless, these increases in G/I ratios are not always clearly seen in fish during long-term fasting experiments 17,65. It is quite apparent that generalizations are difficult at this point and that many other factors, such as the physiological state or previous diet, can influence hormone responses during fasting in fish. It is interesting that in anorexic individuals of brown trout (during the period of reproduction) maintained in captivity, a G/I ratio higher than unity was found in peripheral blood and portal vein 21. Such a high molar ratio was never described before in fish. It is possible that during natural processes of anorexia, the hormonal response is enhanced in comparison to experimental fish. Although it is clear that lipid reserves are mobilized during fasting in fish, the role of glucagon (a strongly lipolytie hormone in mammals) and other members of the glucagon family of hormones in this process needs to be elucidated. An indication of lipolytic action of glucagon has been found in Esox lucius 85 or D. labrax 142 where injections of glucagon induced an increase in plasma free fatty acids. However, administration of glucagon remained without effect in Opsanus tau 176 or eel (A. anguilla) 95. It appears that hepatic lipolysis in fish is mediated by triacylglycerol lipase which hydrolyses stored triacylglycerol to glycerol and fatty acids. Glucagon family peptides have been found to influence lipolysis in salmonids. In vivo administration of glucagon or glucagon-like peptide induces an elevation of plasma fatty acids accompanied by enhanced hepatic triaeylglycerol lipase activity, although the effectiveness of GLP in lipid metabolism in salmon varies depending on the time of the year 152. In vitro experiments support the role of glucagon in lipid metabolism during fasting. It has been also demonstrated that glucagon acts directly on the liver since glucagon stimulates triacylglycerol lipase activity in liver slices as well as fatty acid and glycerol release into the culture medium. More recently, Harmon and coworkers 7~ concluded that the increased lipolytic activity by glucagon in trout hepatocytes is mediated by phosphorylation of the enzyme. The previous nutritional state of fish can modulate hormonal mediated lipolysis: trout basal hepatic lipolysis is enhanced in liver slices from four weeks fasted fish, and glucagon-stimulated lipolysis was more pronounced in liver slices from these food-deprived animals than in liver from fed fish. Surprisingly, glucagon failed to affect hepatic lipolysis in the liver of six-week fasted animals 69, maybe as a result of already advanced lipid store depletion, or alternatively, as a consequence of alterations at the receptor level. Thus, all the above studies support the idea of glucagon and GLP being key hormones in regulating lipid mobilization during fasting in fish. Although plasma glucagon titers give us important information about the role of this hormone during fasting, receptor studies help in understanding the significance of the hormonal regulation. Navarro and Moon 136 have recently characterized, for the first time in fish, specific binding of glucagon in hepatocytes isolated from two teleostean species, the American eel (A. rostrata) and the brown bullhead

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(I. nebulosus). Two classes of binding sites have been described with apparent dissociation constants (Kd) of 1.97 nM (high affinity) and 17.3 nM (low affinity) for bullhead and 2.68 and 22.9 nM, respectively, for eel cells. These values are approximately ten times higher than those generally reported for mammalian hepatocytes 11,8~ This reflects the relatively slow association of glucagon binding in these fish which may be attributed to intrinsic characteristics of ectothermic animals. It is interesting that, a higher number of binding sites was found in the hepatocytes from the eel (10,413/ce11) in comparison to the bullhead (3811/ce11). Mommsen and M o o n 121 reported a higher increase in intracellular cAMP in response to the same glucagon concentrations in eel compared to bullhead hepatocytes. This suggests the existence of a correlation between the responsiveness of liver for glucagon binding and adenylyl cyclase in these fish species. However, it should also be noted that the eel is a species which fasts in captivity, while bullheads adapt to captivity quickly and feed actively. Nevertheless, more studies are needed to elucidate if such differences reflect species characteristics, or nutritional state. Liver cells of both eel and bullhead were able to regulate the abundance of their own receptors, since receptor number decreased by about 55% in both species with incubation of 100 nM glucagon. This 'down regulation' has been described in mammalian species 79,16~ These findings raise the question as to whether plasma glucagon levels could regulate hepatic glucagon receptors in vivo and especially in catabolic situations such as fasting. Variations in the number of receptors could help explain discrepancies described in glucagon sensitivity for fasted chinook salmon (O. tshawytscha) 93, and for sea raven (Hemitripterus americanus) 56. Glucagon-stimulated glycogenolysis in liver pieces isolated from fed and from short-term (one-week) fasted salmon, but failed to stimulate glycogenolysis in liver slices from long-term (three-week) fasted fish. In contrast, epinephrine maintains its glycogenolytic action regardless of nutritional state. In this fish species, a short fast induces an initial liver glycogen degradation while in longer term fasted salmon, mobilization ceases in the face of high glucagon/insulin molar rates. Thus it appears likely that glucagon is modulating fasting-associated adjustments in the metabolism of salmon by decreasing hormone sensitivity. Thus these salmon are able to maintain some minimum pool of glycogen. Distinctive hormonal features of the sea raven (Hemitripterus americanus) fasting strategies distinguish it from those species in which glycogen is partially conserved as in salmon. In six-week fasted sea raven, endogenous carbohydrate stores are used preferentially for the production of glucose instead of gluconeogenic precursors. Hepatocytes isolated from six-week fasted sea raven had an increased apparent sensitivity of rates of total glucose production (presumably by glycogenolysis) to glucagon. This phenomenon would retain the effectiveness of glucagon in the liver and allow for increased glucose production in this species under fasting conditions. All the findings mentioned above show that nutritional state modifies hormone effects and may explain some of the seasonal differences in hormone action previously reported for this and other fish species 52'53. In conclusion, fasted fish have proven to be a good model to study glucagon-like hormone actions. It seems that glucagon family peptides in fish are key hormones

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in regulating energy reserves in fish under fasting conditions. Recent and detailed studies on the glucagon-related hormones reveal fine adjustments to the nutritional state of fish involving tissue receptors and responsiveness of target cells. Clearly such studies are to be favored since they may help to understand the metabollic mechanisms of fish adapting to adverse conditions.

3. Glucocorticoids Administration of adrenocortical hormones, such as cortisol, to higher vertebrates normally stimulates glueoneogenesis, provokes a rise in liver glycogen and leads to an inhibition of glucose uptake in several peripheral tissues. Concomitantly, protein catabolism is stimulated. However, the immediate importance of such hormones under fasting conditions is under debate since generally, a decrease in cortisol secretion in early fasting is observed 46. Nevertheless, these hormones may have a supportive function in the control of normoglycemia, even in fasting conditions. It appears that corticosteroid hormones could have similar actions in teleost fish, but the importance of these hormones during fasting remains controversial. With respect to carbohydrate metabolism, the studies performed in whole animals concluded that cortisol administration resulted in enhanced liver gluconeogenesis based mainly on tissue carbohydrate changes 19,97,132. Since activation of gluconeogenic processes during fasting has been demonstrated also in fish as well as in higher vertebrates, cortisol may contribute to its regulation. In mammals, it has been reported that cortisol may exert, in addition to the direct metabolic effects, an indirect action on other hormones such as thyroid hormones, glucagon or catecholamines. Also, some authors have suggested that the insulin/cortisol ratio could regulate the direction of metabolic fluxes during fasting in fish 132. However, to date none of the in vivo studies analyzing the role of corticosteroids in fish systems have attempted to separate direct from permissive effects. Arguably the best example of such permissive effects in mammals is the potentiating effect of corticosteroids on glucagon's multiple actions, an area that remains to be analyzed with piscine systems. Vijayan and colleagues 182- 184 have looked at the interactive effects of cortisol and other glucoregulatory hormones. These authors demonstrated that cortisol implantation for 7 days in fed sea raven (H. americanus) enhances the responsiveness of hepatocytes to the actions of epinephrine and insulin, but not glucagon, on carbohydrate metabolism. Although hepatocytes from food-deprived (eight weeks) animals showed enhanced responsiveness to pancreatic hormones, these effects were not modified by cortisol implantation. These slow-release implants of steroid hormones have been used successfully to evaluate the chronic effects of cortisol on the physiology of teleosts 181,182,194. The effects on carbohydrate metabolism in isolated fish hepatocytes are not definitive. Recent studies show that cortisol increases hepatic activities of glycerol kinase (GK, EC 2.7.1.30) and fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) in brook charr (Salvelinus fontinalis) indicating enhanced gluconeogenic potential from glycerol 181. Metabolic flux in eel hepatocytes was altered by cortisol administration, shifting the preferred gluconeogenic substrate from lactate to amino acids51

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as previously suggested by other authors 29,s7. Following administration of cortisol, activities of phosphoenolpyruvate carboxykinase and glutamate-oxaloacetate transaminase (AspAT, EC 2.6.1.1) increased, suggesting an enhanced amino acid gluconeogenesis. Surprisingly, the absolute rate of gluconeogenic substrates incorporation into glucose actually declined in hormone-injected fish51. However, repeated injection of cortisol (which is considered a stress hormone itself), requires repeated handling of specimens and may lead to superimposing metabolic stress reactions mediated by catecholamines or permissive effects of cortisol plus catecholamines (or others) skewing the experimental results. In addition, repeated injection of cortisol may actually lead to increased turnover, i.e. speed-up removal of the injected hormone and thus the separation of cortisol-induced effects from those induced by fluctuating hormone titers, or drastically altered endogenous turnover, is difficult. Cortisol is known to stimulate glycogen synthesis in mammals, but in fish the data are not clear-cut. At this point, it is not clear whether species differences hormone application or other parameters are responsible for the observed inconsistent results. In fed goldfish (C. auratus), for instance, cortisol failed to increase liver glycogen concentration, but the hormone seemed to be effective in maintaining liver glycogen content in fasting fish 17~ Administration of cortisol in the Japanese eel, however, resulted in increased liver glycogen levels23. Cortisol has been implicated in the regulation of protein metabolism. Administration of cortisol in fed goldfish resulted in a loss of body weight and the hormone accelerated the rate of loss of body weight in fasted animals 17~ Associated with this observation, a rise in ammonia excretion and a two-fold increase in the concentration of alanine aminotransferase (AIaAT) in the liver were noted for treated fish. This suggests that cortisol is important in controlling the utilization of tissue protein during fasting at least in the goldfish. Similarly, Chart and Woo 23 demonstrated that injections of cortisol induce a rise in liver transaminase activities in the eel, together with an elevation of ammonia excretion, and hyperaminoacidemia; the latter is likely a reflection of increased peripheral proteolysis. These observations coalesce into the suggestion that in teleostean fishes, cortisol shifts the preference towards amino acids as gluconeogenic substrates 29,51, conceivably together with increases in proteolysis in extrahepatic tissues. Studies on the lipid metabolism of the eel indicate that cortisol induces lipolysis, increasing production and utilization of free fatty acids from triglycerides for energy production 36,~~ Implantation of juvenile coho salmon with cortisol resulted in lipid depletion, mainly in triacylglyerols accompanied by elevated lipase activity in the liver, red muscle and mesenteric fat 163. Similarly, one step removed from the immediate effects of cortisol, administration of mammalian adrenocorticotropic hormone (AUrH) increased plasma FFA in goldfish within 6-24 h 116. However, the same treatment failed to affect the concentration of plasma FFA in carp 45 and rainbow trout 174. Unfortunately, no information is available on the interplay of A u r H with lipid metabolism during fasting. In natural fasting situations, such as during the non-feeding migration periods in some fish, glucocorticoids are believed to play an important role, especially in

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enhancing the gluconeogenic processes 132. Plasma glucocorticoid levels increase in sturgeons (Acipenser galdenstadti) during the upstream migration period, particularly in males and this increase in hormone titer is accompanied by a depletion of the interrenal tissue. During the anorexia period of migration of the sockeye salmon (O. nerka), a six-fold increase in the concentration of corticosteroids has been observed. This fact was accompanied by a massive (up to 60%) breakdown of body, primarily muscle, protein 82'84. Other authors have also found high corticoid levels associated with migration in salmon. McBride et al. 111 found that plasma cortisol levels increase in pink salmon (Oncorhynchus gorbusha) during migration, reaching levels similar to those normally associated with stressed fish7. It appears that this hyperadrenocorticism is not directly associated with food deprivation, although it is clear that the catabolic effect of the increased levels of corticosteroids would facilitate the mobilization of energy from body reserves. On the other hand, it has been described that teleost interrenals are able to metabolize androgenic steroid precursors to testosterone and to synthesize progesterone 25,83. The enhanced activity of the adrenal may contribute to the process of sexual maturation. Experimental fasting studies made mainly in salmonids have shown an opposite trend. In O. mykiss (Salmo gairdneri) plasma cortisol levels were not affected by prolonged fasting (65 days), suggesting that this hormone is not involved in the processes of energy mobilization in this species 115. In an attempt to simulate natural migration conditions, continuous swimming of experimentally food-deprived coho salmon (O. kisutch) changed neither plasma cortisol levels nor cortisol binding sites in liver 184. However, plasma cortisol titers decrease with fasting in sea raven 183. Although cortisol levels seem to be representative indicators of the fish responses to stress, these are not greatly affected by fasting. Barton et al. 7, found that plasma cortisol elevations in response to handling stress in juvenile chinook salmon were not appreciably modified in 20-day fasted fish in comparison to controls. Nevertheless, the hyperglycemia response to stress was lower in fasted than in fed fish. In summary, experimental fasting situations have not been conclusive in determining the precise role of cortisol in fish, although some general trends such as the activation of gluconeogenic pathway from amino acids, are apparent. Data on glucocorticoid actions under fasting conditions in fish are scarce and plasma variations are not always clearly correlated with the physiological state of the fish. The interpretation of the data is complicated by the possible permissive effects of cortisol on other hormone actions.

4. Growth hormone Few studies have been performed on the role of growth hormone (GH) during fasting in fish. In mammals, the plasma GH concentration increases with a shortterm fast. Similarly, very large increases in plasma GH levels have been reported for trout after fasting. Periods of fasting of 20-30 days resulted in a 7-fold increase in plasma GH, compared to controls in steelhead trout (Fig. 3) 6. The same species

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fasted for six weeks also maintain higher levels of growth hormone than comparable, fed animals 44. It has been postulated that the increase in lipid mobilization is needed to sustain energy production during periods of continuous activity and food deprivation such during migration time 112. In concordance with this hypothesis, Sheridan 163 has reported that GH stimulates an increase in lipolytic enzyme activity in coho salmon parr. Barret and McKeown 6 also observed an exaggerated GH response when starving fish were exercised for 24 h in comparison with exercised fish maintained on a normal feeding regime (Fig. 3). Obviously fasting leads to a paradoxical situation in which starved animals although having high levels of GH do not grow. GH acts indirectly through insulin-like growth factors (IGFs) in most tissues. Thus, Sumpter and coworkers 172 working on rainbow trout suggest that the most probable mechanism to explain this

Fig. 3. Mean levels of plasma growth hormone (GH) of juvenile steelhead trout (Salmo gairdneti; now: Oncorhynchus mykiss). Some treatment groups were starved for 30 days, others were forced to swim at 1.5 body length per s for 24 h. Growth hormone levels were measured with a radioimmunoassay. Reprinted from ref. 6, with permission.

Fasting and starvation

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paradoxical situation would be a suppression of insulin-like growth factors similar to the situation described for mammals 24. In vitro studies with tilapia (Oreochromis mossambicus), have shown that fasting results in a substantial increase in GH release from pituitary cell incubations 158. An interesting outcome from these studies on the regulation of fish GH secretion during fasting is that the decline in glucose levels would be the signal to induce hormone secretion as it does in mammals. In conclusion, there is a clear response of growth hormone to fasting in fish, although the specific functions of this hormone in the metabolic adjustments demand further investigations.

5. Thyroid hormones It is generally agreed that thyroid hormones, tri-iodothyronine (T3) and thyroxine (T4), play an important role in vertebrates affecting a variety of processes such as metabolism, growth, differentiation and reproduction. Studies on the action of thyroid hormones are complicated because even in higher vertebrates thyroid hormones mostly affect metabolism indirectly being required for the permissive actions of other hormones. It seems that, as in mammals, T4 in fish acts mainly after conversion to T3 which represents the active thyroid hormone at the level of target cells 33. The production of T3 is increased during short-term overfeeding which is correlated with an increased diet-induced thermogenesis in mammals. On the contrary, plasma levels of T3 decrease during fasting. Some of the known metabolic effects of thyroid hormones on mammals are the potentiation of the mobilization of fat reserves, induction of hyperglycemia and activation of protein synthesis. The general picture that emerges from studies on administration of thyroid hormones in fish is that these hormones appear to regulate fish intermediary metabolism in a similar way as in other vertebrates. However contradictory findings have been described depending on the hormonal dose, fish species or season (reviewed in refs. 98 and 153). The thyroid hormone-induced increase in the metabolic rate, consistently reported in the mammalian literature 11, has not been unequivocally proved in fish 145'193 Fasting effects on fish thyroid have been studied primarily in salmonids. Shortterm fasting for several days or more prolonged food deprivation for several weeks induced a decrease of plasma levels of both T3 and T4 in Oncorhynchus mykiss 50'99'115. A similar lowering of plasma thyroid hormone levels was found in starved Platichthys sp. 138. In contrast, no significant changes or even increases in plasma T4 have been described in S. fontinalis 37,74. Decreased thyroid activity in response to fasting in teleost is indicated by histological changes observed in the thyroid of starved Oncorhynchus nerka 11~and Anguilla anguilla 78. Kinetic studies on thyroid hormones are not clearly correlated with changes in plasma hormones although they demonstrate a reduction in thyroid activity in fasted fish as well as in mammals. Since thyroid activity is generally related to a well fed and optimal metabolic state of the animal, this reduction may represent a homeostatic protective mechanism to prolong survival of the organism under conditions of food deprivation. Surprisingly, fasting induces a reduction in T4 and

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/. Navarro and J. Guti~rrez

T3 metabolic clearance similarly in fish or mammals39,74. Thus it appears that fast-induced low levels of plasma T3 could be due to a reduction in peripheral conversion of I"4 to T3 and its release to the plasma. The changes in blood hormone levels may not depend solely on the balance between production and degradation. A major complication is that, at least in mammals, food intake also influences the plasma protein binding of thyroid hormones with attendant changes in total and free hormone levels4~ Some in vivo studies support the hypothesis of decreased mono-deiodination from 1"4 to T3. Injection of 125I-T4 in trout demonstrated that the transformation of T4 to T3 is already reduced after 3 days of fasting3s,s~ The number of putative T3 receptors in the liver nuclei is also reduced by 3 days of fasting is~ At least in salmonids, fasting may produce a peripheral hypothyroidism by decreasing both T3 production and the density of hepatic T3 binding sites. This general decrease in thyroid activity associated with fasting observed in fish and also in higher vertebrates, may reflect the need to limit an exaggerated metabolite mobilization of energy reserves ~5.

VI. General conclusions In conclusion, there is no doubt that fish tissue metabolism is finely regulated under fasting conditions by the actions of many hormones. Interpretation of experimental data have to be done carefully, taking into consideration the previous nutritional and physiological state of the fish. Especially in the case of starvation, laboratory experiments, generally done on heavily inbred fish previously fed on a diet maximizing meat production, give quite different results to those done on naturally starving fish. Thus, understanding how fish respond to fasting requires research at multiple levels from molecular to ecological. Studies involving plasma hormone levels and energy tissue reserves gives us general information on which metabolic processes are favored during a catabolic fasting situation. Bearing in mind that fish metabolism is the result of a complicated integration of multiple processes in the whole animal, and numerous environmental factors, in vitro studies are preferred in order to identify which metabolic pathways are activated during fasting and how they are regulated by the specific actions of the hormones. Piscine cell systems are a relatively new approach for studies at the cellular and metabolic level and could be particularly valuable for integrating molecular and physiological studies. Recent research at the molecular level, the principal ones being, for instance, the characterization of fish tissue hormone receptors and the identification of new regulating peptides, open new questions on the particular strategies of metabolic regulation to survive food deprivation situations, an interesting field of research that demands further investigation in fish models. Acknowledgements This work was supported by grants from CAICYT (PB900471), CICYT (PTR93-0026) and NATO collaborative research programme (CGR.921175). We thank the Departament of Medi Natural (Generalitat de Catalunya) and Zoological Park of Barcelona for their help in supplying fish.

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VII. References 1. Abraham, S., H.J.M. Hansen and EN. Hansen. The effect of prolonged fasting on total lipid synthesis and enzyme activities in the liver of the European eel (Anguilla anguilla). Comp. Biochem. Physiol. 79B: 285-289, 1984. 2. Aster, EL. and T.W. Moon. Influence of fasting and diet on lipogenic enzymes in the American eel, AnguiUa rostrata (LeSueur). J. Nutr. 111: 346-354, 1981. 3. Balage, M., J. Grizard, C. Sornet, J. Simon, D. Dardevet and M. Manin. Insulin binding and receptor tyrosine kinase activity in rat liver and skeletal muscle: effect of starvation. Metab. Clin. Exp. 39: 366-373, 1990. 4. Ballantyne, J.S., H.C. Glemet, M.E. Chamberlin and "I.D. Singer. Plasma nonesterified fatty acids of marine teieost and elasmobranch fishes. Mar. Biol. 116: 47-52, 1993. 5. Ballantyne, J.S., TW. Moon and C.D. Moyes. Compatible and counteracting solutes and the evolution of ion and osmoregulation in fishes. Can. J. Zool. 65: 1883-1888, 1987. 6. Barret, B.A. and B.A. Mckeown. Sustained exercise augments longterm starvation in plasma growth hormone in the steel-head trout, Salmo gairdneri. Can. J. Zool. 66: 853-855, 1988. 7. Barton, B.A., C.B. Schreck and L.G. Fowler. Fasting and diet content affect stress-induced changes in plasma glucose and cortisol in juvenile chinook salmon. Pro~ Fish Cult. 50: 16-22, 1988. 8. Bastrop, R., R. Spangenberg and K. Jfirss. Biochemical adaptation of juvenile carp (Cyprinus carpio L.) to food deprivation. Comp. Biochem. Physiol. 98A: 143-149, 1991. 9. Beis, A., V.A. Zammit and E.A. Newsholme. Activities of 3-hydroxybutyrate dehydrogenase, 3oxoacid CoA-tranferase and acetoacetyl-CoA thiolase in relation to ketone body utilization in muscle from vertebrates and in invertebrates. Eur. J. Biochem. 104: 209-215, 1980. 10. Berges, J.A. and J.S. Ballantyne. 3-Hydroxy-3-methylglutaryl coenzyme A lyase from tissues of the oyster, Crassostrea virginica, the little skate, Raja erinacea and the lake charr, Salvelinus namaycush: a simplified spectrophotometric assay. Comp. Biochem. Physiol. 93B: 538-588, 1989. 11. Bernal, J. and L.J. De Oroot. Mode of action of thyroid hormones. In: The Thyroid Gland, edited by M. De Visser, New York, Raven Press, pp. 123-143, 1980. 12. Bharucha, D.B. and H.S. Tager. Analysis of glucagon-receptor interactions on isolated canine hepatocytes. J. Biol. Chem. 265: 3070-3079, 1990. 13. Bilinski, E. Biochemical aspects of fish swimming. In: Biochemical and Biophysical Perspectives in Marine Biology, edited by D.C. Malins and J.R. Sargent, London, Academic Press, pp. 239-288, 1974. 14. Bilinsld, E., R.E.E. Jonas and Y.C. Lau. Lysosomal triglyceride lipase from the lateral line tissue of rainbow trout (Salmo gairdneri). J. Fish. Res. Bd. Can. 28: 1015-1018, 1971. 15. Black, D. and R.M. Love. The sequential mobilization and restoration of energy reserves in tissues of Atlantic cod during starvation and refeeding. J. Comp. Physiol. 156B: 469-479, 1986. 16. Blasco, J., J. Fernandez and J. Guti6rrez. The effects of starvation and refeeding on plasma amino acid levels in carp, Cyprinus carpio L., 1758. J. Fish Biol. 38: 587-598, 1991. 17. Blasco J., J. Fermtndez and J. Guti~rrez. Fasting and refeeding in carp, Cyprinus carpio L.: the mobilization of reserves and plasma metabolite and hormone variations. J. Comp. Physiol. 162B: 539-546, 1992. 18. Blasco, J., J. Fernandez and J. Guti6rrez. Variations in tissue reserves, plasma metabolites and pancreatic hormones during fasting in immature carp (Cyprinus carpio). Comp. Biochem. Physiol. 103A: 357-363, 1992. 19. Butler, D.G. Hormonal control of gluconeogenesis in the North American eel, AnguUla rostrata. Gen. Comp. Endocrinol. 10: 85-91, 1968. 20. Canals, P., M.A. Oallardo, J. Blasco and J. S~nchez. Uptake and metabolism of L-alanine by freshly isolated trout (Salmo trutta) hepatocytes: the effect of fasting. J. Exp. Biol. 169: 37-52, 1992. 21. Carneiro, N.M., I. Navarro, J. Outi6rrez and E.M. Plisetskaya. Hepatic extraction of circulating insulin and glucagon in brown trout (Salmo trutta fario) after glucose and arginine injection. J. Exp. Zool. 267: 416-422, 1993. 22. Chan, D.K.O. and N.Y.S. Woo. Effect of glucagon on the metabolism of the eel, Anguilla japonica. Gen. Comp. Endocrinol. 35: 216-225, 1978. 23. Chan, D.K.O. and N.Y.S. Woo. Effect of cortisol on the metabolism of the eel, AnguiUa japonica. Ger~ Comp. Endocrinol. 35: 205-215, 1978. 24. Clemmons, D.R. and J.J. Van Wyk. Factors controlling blood concentration of somatomedin C. J. Clin. Endocrinol. Metab. 3: 113-143, 1987.

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157. Renaud, J.M. and T.W. Moon. Characterization of gluconeogenesis in hepatocytes isolated from the American eel, Anguilla rostrata Le Sueur. J. Comp. Physiol. 135: 115-125, 1980. 158. Rodgers, B.D., L.M.H. Helms and E.G. Gordon Grau. Effects of fasting, medium glucose and amino acid concentrations on prolactin and growth hormone release, in vitro, from the pituitary of the Tilapia Oreochromis mossambicus. Gen. Comp. Endocrinol. 86: 344-351, 1992. 159. de Roos R., C.C. de Roos, C.S. Werner and H. Werner. Plasma levels of glucose, alanine, lactate and beta-hydroxybutyrate in the unfed spiny dogfish shark (Squalus acanthias) after surgery and following mammalian insulin infusion. Gen. Comp. Endocrinol. 58: 28-43, 1985. 160. Santos, A. and E. Blazquez. Direct evidence of a glucagon-dependent regulation of the concentration of glucagon receptors in the liver. Eur. J. Biochem. 121: 671-677, 1982. 161. Sargent, J., R.J. Henderson and D.R. Tocher. The lipids. In: Fish Nutrition, 2rid edn., edited by J.E. Halver, pp. 154-209, 1989. 162. Satoh, S., T. Takeuchi and T Watanabe. Bull. Jpn. Soc. Sci. Fish. 50: 79-84, 1984. 163. Sheridan, M.A. Effects of thyroxine, cortisol, growth hormone, and prolactin on lipid metabolism of coho salmon, Oncorhynchus kisutch, during smoltification. Gen. Comp. Endocrinol. 64: 220-238, 1986. 164. Sheridan, M.A. Lipid dynamics in fish: aspects of absorption, transportation, deposition and mobilization. Comp. Biochem. Physiol. 90B: 679-690, 1988. 165. Sheridan, M.A. and W.V. Allen. Partial purification of triacylglycerol lipase isolated from steelhead trout (Salmo gairdnerii) adipose tissue. Lipids. 19: 347-352, 1984. 166. Shibata, N., T Kinumaki and H. lchimura. Triglyceride, cholesterol, free fatty acid, glucose and protein contents in plasma of cultured rainbow trout. Bull. Tokai Reg. Fish. Res. Lab. 77, 77-87, 1974. 167. Simon, J., R.W. Rosebrough, J.P. McMurtry, N.C. Steele, J. Roth, M. Adamo and D. Le Roith. Fasting and refeeding alter the insulin receptor tyrosine kinase in chicken liver but fail to affect brain insulin receptors. J. Biol. Chem. 261: 17081-1788, 1986. 168. Smith, M.A.K. Estimation of growth potential by measurement of tissue protein synthetic rates in feeding and fasting rainbow trout, Salmo gairdneri. J. Fish Biol. 9: 213-220, 1981. 169. Stirling, H.P. Effects of experimental feeding and starvation on the proximate composition of the European bass, Dicentrarchus labrax. Mar. BioL, 34: 85-91, 1976. 170. Storer, J.H. Starvation and the effects of cortisol in the goldfish (Carassius auratus L.). Comp. Biochem. Physiol. 20: 939-948, 1967. 171. Suarez, R.K. and TP. Mommsen. Gluconeogenesis in teleost fishes. Can. I. Zool. 65: 1869-1882, 1987. 172. Sumpter, J.P., P.Y. Le Bail, A.D. Pickering, T G. Pottinger and J.E Carragher. The effect of starvation on growth and plasma growth hormone concentrations of rainbow trout, Oncorhynchus mykiss. Gen. Comp. Endocrinol. 83: 94-102, 1991. 173. Sundby, A., K. Eliansen, A.K. Blom and T Asgard. Plasma insulin, glucagon, glucagon-like peptide and glucose levels in response to feeding and starvation in rainbow trout, and in response to life long exposure to different feed ration levels in Atlantic salmon. Fish Physiol. Biochem. 9: 3-9, 1991. 174. Takashima, E, T Habiya, N. Phan-van and K. Aid. Endocrinological studies on lipid metabolism in rainbow trout II. Effects of sex steroids, thyroid powder and adrenocorticotropin on plasma lipid content. Bull. Jpn. Soc. Sci. Fish. 38: 43-49, 1972. 175. Takeuchi, T., T Watanabe, S. Satoh, T Ida and M. Yaguchi. Changes in proximate and fatty acid compositions of carp fed low protein-high energy diets due to starvation during winter. Nippon Suisan Gakkaishi. 53: 1425-1429, 1987. 176. Tashima, L. and G.E Cahill, Jr. Effects of insulin in the toadfish, Opsanus tau. Gen. Comp. Endocrinol. 11: 262-271, 1968. 177. Thorpe, A. and B.W. Ince. Plasma insulin levels in teleosts determined by a charcoal-separation radioimmunoassay technique. Gen Comp. Endocrinol. 30: 332-339, 1976. 178. Tizley, J.E, V. Waights and R. Holmes. The development of a homologous teleost radioimmunoassay and its use in the study of adrenaline on insulin secretion from isolated pancreatic islet tissue of the rainbow trout, Salmo gairdneri. Comp. Biochem. Physiol. 81A: 821-825, 1985. 179. Unger, R.H. Insulin-glucagon-somatostatin interactions. In: Diabetes Mellitus, Vol. V, edited by V.H. Rifkin and P. Raskin, Bowie, Maryland, Prentice Hall, pp. 43-54, 1981. 180. Van Der Kraak, G.J. and J.G. Eales. Saturable 3,5,3'-triiodo-L-thyronine binding receptors in liver nuclei of rainbow trout (Salmo gairdneri Richardson). Gen. Comp. Endocrinol. 42: 437-448, 1981.

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181. Vijayan, M.M., J.S. Ballantyne and J.E Leatherland. Cortisol-induced changes in some aspects of the intermediary metabolism of Salvenilus fontinalis. Gen. Comp. Endocrinol. 82: 476--486, 1991. 182. Vijayan, M.M., G.D. Foster and T.W. Moon. Effects of cortisol on hepatic carbohydrate metabolism and responsiveness to hormones in the sea raven, Hemitripterus americanus. Fish PhysioL Biochem. 12: 327-335, 1993. 183. Vijayan, M.M. and J.E Leatherland. Cortisol-induced changes in plasma glucose, protein, and thyroid hormone levels, and liver glycogen content of coho salmon (Oncorhynchus kisutch Walbaum). Can. J. Zool. 67: 2746-2750, 1989. 184. Vijayan, M.M., A.G. Maule, C.B. Schreck and T.W. Moon. Hormonal control of hepatic glycogen metabolism in food-deprived, continuously swimming coho salmon (Oncorhynchus kisutch). Can. J. Fish Aquat. Sci. 50: 1676-1682, 1993. 185. Walton, M.J. and C.B. Cowey. Aspects of intermediary metabolism in salmonid fish. Comp. Biochem. Physiol. 73B: 59-79, 1982. 186. Walton, M.J. and R.P. Wilson. Postprandial changes in plasma and liver free amino acids of rainbow trout fed complete diets containing casein. Aquaculture 51: 15-115, 1986. 187. Weatherley, A.H. and H.S. Gill. Recovery growth following periods of restricted rations and starvation in rainbow trout Salmo gairdneri Richardson. J. Fish BioL 18: 195-208, 1981. 188. Weber, J.-M. Effect of endurance swimming on the lactate kinetics of rainbow trout. J. Exp. Biol. 158: 463-476, 1991. 189. Weber, J.-M., R.W. Brill and P.H. Hochachka. Mammalian metabolite flux rates in a teleost: lactate and glucose turnover in tuna. Am. J. Physiol. 250: R452-R458, 1986. 190. Wessels, J.P.H. and A.A. Spark. The fatty acid composition of the lipids from two species of hake. J. Sci. Fd. Agric., 1359-1370, 1973. 191. Wilkins, N.P. Starvation of the herring, Clupea harengus; survival and some gross biochemical changes. Comp. Biochem. Physiol. 23: 503-518, 1967. 192. Wittenberger, C. and R. Giurgea. Transaminase activities in muscle and liver of carp. Rev. Roum. Biol., 18: 441-444, 1973. 193. Woo, N.Y.S. The Effects of Salinity and Hormonal Factors on the Intermediary Metabolism of the Japanese Eel, Anguilla japonica Temminck and Schlegel. D. Thesis. University of Hong Kong, 1976. 194. Woo, ET.K., J.E Leatherland and M.S. Lee. Criptobia salmositica: cortisol increases the susceptibility of Salmo gairdneri Richardson to experimental cryptobiosis. I. Fish Dis. 10: 75-83, 1987. 195. Yang, T.-H. and G.N. Somero. Effects of feeding and food deprivation on oxygen consumption, muscle protein concentration and activities of energy metabolism enzymes in muscle and brain of shallow-living (Scorpaena guttata) and deep-living (Sebastolobus alascanus) scorpaenid fishes. J. Exp. Biol. 181: 213-232, 1993. 196. Zak, R., Martin, A.E and R. Blough. Assessment of protein turnover by use of radioisotopic tracers. PhysioL Rev. 59: 407-447, 1979. 197. Zammit, V.A., A. Beis and E.A. Newsholme. The role of 3-oxoacid-CoA transferase in the regulation of ketogenesis in the liver. FEBS Lett. 103: 212-215, 1979. 198. Zammit, V.A. and E.A. Newsholme. Activities of enzymes of fat and ketone-body metabolism and effects of starvation on blood concentrations of glucose and fat fuels in teleost and elasmobranch fish. Biochem. J. 184:313-322, 1979. 199. Z~bian, M.E and Y. Cr~.ach. Influence du je0ne sur la vitesse de d6gradation de quelques acides amines chez la carpe (Cyprinus carpio L.). lchthyophysiol. Acta 6: 10-27, 1982.

Hochachka and Mommsen (eds.), Biochemistry and molecular biology of fishes, vol. 4 9 1995Elsevier Science B.V. All rights reserved. CHAPTER 18

Origins of luciferins: ecology of bioluminescence in marine fishes ERIC M. THOMPSON AND JEAN-FRANCOIS REES *

Laboratoire de Biologie CeUulaire, Unit~ de Biologic du Dl,veloppement, Institut National de la Recherche Agronomique, ]ouy-en-]osas, France and * Laboratory of Animal Physiology, Catholic University of Louvain, Croix du Sud 5, 1348 Louvain-la-Neuve, Belgium

I. II. III. IV.

Introduction The functions of bioluminescence Organization of the light organs The biochemistry of bioluminescence 1. The components of bioluminescent systems 1.1. Luciferins 1.2. Luciferases 2. Reaction mechanism 3. Energy transfer V. The trophic transfer of marine luciferins 1. Ingestion 2. Intestinal absorption 3. Circulatory transport 4. Storage of luciferin 5. Recycling and de novo synthesis of luciferin 6. Retention 7. Targeting VI. Origins of imidazolopyrazine luciferins in fish bioluminescence VII. Conclusions Acknowledgements VIII. References

L Introduction Enzyme-catalyzed light production, bioluminescence, is a very common property of fishes inhabiting the mesopelagic realm of the oceans. In waters off Bermuda, over 93% of the individuals and 66% of the species were reported to be bioluminescent 4,11. On the other hand, relatively few epipelagic species (1-2%) harbor luminescent organs 82 and no freshwater bioluminescent fish species have been reported. The total absence of luminescent fishes in freshwater is somewhat enigmatic since environmental conditions similar to those of the dysphotic zone occur in some lakes. Interestingly, one report suggested that bioluminescent phenomena might occur in Lake Baikal 13, possibly involving fishes.

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As most luminescent species have evolved sophisticated light organs with differentiated luminous cells (photoeytes) associated with complex lens-like bodies, filters and reflectors 44, it seems evident that the production of light should provide some major selective advantages to biolumineseent fish species. It is of particular interest that research in the last twenty years has revealed that the luminescent system used by a number of species are not strictly endogenous and that either the entire luminescent system (symbiotic luminescent bacteria) or the substrate of the photogenic oxidation could be of exogenous origin. In the latter case, the lueiferins appear to be imidazol0pyrazines derived from either eoelenterazine, a luciferin found in a wide range of biolumineseent organisms or from the luciferin of the ostracod Vargula71. It has been suggested, and demonstrated in the instance of the epipelagie species Potichthys notatus, that fish are capable of utilizing active luciferin obtained 'from the diet 119. This article will review progress in the study of fish bioluminescence and discuss the properties of these systems relative to the suggested roles of light production in fish. The biochemical and evolutionary aspects of bacterial light organs have been recently reviewed 42,76,s7, and these will not be discussed in the present review. We will also describe the problems which fish need to overcome in order to obtain these highly oxidizable substrates from their diet as well as to process and store them. Finally, we will briefly consider the origins of biolumineseence in marine fishes and the possible ultimate sources of the luciferins which they utilize.

II. The functions of bioluminescence The preponderance of luminescent fishes in the deep sea has rendered difficult the study of the roles of bioluminescence in all but a very few species. As a consequence, most hypotheses have been based on morphological and physical parameters such as the anatomical disposition of the light organs and the spectral characteristics of the fight emission 3~ The migration to surface waters of the midshipman fish Porichthys, has allowed some observation on the use of the photophores in this species s2. However, the large discrepancies in light organ morphology, location, and control mechanisms, in different species, mean that generalizations remain speculative. If one considers that the functions of bioluminescence are strictly related to the light emitted and not to any metabolic advantage that might be conferred by luminescent activity, such as the regeneration of metabolic substrates in luminescent bacteria 41, bioluminescent phenomena could be classified into two broad categories. Each of these would place certain constraints on photophore distribution and the physical properties of light production, such as the intensity, emission spectrum, angular distribution, and kinetics. In the first group, photophores are devoted to communication between individuals, that is intra- and interspecific signaling related to feeding, defense, courtship and schooling. One can assume that selection pressure is likely to have favored

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mechanisms enabling fish to emit short-duration flashes of high intensity. A further criterion would be an emission spectrum compatible with the visual acuity of the receiver. The emission spectrum need not exactly match that of the ambient light as contrast would favor detection. However, as not all wavelengths are similarly absorbed, an emission spectrum peaking near the wavelength of optimal transmission in seawater would be beneficial, unless the luminescence is involved in very short-range communication. This would allow the emission to propagate over long distances, a critical parameter in a deep-sea environment characterized by a very low density of organisms. By situating the peak of emission near the wavelength of optimal transmission, modification of the spectral composition with distance is also reduced. In other words, the spectral quality of the signal that could be perceived would remain the same over most of its journey towards potential receivers, with overall intensity decreasing with distance. Thus it does not seem surprising that in most bioluminescent organisms living in the marine pelagic environment, variations in the color of light do not seem to be widely used as an intra- and interspecific recognition parameter. While light emission oriented towards communication relies on detection by other individuals, it has been suggested that light emission could also play a role in making fish less visible to predators 21. CounteriUumination would allow the fish to compensate for the contrast resulting from the absorption of downwelling sunlight by its dorsal surface. In many fish, and also among squid and shrimp, the photophores are located on the lateral and ventral sides of the animal, and light emission is directed ventrally. The hatchetfish Argyropelecus with its large photophores obliterating its ventral surface has been shown to fulfill many conditions that are considered essential for an effective luminescent camouflage, that is, the spectral composition and the angular distribution of the luminescence closely matches that of the downwelling ambient light 29,3~ Further, the fish is equipped with a preorbital photophore directing its emitted light into the eye ~ possibly acting as a standard for comparison with background light. The effectiveness of counterillumination would likely be restricted to regions of the mesopelagic zone where the intensity of light penetration does not demand an excessive energetic cost in operating such a system of camouflage. Optimally, the fish should be able to produce a range of intensities similar to that of the ambient light at the depth range the fish commonly inhabits. Thus the production of bright luminescence would not be of interest for those species which are likely to favor the emission of light with the same spectral composition as the ambient light at a steady matched intensity. A different interpretation for the predominant ventral location of light organs among fishes and oceanic invertebrates has been proposed 7~ According to this hypothesis, communication oriented ventrally may have been selected because of reduced predation pressure as a result of a population decline in the deep sea. Thus, fewer potential predators would be located below the fish to intercept and home in on a luminescent signal. However, this would also imply fewer potential intended receivers for the signal. This hypothesis has not been supported by any solid experimental evidence.

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III. Organization of the light organs Whereas light organs colonized by luminescent bacterial symbionts are open glands connected to the exterior, non-bacterial organs are mostly internal. Only in searsiid fish is the postcleithral photophore a secretory organ expelling luminescent epithelial cells into seawater 4s. Although photophores vary considerably in shape, size, and location among species, their general organization can be schematized as consisting of photogenic tissue surrounded by accessory optical structures. The luminescence originates from highly specialized cells known as photocytes. In some species (e.g. Porichthys, Maurolicus, Gonostoma) photocytes show clear signs of secretory activities 9,~~ but precisely how these relate to luminescence remains to be clearly defined. The photogenic tissue is richly supplied by a capillary network and is innervated. In Porichthys, the nerves are catecholaminergic fibers from the sympathetic system 7,s6. Photocytes are generally surrounded by an interference reflector composed of multiple layers of alternating high and low refractive index. The high refractive index material is usually composed of guanine crystals 44,46 (Fig. 1). In alepocephalid fish, however, the reflectors lack guanine ~2. The reflector directs light emission by the photocytes towards a translucid lens-like body that concentrates the light through the photophore's aperture. In myctophid fishes, this structure occurs as a thickened portion of the overlying scale 66. In some species, photophores completely lack any light-collimating structures and the orientation of the light is a direct function of the shape and position of the reflector. Other accessories include light-channeling structures and filters. Spectrally selective filters are particularly important in adjusting the composition of the fight emission. Since spectral adjustments

Fig. 1. Guanine crystals from the reflector of Porichthysnotatus photophores as seen by scanning electron microscopy.The intracellular crystalswere expelled from damaged reflector cells. Scale bar: 1 /zm (photograph courtesy of T. Smith).

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Fig. 2. Disposition of the light organs of Parapriacanthus ransonneti, tlo - thoracic light organ; st = stomach; pc - pyloric caecae; m = transluscent keel muscle; alo = anal light organ; an = anus. F r o m ref. 36.

are made at the expense of the overall light intensity, filtering is probably not the optimal method for light emission implicated in signaling. For this purpose, energy transfer seems the most cost-effective strategy for altering the emission spectrum with no reduction in the peak intensity (see section IV, part 3). In contrast, selective filtering would be particularly relevant to fish emitting light with a spectral composition closely matching that of low-intensity downwelling light. For example, in the ventrally oriented photophores of the hatchetfish Atg~opelecus, the halfband width is narrowed from 53 to 29 nm through pigmented filters overlying the photogenic tissue with no loss of intensity at the emission maximum 127. Future experiments should provide information on the relative occurrence of these two mechanisms among different species. Apogonid and pempherid fish appear to have evolved a unique type of light organ. The light organs are connected with the exterior via the digestive tract, resembling to some extent, the organization of bacterial light organs. In Parapriacanthus, the luminous organ system consists of a pair of ducts that are connected to the pyloric caeca (Fig. 2) and a single duct adjacent to the anus and communicating with the exterior through a small pore 36. The thoracic organ of Apogon ellioti is an oval-shaped structure directly connected to the intestine. In all cases light passes through the translucent keel muscle 36.

IV. The biochemistry of bioluminescence 1. The components of bioluminescent systems In all fish species studied thus far, light appears to be produced through the oxidation of an imidazolopyrazine substrate. Imidazolopyrazines are lipophilic molecules

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E.M. Thompson and I.-E Rees

A. Vargulalucifedn

B. Coelenterate-type luciferin (coelenterazine)

II

,N

OH

Fig. 3. Chemical structure of imidazolopyrazine luciferins found in Vargula- and coelenterate-like photogenic systems.

characterized by a central fused imidazolone-pyrazine nucleus covalently linked to three side group substituents. Two types of imidazolopyrazine-based systems have been identified among biolumineseent marine organisms (Fig. 3). In the first system, the components cross-react with the luciferins and the luciferases found in luminous coelenterates. In the second, cross-reaction occurs with the luciferin and luciferase of the marine ostracod l~rgu/a (formerly Cypridina). No other system has yet been identified in fish with non-bacterial photophores. The distribution of the two types of imidazolopyrazine systems among fish genera is listed in Table 1. The r system is widely distributed, whereas only a few species utilize a system similar to that of Vargula. It can also be noted that species oxidizing coelenterazine inhabit the mesopelagie realm

Origins of luciferins: ecology of bioluminescence in marine fishes

441

TABLE 1 Distribution of coelenterate-type and Vargula-type luminescent systems among fishes Coelenterate-type

Vargula-type

Salmoniformes Platytroctidae

Batrachoidiformes Batrachoididae

Searsia sp. (E) 16 Stomiiformes Gonostomatidae

Cyclothone braueri (S)a Sternoptychidae

Argyropelecus hemigymnus (E,S) 96.1~176

Porichthys sp. (E,S) 27 Perciformes Apogonidae

Apogon eUioti (E,S) 1~ Pempheridae

Parapriacanthus bercyformes (E,S) 39,59,60

Photichthyidae

Vinciguerria attenuata (E,S) 95 Yarella iUustris (E,S,D) 1~176 Melanostomiidae

Echiostoma barbetum (S)16 Malaeosteidae

Photostomias sp. (E) 16 Myctophiformes Neoscopelidae

Neoscopelus microchir (E,S) 57.100 Myetophidae

Diaphus elucens (E,S,D) 57,1~176 Diaphus coeruleus (E,S,D) 1~176 Diaphus suborbitalis (D) s6 Lampadena sp. (S) 16 Myctophurn sp. (S) 56 Myctophum asperum (D)56 Benthosema fibulata (D) 56 The various components of the luminescent system (E ffi luciferase-like activity; S = luciferin and D -stabilized derivatives of the luciferin) detected in each species are indicated. Superscript numbers refer to the list of references. a J.-E Rees, unpublished.

of the oceans whereas species utilizing Vargula luciferin are restricted to coastal areas.

1.1. Luciferins Both Vargula luciferin and coelenterate luciferin (coelenterazine) emit a bright greenish-yellow fluorescence in organic solvents, such as methanol (~m~ = 535 nm) 1~ In aprotic solvents (DMSO, DMF), both substances are chemiluminescent with very similar emission spectra peaking at 480 nm. However, the quantum yields (~), the number of photons emitted per mole of substrate consumed, are very low for chemiluminescent reactions. For example, the ~ of coelenterazine in DMSO and DMF were shown to be 0.0021 and 0.0001, respectively52,1~ In the coelenterate-type system, the luciferin may be present in a stabilized form. Stabilized derivatives of coelenterazine have been found in the tissues, mostly the liver and pyloric caeca of a few species (Table 1)56,1~176 In Diaphus, Myctophum and

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E.M. Thompson and J.-E Rees

Benthosema, the stabilized derivative is a glucuronide form in which a ketone group is conjugated to D-glucopyranosyluronic acid s6. Coelenterazine has also been found as a luciferyl-sulfate in the biolurninescent anthozoan Renillasl.s4. In this species, the active luciferin can be regenerated by removal of the sulfate group in the presence of phosphoadenosine and luciferyl-sulfokinase 26. Apart from reversibly oxidized luciferinol Hs there are no known stabilized derivatives of Vargula luciferin and it is assumed that the luciferin is stored in its free form in the light organs. Since the luminescence of dried Vargula can be rapidly triggered upon immersion in water it is probable that most of the luminescent substrate is present in a free form that can readily react with the luciferase. However, one cannot exclude the possibility that stabilized forms of Vargula luciferin might exist in Vargula, and in fish, but have not thus far been detected because of the hydrolysis of stabilizing groups during extraction under acidic conditions.

1.2. Luciferases Our knowledge of fish luciferases is extremely limited, when data are not completely lacking. Some coelenterazine-based luciferase activity has been found in a few species (Table 1). In Vinciguerria attenuata, coelenterate-type luciferase activity was found in the body (including the light organs) but not in the digestive tract and glands 95. In this species, weak luciferase activity was associated with cellular membranes and was not affected by EDTA or various potential cofactors such as ATP, or pyridine and flavin nucleotides. In Arg~opelecus, the luciferase activity seems rather specific for coelenterazine as no fight was emitted upon mixing Vargula luciferin and the fish luciferase 96. The highest levels of luciferase activity were recorded in the digestive tract and the light organs of the fish, but luciferase activity was also detected in other tissues. Since the luminescence of isolated luminous organs from Arg~opelecus is completely inhibited by cyanide, a wellknown inhibitor of peroxidases, it is possible that the highly active peroxidase detected in Arg~opelecus tissues 16 could play a role in the luminescence of this species. In Porichthys, Vargula-like luciferase appears to be restricted to the light organs. Very little data is available on this luciferase 1~8. In Diaphus, some luciferase activity against Vargula luciferin and coelenterazine was detected in extracts of the two large nasal photophores 55'1~176 The luminescence reaction induced upon mixing Vargula luciferin and Diaphus photophore extracts is first order and is independent of luciferin concentration, just as in the Vargula reaction n~ Since Diaphus luciferase was inhibited by rabbit antibodies raised against Vargula luciferase, portions of the two enzymes may present similar epitopes n~ Cross-reaction with Vargula luciferin has also been observed in Apogon eUioti,

Arachmia fucata, A. lineolata, Rhabdamia cypselura, Parapriacanthus beryciformes and R ransonneti photophores 34,37- 39,60,104. The luminescence produced by mixing hot- and cold-water extracts of R. cypseluralight organs, and to a lesser extent those of Parapriacanthusransonneti,was found to be potentiated by the addition of N A D

and NADP to the reaction mixture 34'37-39. The luciferases of Porichthys, Vargula and

Origins of luciferins: ecology of bioluminescence in marine fishes

443

Parapriacanthus appear to have similar molecular weights of approximately 65,000 Da, but differences were observed among the chromatographic, immunological and kinetic properties of the enzymes 121.

2. Reaction mechanism Because of the structural similarities between luciferins of the two imidazolopyrazine systems, some weak cross-reactivity between their components may occur. However, the efficiency of the luciferase-catalyzed luminous reaction is much higher with the natural substrate of the enzyme. Weak cross-reactivity may explain conflicting reports on the nature of the luminescent system in the nasal photophores of Diaphus which was first said to be of the Vargula-type12~ and later classified as coelenterate-type 55'1~176 Similarly, the coelenterate-type luciferase of the deedpod shrimp Oplophorus also cross-reacts with Vargula luciferin 12~ Very weak light emission can also be observed when mixing coelenterazine and Vargula luciferase 109. No direct data are available on bioluminescent reaction mechanisms in fish. However, it is likely that they are similar or identical to mechanisms described in other organisms utilizing similar substrates (e.g. Va~ula and Renilla), and for which data are available. All luminescent reactions described thus far involve the oxidation of a substrate by molecular oxygen or, in a few cases, by a derivative such as hydrogen peroxide 41. In imidazolopyrazine-based luminescence, molecular oxygen is required in the reaction. The chemical mechanisms leading to the photogenic oxidation of the luciferin appear very similar in Renilla and Vargula. In both cases, it seems well established that the reaction of the luciferin with oxygen leads to formation of a cyclic peroxide intermediate (dioxetanone) which decomposes to generate sufficient energy for light emission (Fig. 4). The breakdown of the dioxetanone intermediate produces CO2 and the corresponding oxyluciferin. Since one oxygen atom is incorporated into the carbonyl product and another into the oxyluciferin, both Vargula and Renilla luciferase can be considered as monooxygenases 72. Not all coelenterates possess a luminous system similar to that of Renilla. In some species (Aequorea, Clythia, Obelia, etc.), the luminous system consists of a stable complex composed of coelenterazine, oxygen and an apoprotein. The luminescence of this precharged system (photoprotein) is triggered by the binding of calcium ions to specific sites on the proteic moiety of the complex 1~ Although photoproteins are structurally very different from the system found in Renilla, the reaction mechanism is similar. In fish, oxygen appears necessary for all luminescent reactions described thus far, suggesting that precharged systems similar to photoproteins do not occur in this group 34'12~ The wavelength of light emission is not strictly dependent on the nature of the luminescent substrate. It is the structure of the light-emitting complex, the luciferase-bound oxyluciferin, that determines the spectral composition of the luminescence. Physiological conditions (e.g. ions, pH) present during the transition from the excited to the ground state also influence the final emission spectrum.

444

E.M. Thompson and Z-E Rees HO O -

od R1

O

I!

R2

N

N--

N

Rs

C02

N

"~ N

it Fig. 4. Proposed mechanism for the oxidation of coelenterazine resulting in the production of light. From ref. 72.

Thus organisms that share the same luciferin will not necessarily produce light with identical emission spectra. This was demonstrated in an elegant way in click-beetles by Wood and collaborators 12s. In this group of r the luciferin is structurally identical to the benzothiazole molecule described in other insects 74, but the color of the luminescence ranges from green to orange, depending on the species. It was possible to shift the spectral composition of the light towards the lower or the upper end of the spectrum by changing as little as three amino acids in the luciferase amino acid sequence ns. Information on in vitro bioluminescence spectra in fish are limited. An extract of Diaphus elucens light organs was found to catalyze the oxidation of Vargula luciferin with an emission peak at 456 nm n~ whereas similar tests carried out in Apogon revealed an emission maximum at 460 nm 1~ These values are very similar to that of the luciferin-luciferase reaction of Vargula. The bioluminescence spectra of in vitro reactions are very similar in both the coelenterate and Vargula systems. In the Vargula system, the in vitro spectrum peaks at 456-465 nm whereas corresponding values range from 465 to 480 nm for the coelenterate-type system ~s. These values are very similar to that of the emission maxima for the chemiluminescence of both compounds in aprotic solvents 94. In contrast with the paucity of data on in vitro emission spectra, in vivo emission spectra are available for many species 47,n7. Most in vivo bioluminescence spectra are unimodal with the peak emission ranging from 460 to 490 nm. In some cases, the spectrum shows two peaks as observed in Porichthys and Searsia. The bimodal spectrum of Porichthys has been tentatively ascribed to energy transfer or to the protonation of the oxyluciferin anion during the reaction ~7. In most cases, the

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445

emission spectrum of the in vitro luciferin-luciferase reaction cannot be deduced from in vivo data as the presence of filters and reflectors can alter the spectral composition of the light emitted. At the molecular level, energy transfer can also occur, considerably altering the luminescence emission spectrum.

3. Energy transfer As mentioned above, in some species, the spectrum of the light emitted in vitro when mixing luciferin and luciferase in the presence of oxygen does not match that produced in vivo. Although filters and selective reflection can play a role in such spectral shifts these can also result from energy transfer mechanisms. In these systems, the energy liberated by the excited complex returning to the ground state is not emitted directly but is passed on to other molecules. These energy-acceptors are the actual emitters and the spectrum of the light will depend on their structure and environment. Energy transfer can proceed through either radiative or non-radiative mechanisms, that is, the energy produced in the luciferin-luciferase complex is transformed into light prior to its transfer to a fluorescent emitter or, the electronic excitation is directly passed on to the emitter. Non-radiative energy transfer mechanisms can be very efficient with virtually no energy loss during transfer. If the chemiluminescence yield of the emitter is higher than that of the excited oxyluciferin, the overall quantum yield of the reaction can be increased by energy transfer, as observed in the coelenterate Renilla 124. Since these mechanisms render possible the adaptation of a biolumineseent system to the emission of other wavelengths at very low cost, it is not surprising that energy transfer processes are found in many bioluminescent organisms 125. Energy transfer is known to occur in coelenterates using either a photoprotein (e.g. Aequorea) or a conventional luciferin-luciferase system (e.g. Renilla). In fish, energy transfer has been proposed to occur in the deep sea stomiatoid and searsiid fish. In addition to the many small photophores scattered over their body, stomiatoid fishes of the genera Pachystomias, Malacosteus and Aristostomias have two pairs of larger light organs around the eyes. One pair located behind the eyes emit blue light, the other pair underneath the eyes emit red light. The latter pair of photophores contain a red fluorescent protein similar to a phycobiliprotein which is absent from the first pair. Therefore, it has been suggested that the red emission could result from energy transfer to this chromophore 17. As the fluorescence spectrum of this phycobiliprotein does not match that of the in vivo bioluminescence, it remains to be clearly demonstrated whether this pigment functions as the in vivo emitter in these species. In the deep sea fish Searsia, the bioluminescence of excreted luminous cells undergoes spectral changes during the time course of the emission. Just after the onset of the luminescence, the spectrum appears monophasic peaking at 478 nm. As light emission continues, a shoulder in the spectrum appears at 408 nm and its relative intensity gradually increases with time until it predominates 45. Although it has been suggested that these temporal variations could be related to energy transfer mechanisms, it is also possible that

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the changes could be ascribed to progressive alterations in the environment of the emitting species in expelled cells subjected to an hyperosmotic shock in seawater.

V. The trophic transfer of marine luciferins Early biochemical studies revealing cross-reactivity and identity between components of fish luminescent systems and those of invertebrates were quite surprising. Organisms that were not phyllogenetically related were linked by common components of their luminescent systems. The accumulation of examples of various species sharing the Vargula- or the coelenterate-type system among cephalopods, crustaceans, and fish, tended to rule out the possibility of evolutionary convergence. Although the transfer of both the luciferin and luciferase from l~rgu/a to apogonids was proposed in early work34, it now seems that most of these apparent convergences are the result of the transfer of luciferin through trophic chains. Experimental evidence of the occurrence of such a dietary transfer of luciferin was later provided in the fish Porichthys119 and also the mysid shrimp Gnathophausia 31. While possibly explaining the widespread occurrence of imidazolopyrazines among marine bioluminescent organisms, this hypothesis has raised other questions. For example, is it an advantageous strategy for an organism to rely on exogenous sources, sometimes very scarce, for the supply of a highly labile substance that is readily rendered inactive by oxygen? If only the luciferin is transferred, did the various luciferases evolve independently towards the systems found in present species? If so, what were the ancestors of the imidazolopyrazine luciferases? As non-luminescent Porichthys in Puget Sound represent a successful breeding population, what essential functions does bioluminescence serve in this species? These are intriguing questions for the biologist trying to understand the basis for the evolution of bioluminescence in fish and invertebrates. At present, no clear answer to these questions can be formulated, but recent data provide us with clues to pathways which might have been involved. What are the adaptive responses that would be required for such a dietary transfer mechanism to be efficient? These include the ingestion of prey containing luciferin, transfer of luciferin to the light organs, the possible recycling of the oxidized luciferin, the avoidance of loss through body surfaces or the excretory system, and the development of luciferases and bioluminescent control mechanisms. Fig. 5 summarizes the major problems facing a fish using vicarious luciferin. We will now consider some of these possible adaptations in more detail.

1. Ingestion The first problem encountered by a luminescent fish species dependent on an exogenous source of luciferin is ingestion of the source prey. In the case of fishes requiting coelenterate-type luciferin, the dietary supply appears not to be problematic as this

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Fig. 5. The metabolism of exogenous luciferin in fishes. This figure summarizes some of the problems facing a hypothetically luminescent fish. 1 ffi ingesting prey containing the desired luciferin; 2 -absorbing it from the intestines; 3 ffi protecting this very labile luminescent substrate during its transfer via the circulatory system; 4 ffi storing luciferin (e.g. in the liver); 5 ffi recycling the oxidized luciferin or resynthesizing de novo the active luciferin; 6 = avoiding losses through renal excretion and diffusion through the gills; 7 ffi targeting the active luciferin to the light organs.

molecule seems to be abundant in the marine ecosystem. Coelenterazine is a rather widespread molecule that is present in both luminescent and non-luminescent organisms from many phyla 16,1~176176 It has a worldwide geographical distribution and is present in both pelagic and coastal areas of the oceans. Since the highest luciferin concentrations occur in luminescent species ~~176176 it would be more efficient for the fish to feed predominantly on other luminescent organisms using the same luciferin, providing that all other nutritional requirements can be satisfied equally by either luminescent or non-luminescent food items. Do luminescent fish feed selectively on luminous prey organisms? This hypothesis might appear a little paradoxical since the bright luminescent flashes produced by many planktonic organisms were previously assigned roles in predator evasion 1,14,15,2s. Feeding ecology studies analyzing the diet of mesopelagie fishes are few in number and none has thus far investigated the role of bioluminescence in the predator-prey relationship. In an attempt to analyze whether bioluminescent fish prefer luminous to non-luminous prey, we have analyzed the proportion of bioluminescent organisms in the food of mesopelagic fish as reported in ecological studies. Our analysis was restricted to studies reporting the composition of the fish diet down to the genus level (Table 2). It is important to consider this data with caution, as not all species within a genus are luminescent, and not all food items could always be identified. Also, due to the low number of organisms which have been studied for the presence of coelenterazine, the proportion of those actually containing coelenterazine may be underestimated. The data reveal that luminescent prey can make up a large part of the food items

E.M. Thompson and J.-E Rees

448 TABLE 2.

Luminescent organisms in the diet of mesopelagic bioluminescent fish genera Genus

Valenciennellus

% luminescent prey items a 69-86 59-71 47-70*

% prey items with coelenterazine b 84--100 52- 78 33- 44

Reference 81 22 50

Argyropelecus

53-89 64" 12-24

Lampanyctus

22-42 31 * 5- 8

Gonostoma

52 * 60-81

65 36-- 70

50 22

Sternoptyx

10-50

17- 59

49

~ncig~erria

15-80

5- 43

63 22

58- 85 31 92 65 100 100

81 50 63 81 50 62

From data reported in the literature.

a In some cases, large proportions of the food items could not be identified. The bioluminescent nature of prey items was based on systematicdistribution data for bioluminescence4s. Genera were considered luminescent only where the occurrence of bioluminescence has been clearly documented. b From data in reference 16. Since only a few copepod genera were investigated for the presence of coelenterazine in their tissues, and coelenterazine is likely to be the luminescent substrate in many planktonic crustaceans, these values are probably underestimates. *These values correspond to the percentage of luminescent organisms in the three most abundant food items. Dashes indicate the absence of luminescent genera.

taken by these fishes. In some species this can represent 80% of ingested organisms. Most of the luminescent prey consist of copepods, mainly Pleuromamma sp. and euphausiids. While small individuals feed almost exclusively on small zooplankton (mostly copepods), larger individuals tend to feed on euphausiids and even small fishes. Coelenterazine is found to be present in the diet of all fish species for which data are presented. It is remarkable that in some stomach content analyses (Valenciennellus sp.), luminescent prey accounted for more than 70% of the diet and all of these prey items are known to possess a coelenterazine-based luminescent system 16. While these data demonstrate that luminescent fishes ingest luminescent prey, they do not signify that these fishes feed selectively on luminescent organisms. Previous studies of the feeding ecology of fishes reveal large discrepancies in the feeding behavior and regimes of mesopelagic fishes. While some are random feeders, taking the nearest prey available, others show evidence of feeding selectivity. Clarke suggested that sternoptychids and small gonostomatids feed by active, visual searching and preferred prey were probably more visible in terms of body transparency, pigmentation and size 22. He did not take bioluminescence into account in this analysis. In Benthoseraa glaciale, there seems to be a clear preference for the

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luminescent copepod Metridia lucens whereas the predominant copepod species, Clausocalanus, living at the same depth as Gonostoma, appears to be avoided 97. Thus, in summary, a large part of the food taken by mesopelagic fish consists of luminescent organisms, mainly copepods and euphausiids. Although some fish appear to feed randomly, other species selectively feed on particular luminescent organisms from which they can obtain the luciferin for their own luminescence. Since coelenterazine is also present in non-luminescent organisms, though in lower quantities 1~ these might constitute accessory sources of the luminescent substrate, meaning that exclusive feeding on luminescent prey is not essential. The situation of fishes using Vargula luciferin is in sharp contrast with that of those requiting coelenterazine. Although the precise sources of Vm~,ula luciferin remain to be clarified, it seems likely that few organisms biosynthesize Vargula luciferin, and the source may be limited to the ostracods themselves. The known geographic distribution of Vargula luciferin is restricted to areas inhabited by Vargula. The fact that ostracods are scavengers would probably reduce their ability to obtain large amounts of active luciferin from the diet. Finally, Vargula produces bright flashes of luminescence, resulting from the discharge of luciferin and luciferase into seawater, a strategy which does not seem wholly compatible with dependence on an exogenous source of a relatively rare molecule. This is in contrast to the secreted bioluminescence of the mysid shrimp Gnathophausia, a species which is known to acquire its luciferin (coelenterazine) from the diet 31. In this case, the abundance of coelenterazine in the shrimp diet compensates for the loss of luciferin accompanying the activity of the luminous glands. The possibility that no source of luciferin other than Vargula exists is further supported by the sympatry of Vargula sp. and the distribution of fishes using Vargula-like luciferin in their own bioluminescent reactions. In the group perciformes, both luminescent apogonids and pempherids are sympatric with VargUla. However, specimens of Vargula are found only rarely in the stomach contents of these fishes. Only a dozen out of more than 2000 Parapriacanthus beryciformes specimens were found to contain some Vargula specimens 59,114 and Vargula have yet to be found in Apogon stomach contents 36. Quantitative data on the yield of luciferin extracted from the pyloric caeca of 2300 Parapriacanthus specimens revealed that on average, one specimen contained 10/zg of luciferin s9. Considering that the luciferin content of Vargula hilgendorfii is about 1 p,g33, this suggests that the ingestion of a few dozen ostracods should be sufficient to replenish the luciferin stores. While the linkage of luminescent Vargula with apogonid and pempherid bioluminescence seems compelling, the use of an exogenous source of luciferin, most probably luminescent Vargula, has been most extensively studied in the batrachoidid genus Porichthys. Paleontological evidence indicates an Atlantic origin for the genus with a subsequent migration to the Pacific coasts of North and South America. In the Atlantic, all species are functionally bioluminescent. Along the coast of Peru and Chile, one species, Aphos porossus, is given separate generic status because it lacks the photophores characteristic of all other members of the group. All other morphological characters are consistent with inclusion in the genus Porichthys. In the Northern

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E.M. Thompson and J.-E Rees

hemisphere, Porichthys notatus has the greatest range, extending from Vancouver Island to the Southern tip of Baja California. However, this species is divided by a distributional break off the coast of Oregon into two populations. The Northern population, located mainly around Vancouver Island and in Puget Sound, is unable to luminesce due to a lack of luciferin, despite possessing photophores which are ultrastructurally indistinguishable from the Southern population 1~ It is of interest to note that both the Northern population of R notatus and the SouthernA. porossus are separated from their luminescent relatives by regions of cold seasonal upwelling corresponding roughly to the spawning periods of these fishes and which may act as boundaries for the habitat range of the appropriate luminescent prey. In fact, the sparse data available concerning the range of luminescent Vargula tsujii suggest a limit coincident with the Northern extreme of the California population of R notatus 64. Near this distributional boundary both luminescent and non-luminescent individuals of R notatus can be found 126. Furthermore, it is possible to induce sustained luminescence capability in non-luminescent P. notatus by feeding either purified Vargula luciferin or whole luminescent Vargula to the fish 111'119. Feeding of a single luminescent ostracod is sufficient to establish a weak luminescence capability in a previously non-luminescent individual. Despite this strong circumstantial evidence concerning the source of luciferin in the Porichthys diet, considerable stomach content analysis of P. notatus has yet to reveal the presence of luminescent Vargula in the natural luminescent population 2'1~ At the Northern end of the range of the California population of P. notatus, around the San Francisco Bay area, a more detailed analysis of the population structure relative to luminescent capability has suggested some avenues of research 11~ Maternal luciferin is known to be transferred to Porichthys eggs 109'119 and provides an initial luciferin source for developing larvae and juveniles. In juveniles ranging up to 80 mm in standard length (corresponding to the first year class) the relative uniformity in luminescence capability in the San Francisco Bay region may reflect this maternal transfer 11~ However, in the second year class, a distinct gradient of decreasing luminescence capability is found from the South to the North. One explanation may be that Southern juveniles are able to directly feed on luminescent Vargula which are absent further North, thus maintaining sufficient luciferin stores. In this regard, it has also been noted that juvenile P. notatus are attracted by luminescent flashes78. In the third year class, a high proportion of luminescent individuals is found throughout this region. This may result from the attrition of non-luminescent individuals or perhaps an indirect dietary incorporation of Vargula luciferin available to larger but not smaller fish. One possible source would be cannibalism 11~ Young R notatus are an important dietary component of R myriaster, a shallow water relative found further to the South, but there are as yet no data to support the idea that P. notatus cannibalizes its own juveniles.

2. Intestinal absorption Have luminescent fishes evolved specific absorption mechanisms for luciferins and their metabolites? After prey have been ingested, they undergo mechanical and

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451

chemical digestion in the stomach. The acidic conditions present in the stomach should protect luciferins from autoxidation. This seems consistent with previous studies 1~176176 showing that relatively high amounts of active luciferin can be isolated from fish stomachs, irrespective of whether or not these organisms are themselves luminescent. Since enol-sulfate bonds are hydrolyzed at low pH, a proportion of the known stabilized luciferin derivatives may be converted into active luciferin during transit through the stomach. These active molecules would then be unstable at the alkaline pH found in the intestines, and rapid absorption of luciferin would be advantageous. Whether mesopelagic fishes have developed specific absorption mechanisms for coelenterazine is at present very difficult to investigate because of the problem of maintaining these fishes in captivity. On the other hand, experiments carded out on shallow-water fishes suggest that these possess selective absorption mechanisms for Vargula lueiferin. Perhaps the most striking examples of adaptation are found in the pempherid and apogonid fishes of the Indo-pacifie. In these species, the thoracic light organs communicate directly with the digestive tract through small ducts connected either to the intestine (Apogon sp.) or the pylorie caeca (Parapriacanthus sp.). The anal luminous organs of these species, while not connected to the digestive tract, are closely associated with the rectum, and this disposition probably allows fairly rapid transfer of luciferin to these organs 36. The close association of the light organs with the digestive tract probably allows rapid and efficient transfer of luciferin to the light organs, but places constraints on the number, and the localization of the photophores. Thus, such a strategy becomes deficient in the case of a bioluminescent fish such as Porichthys, with more than 700 photophores distributed over the head and trunk. In this genus, luciferin must be absorbed through the intestinal wall and carded to the photophores via the circulatory system. Recent studies suggest that Porichthys may have developed specific intestinal absorption mechanisms for the uptake of Vargula luciferin 112,113. Uptake of orally administered luciferin was compared in luciferin deficient, Puget Sound, P. notatus, a non-luminescent relative, Opsanus beta, and a non-luminescent unrelated fish, Paralabrax clathratus. When fed a single 7.6/.tg dose of luciferin, the blood concentrations of luciferin 12 h later were 2.12, 0.56 and 0 ng/ml respectively. When challenged with 6 such doses of luciferin at 4 day intervals, the blood luciferin levels observed 16 h after the last feeding were 24, 1 and 0 ng/ml respectively. Since the in vitro stability of luciferin in the blood of all three species was the same, the differences in blood luciferin in vivo concentrations seem to reflect differential intestinal absorption. It should be noted, however, that the overall efficiency of luciferin uptake remained low in Puget Sound P. notatus. This was on the order of 0.5%, a figure confirmed when isolating 14C-labeled luciferin from photophores 112. However, it would seem likely that potential natural sources of Vargula luciferin in the diet of P. notatus are present in very low quantities at any given time. Therefore, the evolution of a high affinity-low capacity system of luciferin uptake may have been favored. There are as yet no data on whether naturally luminescent California P. notatus are capable of the uptake and eventual utilization of the oxidized products of Vargula luciferin. Should such a capability exist, the selective

452

E.M. Thompson and J.-E Rees

pressure for the rapid intestinal uptake of active luciferin would be considerably reduced.

3. Circulatory transport Vargula luciferin and coelenterazine are very sensitive to molecular oxygen. Thus, the fish is faced with the problem of protecting luciferin from autoxidation during its circulatory transport to the photocytes. In Porichthys, luciferin is continuously present in the blood 2~'n3. One could propose the involvement of a specific luciferin binding protein, as found in the anthozoan Renilla 25, in the transport of the luminescent substrate to the light organs. Experiments demonstrate that blood components in Porichthys can effectively protect luciferin from autoxidation 113. However, no specific carrier for luciferin was identified in Porichthys blood, independent of whether or not the fish were from a naturally luminescent or non-luminescent population, or originally non-luminescent but subsequently induced with luciferin. Instead, it seems that binding and protection of Vargula luciferin is a non-specific property of fish blood, as this property is also found in the non-luminescent fishes Opsanus and Paralabrax113. Results suggest that Vargula luciferin can bind to either plasma components or red blood cells. It has been calculated that the number of lueiferin molecules binding to the erythroeyte surface was 2.5 x 107per cell. Since the binding capacity of erythrocytes was not affected by a trypsin treatment luciferin appears to bind directly to the membrane surface and not to groups protruding beyond the permeability barrier. The nature of the heat-sensitive plasma components ensuring some protection of the luciferin is not known. No data are available on the stability of coelenterazine in the blood of mesopelagie fishes.

4. Storage of lucifetin It is highly probable that the discontinuous luciferin supply should be compensated for by some ability to store luciferin in a form and location from where it can be rapidly mobilized when needed. The liver appears to contain large quantities of coelenterazine in many mesopelagic fishes and, together with the photophores, are the likely main storage sites for luciferin 1~176 Deep sea fish have a comparatively low resting metabolism 24, and storage involving low energetic cost is likely to have been favored. Among the most cost-effective storage mechanisms for such labile compounds would be to transform the luciferin into a stable, inactive derivative, from which the active form can be easily regenerated, rather than synthesizing binding proteins or maintaining a low pH in intracellular compartments as demonstrated in some other luminescent organisms 43. This seems to be the method selected by organisms using coelenterazine, as natural, stabilized derivatives of this compound have been shown to exist. Two different derivatives have been described, an enol-sulfate form and one in which a keto-group is linked to a gluco-pyranosyl moiety 52,s6. The enol-sulfate form has yet to be clearly identified in fish but its presence is well documented in the anthozoan, Renilla 52'54. It is particularly noteworthy

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453

that transfer of gluco-pyranosyl acid is involved in the detoxification of exogenous substances. Indeed, enzymes involved in glucuronoconjugation play a very important role in vertebrates in neutralizing toxic compounds and other poorly soluble substances prior to excretion through the renal system 1~ Glucoronoeonjugation is known to take place in the liver of vertebrates. Thus it might be that these very systems could be used for preserving labile compounds such as luciferins from autoxidation and allow low-cost storage of these highly valuable substances. The synthesis of enol-sulfate derivatives is also the mechanism apparently involved in the protection of ascorbic acid against autoxidation in fish and crustaceans 77,s3,122. Vertebrates appear to share this property with some invertebrates. Besides Renilla, stabilized forms of coelenterazine, apparently the enol-sulfate derivative, are also present in shrimp, both luminescent and non-luminescent 1~176176 These luciferin derivatives can be synthesized by shrimp, as the non-luminous Crangon septemspinosa and Pandalus danae, when injected with eoelenterazine, accumulated coelenterazine enol-sulfate in the hepatopancreas 1~ Fish which use a luminescent system based on the incorporation of Vargula luciferin, do not have large stores of the luminescent substrate in their hepatic tissues, nor do they seem to possess any stabilized derivative of the luciferin. In Porichthys, the many photophores seem to be the main luciferin storage site, with luciferin content in the liver attaining only 2.5-4% of that of the pooled light organs 27. Although the possible roles of the translucid subocular gels remain enigmatic, these structures were shown to contain rather large amounts of luciferin 27, and might thus constitute important luciferin stores. In other epipelagic species (Apogon, Parapriacanthus), the pyloric caeca and the light organs seem to be the main storage sites for luciferin 36. The nature of the mechanisms utilized in preserving the very labile Vargula luciferin from autoxidation remain obscure. In Porichthys, the luciferin from photophore extracts elutes together with a high molecular weight compound exerting an inhibitory action on the light reaction 118. Although the nature of this compound was not elucidated, it might possibly play some role in the protection of luciferin against autoxidation and also constitute part of the triggering mechanism for luminescence as does the luciferin binding protein in Renilla 2~ Another possibility is that particular ionic conditions (e.g. low pH) might exist in the many large intracellular vesicles suggested to contain the luminescent substrate in the photogenic cells 1~ Porichthys photophores can be dissected out and retain their luminescent capacities for several hours 7,93. When irradiated with near UV-light, they emit a bright greenish fluorescence, the intensity of which seems proportional to the amount of active luciferin present in the photogenic cells 6. Recently, it has been observed that both the fluorescent and luminescent properties of isolated photophores can be irreversibly abolished by treating the light organ with glyceraldehyde 3-phosphate 91,92. This glycolytic intermediate seems to control the luminescent activity of photocytes 92. Since glyceraldehyde 3-phosphate seems to have no inhibitory action on the Vargula luciferin-luciferase reaction in vitro (J.-E Rees, unpublished observation), understanding the action of this compound on the photophores could provide some clues to the storage conditions of the luciferin within the photocytes.

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5. Recycling and de novo synthesis of luciferin We have seen that many bioluminescent fishes are exposed to luciferin in their diet, and are capable of incorporating and possibly modifying this bioactive molecule into stabilized derivatives. To what extent are bioluminescent fish capable of de novo synthesis or recycling of these valuable molecules? Conversion of oxyluciferin to luciferin has been demonstrated in vivo in the firefly ss and it appears that in the bioluminescent squid, Watasenia scintillans, oxyluciferin or etioluciferin may be returned to the liver for resynthesis of luciferin or preluciferin ss. Among bioluminescent fishes the question has been most closely investigated in Porichthys notatus. Although recently, it has been found that luminescent capability can be extinguished in naturally luminescent juvenile California R notatus by massive repetitive stimulations in an epinephrine bath 8~ suggesting, under these conditions, the requirement for a continuous source of luciferin in this population, the finding that feeding of a single luminescent ostracod was capable of establishing luminescence capability in previously non-luminescent R notatus from Puget Sound s, raised the question as to whether this could be explained by a simple incorporation of the luciferin with no subsequent stimulation of de novo synthesis or recycling. A further study was carried out to quantify the light yield from a number of Puget Sound P. notatus, each of which was induced by feeding of a single 6.4/tg dose of purified Vargula luciferin 111. Surprisingly, this single dose was capable of stimulating a luminescence capability which persisted for more than two years and a calculation of the light produced during this period suggested that it surpassed the yield which could be anticipated from the single initial feeding. In order to try and determine whether this resulted from de novo synthesis or recycling of luciferin, ~4C-labeled Vargula luciferin was synthesized and administered to non-luminescent R notatus in. When luciferin was recovered from the photophores 7 weeks later it was found that the labeled luciferin had indeed been directly incorporated and that its specific activity remained unchanged, indicating that no de novo synthesis of luciferin had taken place. To test whether recycling might explain the sustained luminescence capacity, both oxyluciferin and etioluciferin were also administered to non-luminescent fish but failed to stimulate a bioluminescent response ~n. Thus several questions remain to be answered. Are Vargula 0xyluciferin and etioluciferin unable to penetrate to a potential site of luciferin resynthesis in Potichthys? Does Porichthys modify Vargula luciferin or its oxidation products so that Vargula oxyluciferin or etioluciferin are not recognized? Is it first necessary to induce luminescence capability with active Vargula luciferin before the oxidation products can be utilized? Also, it cannot be completely ruled out that de novo synthesis is tied to the presence of active luciferin in the fish but is restricted to certain periods, such as during reproductive activity. There are no indications as to whether mesopelagic fish possess any recycling mechanism for coelenterazine. Nevertheless, one could attempt to compare their theoretical needs with the amounts of coelenterazine that have been detected in their tissues. Let us first consider the hatchetfishArgyropelecus hemigymnus. Accord-

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ing to the angular distribution and the spectral composition of the light produced by their large, ventrally oriented photophores, this species is considered a good example of a counterilluminating fish29'3~ Population studies have shown that this species lives at depths ranging from 100 to 600 rn in the Northeastern Atlantic and 110 to 530 rn in the Mediterranean 3'4'97. The fish migrates to the upper layers (110-270 m) at night 4,32,89,116. In the Strait of Messina, the depth range of A. hemigymnus was reported to be 180-500 m (ref. 5). Light measurements 5,98 showed thatA, hemigymnus are present in an ambient irradiance extending from 1.5 x 10-5 to 8 x 10-3/zW cm -2. Since the optimal use of the photophores for counterillumination requires that the light output equal that of the ambient light level, a one cm 2 photophore should be capable of producing from 3.6 x 107 to 1.9 x 101~photons s -1 (),max = 480 nm). If we assume the quantum yield of the luminescence reaction to be equivalent to that of the coelenterazine-based system of Renilla (~ -- 0.05), the amount of eoelenterazine required would be between 7.2 x 108 and 3.9 x 1011 molecules per second. Thus, a fish with a total emitting area of one cm 2 would consume 0.07 pmol coelenterazine per min when living at the lowest ambient light levels, and 38 pmol min -1 at the brighter end of the inhabited irradiance range. The total coelenterazine content of A. hemigymnus has been reported as ranging from undetectable levels up to 47 pmo116,96,1~ Thus, during the day, the amount of coelenterazine present in the fish would allow the emission of a continuous glow with an intensity matching ambient light for up to about 10 h at the deeper end of its depth range, whereas this could persist for only 70 s at the upper irradiance levels. These values have been confirmed by experiments carried out on isolated photophores. Pharmacological experiments 68 revealed that A. hemigymnus large ventral photophores stimulated by epinephrine can produce a one-hour glow with a total yield of 1012 photons or the equivalent of 0.4 ~W cm 2. No further light response could be elicited following this emission, suggesting that this corresponds roughly to the total luminescence potential of the isolated organ. Thus, the ventral photophores of a fish spending the day at the lower end of the irradiance range could emit a sustained glow for about 8 hours with no need to recycle the oxidized luciferin; on the other hand, camouflage in the upper irradiance zone would only allow for the emission of a few short-duration flashes, and would depend on the existence of recycling mechanisms for the oxidized luciferin. However, at night, light intensities are reduced to 10-6-10 -9 of daytime levels 85. Therefore, if light emission by Argyropelecus was to be used only at night, a single pmol of coelenterazine could allow continuous luminescence for a period of years! Furthermore, if we consider that most fish cannot distinguish a luminescent source from background light when the ratio of the two intensities is as different as 1:10 (ref. 85), this would allow camouflage to be effective at a reduced luciferin consumption rate (up to 90% economy). This strategy is supported by recent observations on the ventrally oriented bacterial photophores of leiognathids. Measurements of the absolute luminescence produced by Gazza minuta in response to increasing levels of downwelling light revealed that, while the luminescence output also increased with that of the background, it never exceeded 20% (range 2-19%) of the ambient light level 7s. If this strategy is effective in dissimulating mesopelagic fishes from predators, this would be a means of conserving luminescent substrates.

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E.M. Thompson and L.F. Rees

Feeding studies on Arg~opelecus showed that the stomach contains an average of 4 prey items per specimen, and that ingestion rates are likely to be the order of a few prey (mostly copepods) per day 22,50,81,97. Considering a coelenterazine content of luminous copepods of 2-35 pmol per organism 16, the fish is likely to dispose of a daily dietary supply of around 10-100 pmol of coelenterazine. This is clearly not sufficient for the production of sustained luminescence at high ambient light intensities unless some recycling of oxidized coelenterazine occurs. However, if the fish were to restrict the use of their photophores to depths corresponding to diurnal periods of low light irradiance, then the dietary supply of coelenterazine would be sufficient and no recycling of the oxyluciferin would be required. Among bioluminescent fishes, Arg~opelecus appears to contain particularly small amounts of coelenterazine 16,96,1~176 The coelenterazine content of myctophids seems to be up to three orders of magnitude greater 1~176 In these fishes, the emission of a sustained glow of the same intensity as ambient light would pose tittle problem, and the photophores would be effective over a wide intensity range. In fact, the quantities of coelenterazine present would allow the fish to emit bright flashes that could be used for communication. The application of electric stimuli to mesopelagic fishes revealed great variations in the intensity and kinetics of the triggered luminescent flashes79. The flash intensities extended from 5 x 10 9 to 8.7 • 1011 q s -1. However, the kinetics of evoked luminescence were very rapid, lasting for no more than 4 seconds. As suggested by the rapid decrease in the intensity of successive flashes, such high intensity luminescence could probably not be produced over long periods, presumably due to the rapid depletion of luciferin stores. Nonetheless, it remains possible that the quantities of luciferin in these fishes would permit use in both counteriUumination and signaling. In this context, it is of interest that among two species of Sternoptyx, S. diaphana, living at depths ranging from 800 to 1500 m, was reported to produce flash intensities ten times that of S. pseudobscura, even though the latter is known to occur at a shallower depth range of 300-1000 m 79. However, one should keep in mind that the light emissions observed were not spontaneous phenomena but resulted from artificial stimulation of perturbed to moribund fishes brought relatively rapidly to atmospheric pressure. Therefore, extrapolation of this data to in situ uses of luminescence remains hazardous. Although the above calculations suggest that numerous mesopelagic species could rely completely on a dietary supply of coelenterazine, the possibility exists that some species may recycle the oxidized luciferin. A possible recycling mechanism for coelenterazine has been proposed involving the etioluciferin (coelenteramine) and p-hydroxyphenylpyruvic acid (Fig. 6) 72. This reaction is the final step in a chemical synthesis of coelenterazine 53. Whether or not this reaction occurs in vivo remains to be demonstrated.

6. Retention Experiments have demonstrated that non-luminescent specimens of Porichthys nora. tus can be made bioluminescent by the ingestion of a few #g of Vargula luciferin, and

Origins of luciferins: ecology of bioluminescence in marine.fishes

IN ~ O H RI~N~Rs

~

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H

jN~ jNH: ~CO2H 20 ~- R.~N~Rs + HO"

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~

[Luciferase

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H

riC02H 0

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"

-,,11o.

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that once induced, this capability persisted for more than 2 years ~ . Since luciferin is found in significant concentrations in P. notatus blood 113, losses are very likely to occur through the gills and kidneys. However, since de novo luciferin synthesis does not seem to occur in this species 111, losses of luciferin would be very disadvantageous and should be prevented to the greatest extent possible. The association of luciferin with blood components described above may in part reduce losses due to diffusion. On the other hand, increases in free luciferin in the circulation may not necessarily result in increased excretion by the kidneys. The kidney structure in Porichthys is likely to resemble that of its close relative Opsanus tau. In this species, the kidneys consist exclusively of tubules with no glomerula 69 and excretory activity relies on active secretion of solutes rather than filtration. The structure of Vargula luciferin, in which a guanido group is attached to the imidazolopyrazine nucleus, makes the molecule reasonably hydrophilic and may also reduce losses by limiting membrane penetration (E.M. Thompson, unpublished observation). Unfortunately, no data concerning coelenterazine retention in mesopelagic fishes are available. In coelenterazine, the guanido group of Vargula luciferin is replaced by a phenyl residue rendering the molecule more hydrophobic. Recent reports have demonstrated that coelenterazine can easily diffuse across membranes 61. Thus, this molecule would theoretically be free to diffuse across surfaces such as the gills and be irreversibly lost in the surrounding water. However, since the abundance of coelenterazine in the fish diet appears much higher than for Vargula luciferin, one could expect that the mechanisms implicated in the retention of the luciferin and/or its oxidation products should not necessarily be as performant as in the case of

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Porichthys. Also, the transformation of coelenterazine into a more stable, and more hydrophilic form, by sulfotransfer or glucuronoconjugation, could, by decreasing coelenterazine's hydrophobicity, reduce membrane transit, and aid in retention of the luminescent substrate. Z Targeting Once present in fish blood, luciferin must be transferred into the photogenic cells where it can be used for light production. As coelenterazine is hydrophobic it is able to diffuse across the cell membrane. However, passive diffusion of the lucfferin would be slow and non-specific, and is unlikely to be the only mechanism of luciferin uptake in the photocytes. Studies in Porichthys notatus, suggest that highly specific lucifcrin uptake systems are present on the photocyte cell membrane. In non-luminescent Puget Sound specimens, there is a delay of several days after feeding with Var~/a luciferin, before lucifcrin begins to accumulate in the photocytes, as judged both by bioluminescence capability and the onset of photophore fluorescence in near ultraviolet lights,111,119. Since Vargu/a luciferin and Podchthys luciferin appear to be very similar, if not identical 27,1~ it is unlikely that this delay reflects a conversion of the luciferin, and it is more probable that the presence of lucifcrin in the blood may be necessary to stimulate the synthesis of lucifcrin receptors on the photocytes. The uptake mechanism appears to be highly selective, as two lucifcrin analogues, one of them differing from Vargula luciferin only by a guanido group, were unable to induce luminescence or photophore fluorescence in Puget Sound E notatus in. Similar results were obtained by either oral or intraperitoneal administration of these compounds, ruling out the possibility that this only reflects specificity at the level of intestinal absorption. Since the amounts of lucifcrin in P. notatus blood are in the ng/ml range, the photocytes are likely to have a very high affinity uptake mechanism for luciferin. Photophorcs are not the only targets for luciferin uptake. Female Porichthys notatus lay over 100 eggs on the underside of rocks. Early observations demonstrated that Vargula luciferin can be found in newly laid eggs and was detectable throughout all developmental stages of luminous R notatus 119. At that time, the presence of Vargula lucifcrin in eggs and prefeeding larval stages was interpreted as a reflection of the capacity of the fish for de novo synthesis of luciferin. However, subsequent work suggests that the presence of lucifcrin in P. notatus eggs more likely involves the transfer of maternal lucifcrin stocks into the eggs Ill. When female E notatus were induced with Vargula luciferin, luminescence intensity showed a cyclic pattern with low points corresponding to the spawning season. During this period the females had swollen abdomens, characteristic of egg formation and the few eggs which were laid were found to contain lucifcrin. Following this period, the luminescence intensity of these females was found to recover, coincident with the reabsorption of unlaid eggs. No similar cyclical pattern of luminescence intensity was observed in induced males 111. The presence of maternal lucifcrin in the eggs, and the detection of lucifcrase on day 28, just prior to release of the juveniles from the substrate 119, means that the young juveniles possess a fully functional luminescent system when

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459

they become free-swimming. Maternal transfer of luciferin does not appear to be restricted to P. notatus, as in the hatchetfishArgyropelecushemigymnus, not only was coelenterazine present in the egg bags, but its concentration was the highest among all fish tissues, about twelve times that found in the light organs 96.

VI. Origins of imidazolopyrazine luciferins in fish bioluminescence Earlier, we divided the functions of bioluminescence in fish into two broad categories. However, counterillumination and signaling need not necessarily be mutually exclusive functions, both could be used in a given species, simply by varying the intensity and kinetics of light emission. As previously discussed, the properties of light propagation in seawater may have led to similar selective pressures on the wavelength of emission for either function. Thus, it is important to note that the chemiluminescent properties of both Vargula luciferin and coelenterazine correspond well with the constraints imposed by transmission in the seawater medium. The emission spectrum of imidazolopyrazine chemiluminescence is affected by the electronic charges of the imidazolopyrazine moiety and of the substituent side groups. At physiological pH, the emitter is the monoanion form of the oxyluciferin 72 and the luminescence maximum is from 470 to 480 nm. The various luciferases which have evolved do not appear to have significantly modified this spectral distribution as many bioluminescent organisms, using either eoelenterazine or Vargulaluciferin, have very similar emission spectra. In organisms which do emit considerably different wavelengths of light, this seems to be achieved by transferring the energy of the excited imidazolopyrazine luciferin to a secondary emitter such as a fluorescent protein 124. Thus imidazolopyrazines are well suited for use in marine bioluminescence, but do they have other properties that might account for their widespread occurrence in the marine environment? Besides the production of light, bioluminescent reactions using imidazolopyrazines also consume oxygen. It has been suggested that primitive luciferases may have evolved in order to utilize molecular oxygen directly as an electron acceptor at the low oxygen tensions present in the primitive atmosphere 73,99. Initially, oxygen would have been toxic to organisms which had evolved in an anaerobic environment. Therefore, the presence of molecules with a high affinity for the neutralization of oxygen would have become important. Further data have shown that imidazolopyrazines also react with oxygen derivatives such as the superoxide anion (O2), and singlet oxygen (102)67'84. These molecules are highly reactive and are deleterious for many cellular functions. In seawater, significant concentrations of superoxide anion and hydrogen peroxide are generated by photochemical processes 9~176 The highest levels of these molecules are found in the surface layers of the ocean and decrease with depth 13~ In coastal waters at midday, the rate of superoxide production 9~ can reach 5 x 10-7 mol 1-1 h -1 and its steady-state concentration is 2 x 10 -8 mol 1-1. Therefore, an effective defense against oxidative processes is essential for marine organisms. A byproduct of the proposed use of imidazolopyrazines in these reactions would have been the emission of a weak chemiluminescence.

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When Vargulaluciferin is placed in physiological media containing bovine serum, there is a very weak catalysis of luminescence. This produces a very broad spectral distribution with no distinct peak shape (Thompson, unpublished results). Thus, to make effective adaptive use of the low light level produced in antioxidative processes, would have involved the evolution of enzymes capable of: (1) improving the quantum yield of the chemiluminescent reaction; and (2) reducing the interaction of the excited state oxyluciferin with other molecules in counterproductive energy transfer resulting in a degradation of spectral quality. The present day luciferases are the fruit of this process and it will be of considerable interest to unravel the various evolutionary origins of these enzymes. It has been suggested that bioluminescence may have as many as 30 independent origins during the course of evolution 41. We propose that antioxidative processes are the evolutionary roots of fish bioluminescence and that counterillumination was likely to be the first bioluminescent function to arise from this origin. Selection based on predator avoidance would have acted to increase the quantum yield of the chemiluminescent reactions and to orient light emission in a ventral direction. Once established, further gains in luminescence intensity, surpassing ambient light levels, and the development of finer controls on the kinetics of bioluminescent emission, would have made communication possible. The imidazolopyrazine luciferins are at the heart of this process, but are they synthesized by the majority of marine bioluminescent fishes, or is the dietary transfer we have discussed the predominant route accounting for the widespread use of these molecules? If the latter explanation is correct, what are the ultimate sources of these molecules in the marine environment? As considered in section V, luminescent Vargula are most probably the source of substrate for fishes exploiting Vargula lueiferin in their luminescent reactions. However, the number of fish species using this system is small in comparison to the number of marine fishes which are likely to make use of eoelenterazine. The ultimate source(s) of coelenterazine are more difficult to pinpoint. However, the organism(s) are likely to have a wide oceanic distribution, both coastal and open water, and occupy a relatively low trophie level. It is not essential that the organism(s) be bioluminescent themselves. Copepods fit well with the above criteria and bioluminescenee depletion studies seem to support this possibility. The ability of copepods (Pleuromamma, Gaussia) to recover biolumineseence ability after their luminescence potential had been completely exhausted by overstimulation has recently been investigated 65. Since both species excrete all components of their luminous system into seawater 23 ,28 ,65 , it is noteworthy that unfed individuals of both species fully recovered their luminescent potential within 24 hours. Similar experiments carded out on the deeapod Gnathophausia revealed that this crustacean was unable to synthesize coelenterazine in order to reestablish bioluminescence in the absence of a dietary source of this molecule 31. In these experiments, one cannot be certain that the depletion of copepod luminescence ability is due to the depletion of luciferin, but these observations do suggest that luminescent eopepods synthesize 1ueiferin. The luciferin in these eopepods has been characterized as coelenterazine 16. As luminescent copepods are consumed by a wide range of organisms, they could

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constitute the bulk of the luciferin sources for many luminescent fishes and invertebrates. In opposition to this proposal is a report that the luminescence of the copepod Metridia lucens was affected by its nutritional status 28. The intensity of flashes decreased in starved specimens while remaining rather stable in individuals which continued to feed. This observation does not rule out the possibility that M. lucens is able to synthesize luciferin as the diminishing luminescence capability of unfed specimens could also have resulted from overall physiological deterioration of the animal. It is of interest that in this study, the control group was fed exclusively on diatoms (Thalassiorira sp.). Thus, the possibility exists that diatoms could be the source of luciferin, either directly or through the supply of some precursor, as previously suggested 16.

VII. C o n c l u s i o n s

The relative scarcity of bioluminescence among terrestrial organisms has probably contributed to a general view of such displays as aesthetically pleasing oddities. However, relative to the terrestrial environment, the marine world presents a much greater diversity of light regimes. For those fortunate enough to have studied bioluminescence in the oceans, the pervasive importance of these phenomena in the interaction of marine organisms with their environment is clear. Probably arising initially as a response to environmental oxidative stress, the weak chemiluminescent byproduct has been harnessed and developed into a variety of functions essential for the survival and reproduction of a large number of marine fish species. The more or less random phylogenetic distribution of bioluminescent organisms, led to an initial view that bioluminescent systems had likely evolved completely independently on a number of occasions. The subsequent discovery that many of these organisms were linked by the use of common luminescent substrates has stimulated new and interesting avenues of research. Understanding of the trophic relationships between bioluminescent fishes and source organisms containing luciferin, and the characterization of the biochemical and physiological adaptations required for the effective use of these bioactive exogenous molecules, have become important areas of investigation. In this regard it is interesting to compare the imidazolopyrazine luciferins with vitamins. As is the case with vitamins, at least some fishes must acquire luciferins nutritionally 41. There are further similarities between luciferins and vitamin C. Both imidazolopyrazines and ascorbic acid are susceptible to autoxidation, stabilized forms of both types of molecule include enol-sulfate derivatives, and both react with the superoxide anion 19,84. Although these similarities may be coincidental, they might also indicate a more general strategy among organisms for the use of certain exogenous compounds against undesired oxidative processes. Thus, the study of bioluminescence in marine fishes presents challenging problems in a process which evolved as a response to environmental conditions, and has, over evolutionary time, been exploited by fishes for the manipulation of their surroundings. The knowledge we gain of these events will extend well beyond the comprehension of the production of light.

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Acknowledgements. This work was supported by the Fonds National de la Recherche Scientifique (FNRS), Belgium, and the Institut National de la Recherche Agronomique (INRA), France. Jean-Francois Rees is a Research Associate of the FNRS.

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24. Cocker, J.E. Adaptations of deep sea fishes. Env. Biol. Fish. 3: 389-399, 1978. 25. Cormier, M.J. and H. Charbonneau. Isolation, properties and functions of a calcium-triggered luciferin binding protein. In: Calcium Binding Proteins and Calcium Function, edited by R.H. Wasserman, A. Corradino, E. Carafoli, R.H. Kretsinger, D.H. McLennan and EH. Siegel, Amsterdam, Elsevier, pp. 481-490, 1967. 26. Cormier, M.J., K. Hori and Y.D. Karkhanis. The conversion of luciferin to luciferylsulfate by luciferin sulfoidnase. Biochemistry 9:1184-1190, 1970. 27. Cormier, M.J., J.M. Crane and Y. Nakano. Evidence for the identity of the luminescent systems of Porichthys porosissimus (fish) and Cypridina hilgendorfii (crustacean). Biochem. Biophys. Res. Commun. 29: 747-752, 1967. 28. David, C.N. and R.J. Conover. Preliminary investigation on the physiology and ecology of luminescence in the copepod, Metridia lucens. Biol. Bull. 122: 92-107, 1961. 29. Denton, E.J., J.B. Gilpin-Brown and B.L. Roberts. On the organization and function of the photophores of Argyropelecus. J. Physiol. 204: 38-39, 1969. 30. Denton, E.J., J.B. Gilpin-Brown and P.G. Wright. The angular distribution of the light produced by some mesopelagic fish in relation to their camouflage. Proc. R. Soc. Lond. B. 182: 145-158, 1972. 31. Frank, TM., E.A. Widder, M.I. Latz and J.E Case. Dietary maintenance of bioluminescence in a deep-sea mysid. J. Exp. Biol. 109: 385-389, 1984. 32. Goodyear, R.H., B.J. Zahuranec, W.L. Pugh and R.H. Gibbs. Ecology and vertical distribution of Mediterranean midwater fishes. In: Mediterranean Biological Studies, Arlington, Virginia, Office of Naval Research, Dept. of the Navy, pp. 91-133. 1972. 33. Haneda, Y., EH. Johnson, Y. Masuda, Y. Saiga, O. Shimomura, H.-C. Sie, N. Sugiyama and I. Takatsuki. Crystalline luciferin from live Cypridina. J. Cell. Comp. Physiol. 57: 55-62, 1961. 34. Haneda, Y., El. Tsuji and N. Sugiyama. Luminescent systems in Apogonid fishes from the Philippines. Science 165: 188-190, 1969. 35. Haneda, Y. and El. Tsuji. The luminescent systems of pony-fishes. J. Morph. 150: 539-552, 1976. 36. Haneda, Y. and EH. Johnson. The photogenic organs of Parapriacanthus beryciformes Franz and other fish with the indirect type of luminescent system. J. Morph. 110: 187-198, 1962. 37. Haneda, Y., El. Tsuji and N. Sugiyama. Newly observed luminescence in Apogonid fishes from the Philippines. Sci. Rep. Yokosuka City Mus. 15: 1-9, 1969. 38. Haneda, Y. and EH. Johnson. The luciferin-luciferase reaction in a fish, Parapriacanthus betyciformes, of newly discovered luminescence. Proc. Natl. Acad. Sci. USA 44: 127-129, 1958. 39. Haneda, Y., EH. Johnson and H.-C. Sie. Luciferin and luciferase extracts of a fish, Apogon marginatus, and their luminescent cross-reaction with those of a crustacean, Cypridina hilgendorfii. Biol. Bull. 115: 336, 1958. 40. Hart, R.C., K.E. Stempel, P.D. Boyer and M.J. Cormier. Mechanism of the enzyme-catalyzed bioluminescent oxidation of coelenterate-type luciferin. Biochem. Biophys. Res. Commun. 81: 980986, 1978. 41. Hastings, J.W. Biological diversity, chemical mechanisms and the evolutionary origins of bioluminescent sytems. J. Mol. Evol. 19: 309-321, 1983. 42. Hastings, J.W. and K.H. Nealson. Exosymbiotic luminous bacteria occurring in luminous organs of higher animals. In: Endocytobiology, Endosymbiosis and Cell Biology. Vol. l, edited by W. Schwemmler and H.E.A. Schenk, New York, de Gruyterand, pp. 467-471, 1980. 43. Hastings, J.W. Bacterial and dinoflagellate luminescent systems. In: Bioluminescence in Action, edited by P.J. Herring, London, Academic Press, pp. 129-170, 1978. 44. Herring, P.J. How to survive in the dark: bioluminescence in the deep sea. Soc. Exp. Biol. Symp. 39: 323-350, 1985. 45. Herring, EJ. Bioluminescence of Searsiid Fishes. J. Mar. Biol. Ass. U.K. 52: 879-887, 1972. 46. Herring, EJ. and J.G. Morin. Bioluminescence in fishes. In: Bioluminescence in Action, edited by P.J. Herring, London, Academic Press, pp. 273-329, 1978. 47. Herring, P.J. The spectral characteristics of luminous marine organisms. Proc. R. Soc. Lond. B. 220: 183-217, 1983. 48. Herring, EJ. Systematic distribution of bioluminescence in living organisms. J. Biolum. Chemilum. 1: 147-163, 1987. 49. Hopkins, TL. and R.C. Baird. Diet of the hatchetfish Stemoptyx diaphana. Mar. Biol. 21: 34-46, 1973. 50. Hopkins, TL. and R.C. Baird. Net feeding in mesopelagic fishes. Fish. Bull. 73: 908-914, 1975.

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Species Index Acanthopagms schlegeli, 138 Acanthustius brasilianus, 129 Acerina cernua, 104 Acipenser galdenst~dti, 423 Acipenser sp., 230 Acipenser transmontanus, 145 Aequorea sp., 443, 445 Alosa pseudoharengus, 403 Amia calva, 245 Ammodytes lancea, 128 Anguilla anguilla, 25, 36, 43ff., 50, 52, 55f., 103, 105, 107f., l lOf, 138, 163ff., 227, 395f., 401f, 419, 425 Anguilla japonica, 166f., 172, 315, 418 Anguilla rostrata, 19f., 46, 50, 55f., 69, 139, 170, 175, 181,404, 406, 414, 419 AnguUla sp., 19, 36, 102 Aphos porossus, 449f. Apogon eUioti, 439, 441f. Apogon sp., 444, 451, 453 Apteronotus albifrons, 267 Apteronotus leptorhynchus, 267 Arachmia fucata, 442 Arachmia lineolata, 442 Arapaima gigas, 76 Argyropelecus hemigymnus, 441, 454f, 459 Argyropelecus sp., 437, 439, 442, 448, 455f. Aristostomias, 445

Balaenoptera sp., 310 Benthosema fibulata, 441 Benthosoma glaciale, 448 Benthosoma sp., 442 Boleophthalmus boddaerti, 179f. Boops salpa, 222 Brevoortia tyrannus, 348 Brienomyrus brachyistius, 266 Brienomyrus sp., 266, 268 Busycon caniculatum, 299 CaUionymus lyre,343 Camssius auratus,36, 46f.,50, 52, 121, 167f.,176, 179, 292, 403, 412f.,417, 422 Carassius camssius, 46f.,52, 68, 372 Carassius sp.,52 Centrophorus squamosus, 26 Chaenocephalus aceratus,343 Channa punctata, 74 Channa sp., 128 Chanos chanos, 230

Chimaera monstrosa, 24 Chondrostoma nasus, 210 Chrysophrys major, 76, 145 Clarias batrachus, 138 Clarias lazera, 398 Clausocalanus sp., 449 Clupea harengus, 25, 128, 342 Clythia sp., 443 Cobitis biwae, 43 Columbia livia, 6 Conger sp., 26 Conger vulgaris, 26 Coregonus sp., 144 Coryphaenoides armatus, 355 Crangon septemspinosa, 453 Ctenopharyngodon ideUa, 201, 206, 210 Ctenophatyngodon sp., 144 Cyclothone braueri, 441 Cynoscion regalis, 281 Cypridina sp., 440 Cyprinodon macularius, 343 Cyprinus carpio, 46f., 5Of., 55, 89, 123, 161, 169ff., 174, 182, 230, 292, 343, 346, 352, 371, 374, 394f., 398f., 401, 404f., 408f., 413, 415f.

Diaphus coeruleus, 441 Diaphus elucens, 441, 444 Diaphus suborbitalis, 441 Dicentrarchus labrax, 20, 24f., 126, 196, 210, 341, 396, 398, 400, 402, 413, 416ff.

Dicentrarchus sp., 21 Discopyge tschudii, 143 Echiostoma barbetum, 441 Eigenmannia, 265, 267 Electrophorus electricus, 261 Electrophorus sp., 259, 263, 265, 269, 271ff. Embiotaca lateralis, 89 Engraulis mordax, 129, 348 Eptatretus stouti, 68, 89 Eptesicus ~scus, 6 Escherichia coli, 224 Esox lucius, 129, 384, 400, 403f., 419 Etmopterus spinax, 24 Euthynnus affinis, 248, 318 Euthynnus pelamis, 129 Fugu vermicularisporphyreus, 402 Fundulus heteroclitus, 55, 104, 226f., 353, 403

Species Index

468

Gadus aegle]inus, 24f. Gadus callarias, 321 Gadus morhua, 24f., 36, 46, 50, 73, 105, 107f., 122, 207, 210, 337, 398ff., 405, 407f., 413, 417f. Gadus poUachius, 122 Gadus sp., 103 Gambusia affinis, 344 Gaussia sp., 460 Gazza minuta, 455 GiUichthys mirabilis, 163 Gnathonemus petersii, 266 Gnathonemus sp., 266 Gnathophausia sp., 446, 449, 460 Gobius minutus, 344 Gobius sp., 103 Gonostoma sp., 438, 448f. Gymnarchus niloticus, 265 Gymnarchus sp., 265 Gymnotus carapo, 264 Gymnotus sp., 273 Hemitripterus americanus, 20, 66, 348, 357, 384, 412, 418, 420f. Hemitripterus sp., 20 Heteropneustesfossilis, 138 Hippoglossoidesplatessoides, 341, 347 Hippoglossus hippoglossus, 128 Hoplias malabaricus, 20, 343 Hoplias sp., 21 Hypopomus brevirostris, 262 Hypopomus occidentalis, 266 Hypopomus pinnicaudatus, 272 Hypopomus sp., 266, 268 Ictalurus melas, 418 lctalurus nebulosus, 46, 72, 403, 420 lctaluruspunctatus, 19, 122, 16Of., 168, 173f., 205, 382, 404 Ictalurus sp., 230 Isurus oxyrhinchus, 243f. Katsuwonus pelamis, lf., 6, 18ft., 36, 38, 44, 69, 248, 314, 318f., 374, 382, 284, 398 Labio rohita, 1, 129 Lampadena sp., 441 Lampanyctus sp., 448 Lampetra fluviatilis, 68, 89 Latimeria chalumnae, 26, 128 Latimeria sp., 26 Lepisosteus osseus, 129 Lepomis macrochin~s, 112, 131 Lepomis sp., 46, 52 Leuciscus hakonennsis, 316 Leuciscus idus melanotus, 75 Limanda limanda, 212, 402

Makaira nigricans, 314, 383 Malacosteus sp., 445 Malaptemrus sp., 261 Mallotus villosus, 128 Maurolicus sp., 438 Melanogrammus aeglefinus, 128 Merlangus merlangus, 128 Meduccius capensis, 402 Merluccius hubbsi, 129 Metridia lucens, 449, 461 Micropterus salmoides, 341, 352 Microstomuspaciftcus, 342, 354 Misgurnus anguiUacaudatus, 138 Miatichthys luzonensis, 343 Monopterus albus, 89 Morone saxatilis, 230, 233 Mullus surmuletus, 24 Mustelus canis, 129 Myctophum asperum, 441 Myctophum sp., 441f. Mytilus edulis, 196 Myoxocephalus scorpius, 370 Myxine glutinosa, 26, 66, 396, 411 Neoscopelus microchir, 441 Obelia sp., 443 Oncorhynchus clarki, 46, 52, 341, 347 Oncorhynchusgorbusha, 125f., 414, 423 Oncorhynchus keta, 50, 125 Oncorhynchus kisutch, 19f., 47, 128, 161,233, 382, 404, 41 lff., 418, 423 Oncorhynchus masou, 128 Oncorhynchus mykiss, 18ft., 24ff., 36, 46f., 5Of., 55, 68, 121, 161, 172, 174, 178, 192, 195, 197, 210, 213, 226f., 230, 293, 293, 295f., 314f., 319, 321, 323, 341, 348, 355f., 371, 384, 396, 398, 400, 409, 413, 418, 423ff. Oncorhynchus nerka, 22, 25f., 50, 55f., 170, 182, 315, 371, 395, 399, 423, 425 Oncorhynchus sp., 170 Oncorhynchus tshawytscha, 131, 315, 418, 420 Ophiocephalus sp., 341 Ophiophagus sp., 310 Oplophorus sp., 443 Opsanus beta, 69, 105, 107f., 116, 181, 283ff., 345, 349ff. Opsanus sp., 452 Opsanus tau, 167, 280ff., 286, 402, 412, 419, 457 Oreochromis alcalicus grahami, 181 Oreochromis [Sarotherodon] mossambicus, 53, 73, 138, 163ff., 171, 207, 225, 227f., 230f., 341, 425 Oreochromis niloticus, 139, 340, 342 Oreochromis sp., 50, 212 Osmerus mordax, 403 Pachystomias sp., 445 Pagrus major, 138

Species Index Pandalus danae, 453 Paralabrax clathratus, 170, 355, 451 Paralabrax nebulifer, 407f. Paralabrax sp., 20ft., 347, 452 Paralichthys califomicus, 346f., 355 Paralichthys olivaceus, 138 Parapriacanthus beryciformes, 441f., 449 Pampriacanthus ransonneti, 439, 442f. Parapriacanthus sp., 439, 443, 449, 451,453 Parasilurus asotus, 138 Perca flavescens, 396, 401f., 406 Perca fluviatilis, 47, 103f. Perca sp., 36, 103 Periophthalmodon schlosseri, 179f. Periophthalmus chrysospilos, 178ff. Petromyzon marinus, 66, 87, 245 Photostomias sp., 441 Phoxinus phoxinus, 344 Pimephales promelas, 131 Platichthys flesus, 47, 52 Platichthys sp., 425 Platichthys stellatus, 19, 69, 384 Plecoglossus altivelis, 145, 315 Pleuromamma sp., 448, 460 Pleuronectes platessa, 20, 56, 138, 177, 341, 401, 407

Poecilia reticulata, 128 PoUachius virens, 128 Pomoxis nigromaculatus, 403 Porichthys myriaster, 450 Porichthys notatus, 280, 282, 284ff., 436, 438, 450ff., 454, 456ff. Porichthys sp., 436, 438, 441ff., 446, 449ff., 457 Pseudemys scripta, 298 Pseudopleuronectes americanus, 122, 173

Raja clavata, 403 Raja erinacea, 71, 170, 244f., 345 Raja radiata, 24 Renilla sp., 442ff., 452ff. Rhabdamia cypselum, 442 Rhamdia hilarii, 73, 400, 402 Rutilus rutilus, 52, 55f. Salmo clarki, see Oncorhynchus clarki Salmo gairdneri, see Oncorhynchus mykiss Salmo salar, 131, 176, 201, 210, 349 Salmo tmtta, 55, 73, 115, 127, 161, 410, 413, 415, 417

Salmo trutta fario, 395, 397f., 400, 406, 408, 416 Salvelinus alpinus, 24, 125 Salvelinus fontinalis, 138, 173, 421, 425 Salvelinus namaycush, 174, 177, 181, 245

469 Salvelinus sp., 50 Sardinops caerulea, 26, 125 Sardinops sagax, 348 Sarotherodon, see Oreochromis Scardinius erythrophthalmus, 139, 182 Scomber japonicus, 327 Scomber scombms, 24f., 128, 405 Scophthalmus maximus, 131, 169 Scyliorhinus canicula, 24, 26, 129, 396, 401, 403, 411, 413

Scyliorhinus sp., 26 Searsia sp., 441, 444f. Sebastes schlegeli, 138 Sebastodes miniatus, 106, 108 Selaphorus rufus, 6 Sparus aurata, 415 Sphymena barracuda, 107 Squalus acanthias, 24, 122, 176, 182, 244f., 410ft. Steatogenys elegans, 264 Stenotomus chrysops, 106f. Sternoptyx diaphana, 456 Stemoptyx pseudobscura, 456 Stemoptyx sp., 448, 456 Stemopygus macrums, 262 Stemopygus sp., 265ff., 270ff. Stizostedion vitreum, 176 Stomatorhinus cometi, 256 Syngnathus sp., 103 Synodontis sp., 261 Thalassiorira sp., 461 Themgra chalcogramma, 137 Tilapia, see Oreochromis Tinca tinca, 83, 124 Torpedo sp., 259, 262, 269ff. Trachums sp., 46 Trachurus symmetricus, 54 Trachurus trachurus, 343 Valenciennellus sp., 448 Vargula hilgendorJii, 449 Vargula sp., 436, 440ff., 449ff., 454, 456ff. Vargula tsujii, 450 Vinciguerria attenuata, 441ff. Vinciguerria sp., 448 Watasenia scintiUans, 454 Xenopus sp., 270 Xiphias gladius, 250f. Yarella illustris, 441

Subject Index A23187 eicosanoid production, 140 leukotrienes, 142 platelet-activating factor synthesis, 143 protein kinase C, 143 steroidogenesis, 143 Absorption glucose, 225 Absorptive cells intestinal transport, 222 Accessory organs electric organ, 264 Acetate anoxia, 75 flux into fatty acids, 404 flux into lipids, 404 Acetazolamide gas gland metabolism, 114 Acetoacetate utilization in muscle, 377 Acetoacetyl CoA acetyltransferase thermogenic tissue, 247 N-Acetyl-L-histidine deacetylase anserinase, 322 N-Acetylation histidine, 329 Acetyl-Coenzyme A carboxylase covalent modification, 404 starvation, 404 Acetylcholine gas gland lactate formation, 105 salt secretion, 143 sonic motoneuron, 281 Acetylcholine esterase gas gland tissue, 112 Acetylcholine receptor (AChR), 259ff. antibodies, 269 asymmetry in electric organ, 273 clustering, 270 developmental dynamics, 270 effects of electric organ denervation, 269 localization in electrocyte, 271 mRNA injection, 270 mRNA, 270 subunit composition, 269 Acid lipase, 403 Acid protease starvation, 56 Acid secretion hypoxia, 112 Acid-base

cannulation, 37 gas gland, 112 Acidification gas gland, 111 vs. lactate release, 111 Acidosis ammoniogenesis, 176 ATP/ADP ratio, 385 mitochondrial respiration, 386 Aconitase thermogenic tissue, 247 ACITI effects on plasma fatty acids, 25 plasma fatty acids, 422 starvation, 422 F-Actin effects of enzyme binding on Kin, 301 enzyme association, 303 enzyme binding, 292 in electric organ~ 271f. kinetic effects on glycolytic enzymes, 302 muscle, 408 phosphofructokinase binding, 299 protein synthesis, 192 starvation, 408 Actinopterygii electric discharge, 260 families of electric fish, 260 Acyl-CoA: 1-acyl-sn-glycero-phosphorylcholine acyltransferase, 122 Adenylate kinase calculation of free ADP equilibrium, 386 Adenylate status muscle recovery, 380ff. Adipose tissue, 47 acid lipase, 403 amino acids, 369 catfish, 400 fatty acids, 369 gadids, 399 glucose, 369 glycerophospholipid storage, 128 glycogen, 369 lactate, 369 neutral lipase, 403 protein, 369 salmonids, 399 triacylglycerols, 369 ADP calculation of free ADP from NMR, 43

472 ADP (continued)

muscle pool, 385 muscle recovery, 381 Adrenalin, see epinephrine Adrenergic fibers gas gland, 112 Adrenoceptors signal transduction, 77 Aerobic capacity, 346, 358 effects of 17a-methyltestosterone, 287 endurance training, 83 mass-specific, 284 red muscle, 250 scaling, 346 sonic muscle, 284 tissue-specific partitioning, 352 Aerobic exercise, 377 glucose utilization, 373 Aerobic fibers red muscle, 1 Aerobic metabolism, 335ff. capacity, 338 gas gland, 109 red blood cells, 90 respiration, 340 scaling, 336ff. ~ Aerobic scaling exponent, 359 Aerobic scope body size, 359 Aerobic swiming, 20 Aglomerular kidney taurine secretion, 174 role in luciferin loss, 457 Air sac cyclooxygenase, 138 prostanoid synthesis, 139 Air-breathing fish ammoniogenesis, 176 Alanine brush border uptake, 227 flux, 22 flux to C6-products, 71 gluconeogenesis, 410, 414 migration, 55 QIO, 75 hepatic uptake, 410 hypoxia, 52 inhibitor of amino acid uptake, 227 muscle proteolysis, 170 oxidation, 22, 371 fasting, 73 kidney, 79 QIO, 75 red blood cells, 162 release from muscle, 182, 410 spawning migration, 22 starvation, 409 stimulation of intestinal ion transport, 166 turnover, 22

Subject Index

Alanine aminotransferase (GP'P, AAT), see also Aminotransferases brain, 87 effects of cortisol, 422 gut, 166 heart, 84 kidney, 78 liver, 70, 166, 396 red blood cells, 162 red muscle, 82 starvation, 406 white muscle, 81 D-Alanine stimulation of intestinal ion transport, 166 Albumin elasmobranchs, 243, 403 starvation, 408 Albumin-like protein phospholipid transport, 125 Aldolase association with F-actin, 303 binding in turtle brain, 297 binding to subcellular muscle structures, 292 effects of metabolites on enzyme binding, 295, 300 kinetic effects of F-actin, 302 loss during muscle preparation, 296 red blood cells, 90 Algae effects on glycerophospholipid composition, 133 1-O-alkyl- l'-enyl-2- acylglycerophospholipid structure, 120 1-O-alkyl-2-acylglycerophospholipid structure, 120 Alkylacylphosphofipids olfactory nerve, 129 Alkyiglycerophosphate, 122 Allometry, 340 lactate turnover, 19 metabolic, 336ff. spleen, 340 Allosteric activators glycolytic flux, 292 a-Amino-carboxylic acids brush border uptake, 227 t~-Aminoisobutyrate intestinal transport, 163 a-Bungarotoxin localization of AChR, 271 a-Globulins starvation, 408 a-Glycerophosphate elasmobranch muscle mitochondrial substrate, 244 oxidation in thermogenic tissue mitochondria, 251 a-Glycerophosphate dehydrogenase vs. lactate dehydrogenase, 249

473

Subject Index o~-Glycerophosphate dehydrogenase (continued) brown adipose tissue, 249 ot-Glycerophosphate shuttle, 247, 249 thermogenic tissue, 255 a-Ketoglutarate mitochondrial transport, 245 a-Ketoglutarate dehydrogenase control by calcium, 252 ot-(Methylamino)isobutyrate brush border uptake, 227 intestinal transport, 163 ct-Methyl-glucosides glucose transport, 223 ot-Methyltestosterone glucose permeability, 232 leucine transport, 232 Na+-glucose cotransport, 232 Na +/K+-ATPase, 232 nutrient absorption, 232 transporter induction, 232 Amino acid flux androgen action, 22 measurement, 23 protein degradation, 196 protein synthesis, 192 spawning migration, 22 Amino acid homeostasis role of intestine, 162f. Amino acid metabolism, 159ff. anoxia, 53 Km of enzymes, 205 muscle, 11, 177ff. postprandial, 48 regulation, 248 starvation, 177 D-Amino acid oxidase intestine, 166 Amino acid oxidation, 22 elasmobranch muscle mitochondria, 245 flux, 22 intestine, 165 nitrogen loss, 198 starvation, 409 thermogenic tissue, 251 vs. oxygen consumption, 22 Amino acid pool homeostasis, 199, 205 postprandial, 204 protein synthesis, 198 Amino acid recycling prelabelled proteins, 196 protein synthesis, 193 Amino acid transport basolateral membrane, 164 between red blood cells and plasma, 161 brush border membrane vesicles, 163 induction, 235 limits to hepatic protein synthesis, 167 Na+-dependence, 163

vs. protein synthesis, 165 Amino acid uptake protein synthesis, 198 stimulation by growth hormone, 233 Amino acid utilization deamination, 205 oxidation, 205 protein synthesis, 205 Amino acids adipose tissue, 369 as endogenous fuel, 48 brush border uptake, 226f. carbon dioxide prodcution, 22 concentration in plasma, 23 concentration in red blood cells, 23 content of muscles, 50 cortisol, 69 extracellular fluid, 369 gluconeogenesis, 22, 422 gradient from liver to plasma, 167 hepatectomy, 167 homeostasis, 199 imbalance, 206 infusion, 371 interorgan flux, 180f. intestinal transport, 222 ketogenesis, 244 kidney, 49, 173 liver, 49, 166, 369 maintenance cost, 370 mucus production, 22 muscle, 49 Na-amino acid coupling ratios, 228 Na+-dependent uptake, 226 Na+-independent transport, 228 oxidation in elasmobranchs, 22 oxidation, 21, 370 plasma concentration at rest, 17 postprandial, 161, 199 red muscle, 369 renal absorption, 173 renal levels, 173 starvation, 407 stereospecific uptake, 226 stimulation of protein synthesis, 199 tissue pools in fasting, 160 transport, 226 utilization, 371 white muscle, 369 2-Amino-2-norbonane carboxylic acid brush border uptake, 227 Aminoacyl-tRNA synthetases Kms, 167, 205 Aminooxyacetate alanine transport, 166 aminotransferases, 168 ammonia production, 180 glutamate oxidation, 245 Aminotransferases, 171

474 Aminotransferases (continued) aminooxyacetate, 168 compartmentation, 168 diet, 172 kidney, 174 starvation, 171 Ammonia amino acid oxidation, 205 during kidney passage, 176 excretion, 371 hepatic portal vein, 166 muscle, 380 nitrogen loss, 198 protein synthesis, 196 removal, 338 Ammonia excretion, 405 cydoheximide, 106, 205f. effects of cortisol, 422 exercise, 53 indirect calorimetry, 41 postprandial increase, 205f. postprandial, 214 starvation, 167, 171 Ammonia production aminooxyacetate, 180 bromofuorate, 180 from glutamine, 180 hepatocytes, 162 vs. glutamate oxidation, 162 vs. glutamine oxidation, 162 Ammonia quotient, 42 Ammoniogenesis acidosis, 176 air-breathing fish, 176 intestine, 166 liver mitochondria, 167 muscle, 179 AMP deaminase, 380, 386 muscle recovery, 381, 385 muscle, 380 AMP deaminase, 386 purine nucleotide cycle, 386 Amphibians distribution of histidine-related dipeptides, 312 Amplitude electric organ discharge, 263 Amylase intestine, 223 Anabolic steroids regulation of nutrient transport, 232, 235 Anabolic stimulation efficiency definition, 193 protein synthesis, 198 Anabolism scaling exponents, 212 Anaerobic capacity correlation with muscle buffering, 327 Anaerobic energy production

Subject Index red muscle, 370 white muscle, 370 Anaerobic glycolysis, 106 burst swimming, 314 gas gland tissue, 106 rete mirabile, 115 Anaerobic metabolism, 359 burst swimming, 80 limitations, 358 Anaerobic potential muscle, 355 Anaerobic scope, 357 Anaesthesia, 35 erythrocyte swelling, 37 Androgens, 232 amino acid flux, 22 effects on nutrient transport, 233f. influence on electric organ discharge, 266ff. sonic muscle, 287 spawning migration, 22 white muscle proteins, 22 Anoxia, 87 acetate, 75 amino acid metabolism, 53 brain ketone bodies, 88 enzyme binding, 298 ethanol production, 75, 355 fatty add levels, 52 fructose 2,6-bisphosphate, 74 glucose production, 74 glycogen phosphorylase activity, 74 lipogenesis, 52 liver glycogen, 75 metabolic rate, 291 oxygen debt, 75 Pasteur effect, 74 protein degradation, 53 seasonality, 77 tolerance in carp, 74 Anoxic stress brain, 87 Anserinase, 321f. anserine degradation, 322 brain, 322 muscle, 322 Anserine biosynthesis, 321 degradation, 322f. from carnosine, 322 from injected histidine, 320 growth, 318 heart, 329 imidazole dipeptides, 309 imidazole pK value, 325 incorporation into kidney, 324 incorporation into liver, 324 interorgan transport, 323 red muscle, 313 structure, 309

475

Subject Index

Anserine (continued) white muscle, 313 Anterior intestine amino acid absorption, 163 peptide absorption, 163 Anti-dystrophin antibody immunocytochemistry of electrocyte, 271 Antioxidants gas gland, l15f. origin of luminescence, 459 Aperture photophore, 438 Apoproteins enzyme activation, 126 in luminous system, 443 lipid transport, 124 lipoproteins, 126 receptor binding, 126 Arachidonate eicosanoid metabolism, 138f. Arachidonic acid cascade, 138 Arginase kidney, 175 liver, 175 Arginine brush border uptake, 227 intestinal transport, 163 osmoregulation, 316 Arginine vasotocin effects on plasma fatty acids, 25 L-U-14C-Arginine protein synthesis, 195 Aromatase electric organ discharge frequency, 267 Ascorbate autoxidation, 453, 461 Asparaginase kidney, 176 liver, 169 muscle, 178 Asparagine synthetase kidney, 176 muscle, 178 liver, 176 Aspartate conversion to succinate, 53 liver mitochondrial substrate, 167 muscle, 160 purine nucleotide cycle, 386 red blood cells, 161 turnover, 22 Aspartate aminotransferase (GOT, DAT), see also Aminotransferases brain, 87 effects of cortisol, 422 elasmobranch muscle mitochondria, 245 glutamate oxidation, 245 gut, 166 heart, 84, 285

in glutamate metabolism, 246 kidney, 78 liver, 70, 166, 396 red blood cell, 162 red muscle, 82, 285 sonic muscle, 285f. starvation, 406 toadfish heart, 285 toadfish white muscle, 284ff. white muscle, 81, 284ff. Aspartate-glutamate carrier in glutamate metabolism, 246 ATP effects on glycolytic enzyme binding, 295 hypoxic muscle, 51 muscle recovery, 381 ATP buffering role of phosphocreatine, 380 ATP hydrolysis, 34 heat production, 255 during tissue extraction, 39 ATP production burst swimming, 291 ATP synthesis, 34 ATP utilization red muscle, 370 white muscle, 370 ATP-citrate lyase starvation, 404 ATP/ADP ratio glycogenesis, 82 in acidotic tissue, 385 ATP synthesis, 386 ATPase thermogenesis, 242 Atropine gas deposition, 112 Autoxidation ascorbate, 453, 461 imidazolopyrazines, 461 luciferins, 461 Back diffusion rete mirabile, 112 swimbladder, 102 Balenine degradation, 322 HPLC, 311 imidazole dipeptides, 309 imidazole pK value, 325 red muscle, 313 structure, 309 vertebrate muscle, 309 white muscle, 313 Band Ill anion exchanger red blood cell, 114 Basolateral glucose transport, 225, see glucose transport Basolateral membrane

476 Basolateral membrane (continued) amino acid release, 162ff. transport processes, 222 Na +/K+-ATpase, 222 Basolateral membrane vesicles amino acid transport, 164 Basolateral Na +/K+-ATpase, 163 Bat flight muscle characteristics, 2 muscle morphometric data, 6 Benzothiazole in click beetleluciferin, 444 fl-Alanine carnosine synthesis, 320 incorporation into carnosine, 320 intestinal transport, 226 17fl-Estradiol effect on electric organ discharge, 266f. ,0-Globulins, 408 starvation, 408 fl-Hydroxybutyrate elasmobranch muscle mitochondrial substrate, 244 elasmobranch muscle, 246 monocarboxylate carrier, 246 oxidation in teleosts, 248 oxidation in thermogenic tissue mitochondria, 251 utilization in muscle, 377 fl-Hydroxybutyrate dehydrogenase mitochondria, 246 teleosts, 248 fl-Methyl-glucosides glucose transport, 223 fl-Oxidation fatty acids, 34 fl-Receptors heart, 85 BF-2 cell line cost of protein synthesis, 208f. fractional protein synthesis rate vs. oxygen consumption, 208 glycerophospholipid fatty acids, 131 Bicarbonate gas gland entry, 114 proton transfer, 114 source of carbon dioxide in gas gland, 108 Bile salts lipase, 123 phospholipase A2 Billfishes thermogenic tissue, 242 Binding protein fatty acids, 86 luciferin, 452 Bioluminescence, 435ff. energy transfer, 445 origins, 459 spectral shifts, 445

Subject Index Birds distribution of histidine-related dipeptides, 312 muscle buffering capacity, 326 Blood acidification by gas gland, 111 circulation time, 339 glucose after hepatectomy, 68 histidine-related dipeptides, 314 6-keto prostaglandin Fla, 139 lipoxygenase products, 139 nutrient transport from gut, 221 velocity, 339 volume scaling, 339 Boatwhistle, 279 Body mass lactate turnover, 19 Body weight scaling with protein metabolism, 212 Bohr effect gas gland, 111 Bolus (flooding dose) injection fractional rate of protein synthesis, 195 isolated cells, 196 isolated organs, 196 metabolite turnover, 16f. protein synthesis, 192 Bouton glycogen, 281 mitochondria, 281 sonic muscle, 281 Boyle's law, 101 Bradycardia hypoxia, 50 Brain amino acids, 53, 160 exercise, 53 amino acids in fasting, 160 anoxia, 88 anoxic stress, 87 anserinase, 322 carbon dioxide production, 89 cyclooxygenase, 138 glucose uptake, 86 glucose utilization, 66 glycerophospholipid composition, 128f. glycogen content, 46, 66f., 86 glycolytic enzyme binding, 297 histidine-related dipeptides, 314 3-hydroxybutyrate dehydrogenase, 403 hypoglycemic stress, 87 insulin hypoglycemia, 86 ischemia, 87 ketone bodies, 88 lactate dehydrogenase scaling, 355 lipoxygenase products, 139 oxygen consumption, 89 prostanoid synthesis, 139

477

Subject Index

Brain (continued) succinate dehydrogenase, 346 Brain heater, see also thermogenic tissue, 243 ATPases, 253 heat production, 252 metabolism, 250ff Na +/K+-ATl'ase, 254 Branched-chain or-ketoacid dehydrogenase localization, 175 phosphorylation/dephosphorylation, 175 Branched-chain amino acid aminotransferase influence of diet, 177 tissue distribution, 174 Branched-chain amino acids liver metabolism, 181 muscle metabolism, 181 Branchial pump metabolic cost, 345 Bromofuorate ammonia production, 180 Brown adipose tissue, 249 heat production, 252 ketone body oxidation, 251 proton channel, 241 Brush border amino acid uptake, 226f. Na/glucose transporter, 223 transport amino acids, 165 glucose, 165 Brush border membrane amino acid uptake, 162 glycerophospholipid headgroup composition, 129 phospholipid changes with seawater adaptation, 135 vesicles, 227, 231 amino acid transport, 163 dipeptide transport, 164 proline transport, 231 renal amino acid uptake, 173 intestinal amino acid uptake, 173 Buffering histidine-related dipeptides, 324 non-bicarbonate, 325 Buffering capacity correlation with anaerobic capacity, 327 correlation with lactate dehydrogenase, 327 correlation with myoglobin content, 327 gas gland, 112 heart, 85 muscle homogenates, 328 white muscle, 356 Burst exercise, 367, 375, 378 anaerobic metabolism, 80, 359 ATP demand, 378 ATP production increase, 291 creatine phosphate, 45 glycogen, 80

glycolysis, 314 scaling, 358 Ca 2+ -ATPase elasmobranch muscle, 243 sonic muscle, 282 thermogenesis, 242, 252 Calcium control of mitochondrial enzymes, 252 during tissue extraction, 39 eicosanoid production, 140 enzyme binding, 299 in luminous systems, 443 inhibition of enzyme binding, 300 phospholipase A2, 138 protein kinase C, 143 sonic muscle, 282 Calcium ionophore, see A23187 Calorimetry, 41f. Camouflage bioluminescence, 455 counterillumination, 437 photocytes, 437 cAMP enzyme binding, 299 glucagon, 77 isotocin, 77 phosphoinositide cycle, 142 seasonal response, 77 vasotocin, 77 Cannibalism source of luciferin, 450 Cannulation acid-base, 37 blood gas analysis, 37 dorsal aorta, 37 turnover studies, 41 Capacity for protein synthesis definition, 193 Capillaries degree of orientation, 7 density, 3 gas gland rete mirabile, 115 geometry, 8 length density, 5 length per fiber volume, 8 manifolds, 3f., 11 orientation, 3, 5 surface per fiber volume, 10 tortuisity & branching, 7 Capillarity vertebrate muscles, 6 Capillary density, 7 length density, 7f. Capillary manifolds, 3ft. hummingbird flight muscle, 4 muscle, 11 red muscle, 3f., 11 Capillary network

478 Capillary network (continued) gas gland, 103 photogenic tissue, 438 Capillary to fiber ratio, 3 Capillary-fiber surface ratio, 11 Carbohydrate homeostasis, 66 Carbohydrate metabolism, 65ff. red blood cells, 89f. thermogenic tissue, 251 Carbohydrates as endogenous fuel, 45ff. Carbon dioxide from bicarbonate, 108 gas gland metabolism, 109 production indirect calorimetry, 41 swimbladder, 106 amino acids, 22 brain, 89 removal, 338 Carbonic anhydrase gas gland, 109 gas gland metabolism, 113ff. red blood cell, 114 rete mirabile, 113 Carboxypeptidase A starvation, 407 Cardiac myoglobin scaling, 342 Cardiac output, 340 contraction frequency, 344 stroke volume, 344 vs. lactate flux, 18 Cardiac pump metabolic cost, 345 Cardiolipin, 120f. brush border membranes, 129 gill, 129 gill mitochondria, 129 liver, 129 muscle mitochondria, 129 Cardiomyocytes cyclooxygenase, 138 6-keto prostaglandin Fla, 139 prostaglandin synthesis, 140 prostanoid synthesis, 139 thromboxane synthesis, 140 Cardiovascular system prostanoids, 142 Carnitine acyl transferase elasmobranchs, 243 Carnitine palmitoyl transferase, 377 thermogenic tissue, 251 Carnivores, 211 glucose uptake, 230 intestinal nutrient transport, 230 Carnosinase, 322 muscle, 321 Carnosine, 318

Subject Index as neurotransmitter, 329 biosynthesis, 320 from histidine, 320, 322 heart, 329 imidazole dipeptides, 309 imidazole pK value, 325 N-methylation, 321 osmoregulation, 315f. red muscle, 313 starvation, 317f. structure, 309 synthetase-like enzyme, 321 white muscle, 313 Carnosine N-methyltransferase carnosine biosynthesis, 320f. Carrier-mediated transport, 222f. Carriers basolateral membrane, 223 brush-border, 223 Catabolism scaling exponents, 212 Catalase gas gland, 115 Catecholaminergic fibers photogenic tissue innervation, 438 Catecholamines, 68 anoxia, 52 effects on plasma fatty acids, 25 gas gland lactate formation, 105 gluconeogenesis, 68 glucose turnover, 21 glycogenolysis, 68 lipolysis, 52 red blood cells, 91 role in thermogenic tissues, 255 signal transduction, 77 swimbladder perfusion, 112 Catfish Na+/glucose transporter, 224 Cathepsin spawning migration, 22 starvation, 56, 396, 405, 407 Catheterization, flux measurements, 17 cDNA Na+/glucose transporter, 223 CDP-choline-l,2diacylglycerol choline phosphotransferase brain microsomes, 121 liver microsomes, 121 temperature acclimation, 135 CDP-ethanolamine phosphotransferase hepatocytes, 121 temperature effects, 135 Cell lines glycerophospholipid fatty acids, 131 Cell proliferation hyperoxia, 115 Cephalopods

Subject Index Cephalopods (continued) luminescent system, 446 cOMP enzyme binding, 299 Chemiluminescence bioluminescent systems, 444 Chicken cost of protein synthesis, 209 Chloride cells phosphoinositide cycle, 142 Chloride dependence taurine secretion, 174 transport, 222 Cholesterol implants control for experiments with steroids, 233 Choline diet, 145 in glycerophospholipids, 120 Cholinergic fibers gas gland, 112 CHSE-214 glycerophospholipid fatty acids, 131 Chylomicron-like particles lipid transport, 124 Chylomicrons composition, 25, 124 hydrolysis, 126 intestinal production, 125 Circulation convective circulation, 338 lactate exchange, 19 metabolic substrates, 15 oxygen delivery, 338 Citrate, 113 Citrate synthase brain, 87 gas gland tissue, 105 heart, 84, 285 liver, 70 mass specific activity, 285 muscle, 81f., 250, 285ff., 35411". red blood cells, 90 red muscle, 82, 250, 355 scaling in white muscle, 354 sonic muscle, 284ff. thermogenic tissue, 247, 251 toadfish heart, 285 toadfish white muscle, 284ff. white muscle, 81, 284ff., 354f. Click beetle luciferin, 444 recycling of luciferin, 454 Coelacanth brain glycerophospholipid composition, 128 plasma lipoproteins, 25 Coelenteramine recycling of coelenterazine, 456 Coelenterazine, 436, 460 cephalopods, 446

479 content of copepods, 456 crustaceans, 446 decarboxylation, 444 diffusibility, 457 distribution in fishes, 441 enol-sulfate, 452 glucuronidation, 442 liver, 442 luciferin regeneration, 442 luciferyl-sulfate, 442 mechanism of oxidation, 444 peroxide formation, 444 pyloric caeca, 442 recycling mechanism, 454, 456f. stable derivatives, 452 storage, 452 structure, 440 Collagen percent of whole animal protein, 213 Communication bioluminescence, 436 photophores, 436 short-range, 437 Compartmentation aminotransferases, 168 glutamate metabolism - schematic, 245 Consumption-growth relationship growth rates, 202 Continuous infusion metabolite turnover, 16f. protein synthesis, 192 Contractile protein percent of whole animal protein, 213 Contraction cycle parvalbumin content, 283 Contraction frequency, 344f. negative allometry, 345 Convective transport, 340 amino acids, 161f. oxygen, 339 Copepods bioluminescence, 448 coelenterazine content, 456 fish diet, 448 luciferin synthesis, 460 Copper transport histidine-related dipeptides, 324 Cori cycle, 69, 71 Cortisol activation of liver enzymes, 69 amino acid availability, 69 carbohydrate metabolism, 76 chronic effects, 421 effect on liver enzymes, 69, 421 effects on other hormones, 421 effects on plasma fatty acids, 25 gluconeogenesis, 69, 421 glucose turnover, 21 glucose uptake, 421

480 Cortisol (continued) handling stress, 37 lipolysis, 52 liver binding, 423 liver enzymes, 69, 76, 421 parr-smolt transformation, 233 proline transport, 233 protein catabolism, 202, 421 protein synthesis, 202 seawater adaptation, 233 spawning migration, 423 starvation, 421 stress, 202, 232 during perfusion experiments, 232 Cost of protein synthesis, 208ff. Counter-current heat exchange red muscle, 1 Counter-current system gas gland, 110, 112 single concentrating effect, 110 swimbladder, 102 Counterillumination, 455 photophores, 437 Creatine muscle recovery, 381 phosphorylation, 43, 381 Creatine phosphate exhausting exercise, 54 hypoxic muscle, 51 muscle, 45 Creatine phosphokinase, 34 binding to subcellular muscle structures, 292 calculation of free ADP, 386 mass action ratio, 379 Creatinine HPLC, 311 Critical speed, see Ucri, Crocodiles distribution of histidine-related dipeptides, 312 Cross-over plot, 287 Crustaceans luminescent system, 446 Current electric organ discharge, 263 Cyanide gas gland cytochrome oxidase, 115 inhibition of luminescence, 442 a-Cyano-3-hydroxycinnamate pyruvate transport, 90 Cycloheximide ammonia excretion, 205f. protein synthesis, 205 Cycloleucine intestinal transport, 226 Cyclooxygenase eicosanoid metabolism, 137f. dihomo-y-linolenic acid, 141 tissue distribution, 138 Cysteine

Subject Index

brush border uptake, 227 Cytochalasin B glucose transport, 225 C~ochrome c oxidase aerobic capacity, 352 carp muscle, 352 carp viscera, 352 copper, 329 gas gland tissue, 105, 115 raven heart, 357 Cytoplasmic actin, 271 D5 desaturase, 134 Deiodinase diet, 426 2-Deoxyglucose, 379 glucose transport, 223 glucose uptake, 21 transport by white muscle, 382 uptake in red muscle, 373 Dephosphorylation activation of pyruvate dehydrogenase, 380 Depolarization electric organ, 263 Desaturase, 134f. temperature acclimation, 135 Desaturation fatty acids, 134 Desmin electrocyte, 271 Detergent solubilization bound vs. free enzymes, 294 Development AChR, 270 Diacylglycerol phosphoinositide cycle, 142 protein kinase C, 143 structure, 120 Diacylglycerol lipase eicosanoid metabolism, 138 Diacylglycerophospholipid structure, 120 Diet choline, 145 effects on glycerophospholipid composition, 133 glycerophospholipids, 145 inositol, 145 intestinal transport, 230 luciferin, 436 natural vs. artificial, 76 renal aminotransferase, 177 renal glutamate dehydrogenase, 177 Dietary lipid protein sparing, 206 Diffusion amino acid uptake, 164 coefficient, 338 luciferin, 458

Subject Index

Diffusion distance, 338 intrafiber, 9 oxygen flux, 9 Digestion heat production, 242 Digestive enzymes nitrogen loss, 198 Digestive tract luciferase, 442 Dihomo-F-linolenic acid cyclooxygenase, 141 Dihydrotestosterone effect on electric organ discharge, 266f. Dihydroxyacetone phosphate pathway ether-linked glycerophospholipids, 122 Dilution method enzyme binding to subcellular muscle structures, 293f. Dioxygenase eicosanoid metabolism, 137f. Dipeptide transport brush-border membrane vesicles, 164 intestine, 163f. Dipeptides, see also histidine-related dipeptides brush border uptake, 227f. Diurnal variation protein synthesis, 196 DNA liver content with starvation, 395 muscle content with starvation, 395 starvation, 406 Dominance hierarchy growth, 201 protein synthesis, 201 Dormancy low temperature, 72 Dorsal aorta cannulation, 37 Down-regulation metabolic, 401 glucagon receptors, 420 Drag, 344, 356 Duchenne muscular dystrophy dystrophin, 270 Duration electric organ discharge, 263 Dystrophin in electric organ, 270 localization in electrocyte, 271 Dystrophin-related protein in electric organ, 270 Edema hyperoxia, 115 Eggs glycerophospholipid composition, 128 luciferin, 458 reabsorption, 458 transfer of luciferin, 450 Eicosanoid metabolism, 137ff.

481

phosphatidylinositol fatty acids, 131 Eicosanoids, 24; see also individual group distribution, 138 fatty acid precursors, 139f. function, 142 metabolism 137ff. production, 139 sources, 141 thrombocyte aggregation, 142 tissue distribution, 138 Elasmobranchs albumin, 243, 403 amino acid oxidation, 22 carnitine acyl transferase, 243 distribution of histidine-related dipeptides, 312 electric discharge, 260 erythrocyte plasmalogens, 129 ether-linked glycerophospholipids, 122 fasting, 397, 411 fatty acid carrier protein, 243 fatty acid oxidation, 243, 377 glutaminase, 244 glutamine oxidation, 22 glycemia, 397, 412 ketone bodies, 243, 377, 402f., 411 kidney, 79 glutaminase, 176 glutamine synthetase, 176 leukocyte prostacyclin, 139 lipid oxidation, 22 location of electric organs, 260 mitochondrial substrates, 244 muscle ATPases, 243 carnitine acyl transferase, 243 lipid content, 401 mitochondria substrates, 244 phosphoinositide cycle, 142 plasma fatty acids, 24, 243 plasma glucose, 247 plasma glutamine, 244 red muscle mitochondria hexokinase, 247 respiratory control ratio, 247 role of insulin, 411 Elastin percent of whole animal protein, 213 Electric eel electric organ, 261 Electric fish distribution, 260 location of electrocytes, 260 Electric organ (EO), 259ff. discharge, 260 evolution, 261 F-actin, 271 from oculomotor muscle, 261 GTP binding protein, 273 innervation, 269

482 Electric organ (EO) (continued) keratin, 272 microtubules, 273 myogenic, 260, 265 nicotinic cholinergic neurons, 262 pacemaker nucleus, 262 Electric organ discharge (EOD) amplitude, 263 diphasic, 264 duration, 263 effects of steroid hormones, 266f. frequency, 265 in fish families, 260 in pulse-type fish, 262. in wave-type fish, 262 monophasic, 263 pulse-type, 262f. sex differences, 262f. triphasic, 264 waveform, 262 Electric ray inositol lipid metabolism, 143 phospholipase C, 142 Electrocytes, 263 anatomy, 264 desmin, 271 EPSP, 264 expression of my5, 270 expression of MyoD, 270 expression of myosin, 270 expression of tropomyosin, 270 F-actin, 271f. inositol phospholipid metabolism, 143 keratin-like protein, 272 Li-sensitive phosphatase, 143 location in fish, 260 myofibrils, 271 myosin, 270, 272 Na + current, 268 phospholipase C, 143 plasmalemma, 272 sarcomere, 261 T-tubule system, 261 voltage-clamp, 268 Z bands, 272 Electrolocation, 261 Electromotor neuron, 265 location in electric fish, 260 innervation of electric organ, 262 Electroreception, 259 Elongation fatty acids, 134 Emission spectrum photophores, 437 Endogenous fuels amino acids, 48 carbohydrates, 45ff. high-energy phosphates, 45 lipids, 47

Subject Index

proteins, 48 Endoplasmic reticulum gas gland cells, 103 Endothermic tissue fishes, 242 heat production, 242 Endothermy, 242 Endurance swim, 375 Endurance training effects on enzymes, 83 aerobic capacity, 83 Energy minimization protein synthesis, 214 Energy transfer photoproteins, 445 Energy use, 337 Enterocytes basolateral membrane, 229 transport, 229 turnover, 162 Enzyme association synaptosomes, 296 Enzyme binding artifacts, 298 metabolic triggers, 298f. Epinephrine, see also catecholamines effects of glucocorticoids, 421 effects on luminescence, 454 gas gland lactate formation, 112 gill inositol lipid turnover, 142 glycogenolysis, 73, 420 handling stress, 37 Epithelial cells photophore secretions, 438 Epoxides eicosanoids, 139 EPSP electrocytes, 264 Erythrocytes, see also Red blood cells concentration, 340 release from spleen, 341 storage, 338 Escherichia coli

Na +/proline transporter, 224 Essential amino acids fasting, 409 oxidation, 22 plasma, 161 postprandial, 161, 204 Estradiol leucine transport, 232 regulation of nutrient transport, 232 sonic muscle, 287 Estrogen, 232, 287 effect on electric organ discharge, 266tf. sonic muscle, 287 vitellogenin, 127 VLDL, 127 Ethanol

483

Subject Index

Ethanol (continued) anoxia, 75, 354 cyprinids, 354 hypoxia, 52, 354 Ethanolamine in glycerophospholipids, 120 Ether-linked glycerophospholipids, 122 dihydroxyacetone phosphate pathway, 122 elasmobranchs, 122 Etioluciferin, 454 recycling of coelenterazine, 456 Euphausids fish diet, 448 Everted gut sac nutrient transport, 232 Evolution electric organs, 261 luminescence, 459ff. Na +/glucose transporter, 224 Excretion ammonia, 41, 53, 106, 167, 171, 205ff., 214, 371, 405, 422 Exercise, 374 aerobic, 377 ammonia excretion, 53 carbohydrate utilization, 371 catecholamines, 91 dephosphorylation of pyruvate dehydrogenase, 380 enzyme binding, 298 fatty acids, 53 free fatty acids, 376 fuel selection, 368, 371 glucose turnover, 21 glycogen repletion, 82 glycolytic enzyme binding, 292, 303 glycolytic flux, 303 lactate load, 81f. lactate removal, 82 lactate turnover, 19 lipid oxidation, 53 metabolic acid load, 81f. metabolic control, 368 metabolism, 369 metabolite levels, 368 phosphagens, 385 plasma lactate, 18 protein fuels, 53 purine nucleotide cycle, 179 Exhausting exercise, 378 creatine phosphate, 54 dipeptides, 318 glycogen, 54, 380 glycogenolysis, 54 histidine, 318 lactate, 54 lactate release, 55 phosphocreatine, 380 purine nucleotide cycle, 179

Exocytosis electric organ, 273 Golgi apparatus, 273 Extracellular fluid amino acids, 369 fatty acids, 369 glucose, 369 glycogen, 369 lactate, 369 protein, 369 triacylglycerols, 369 Extraocular muscle thermogenic tissue, 250 Eye anserinase, 322 phospholipid changes with seawater adaptation, 135 heater, 243 F-Actin, see actin Facilitated glucose transport, 225 Faecal nitrogen loss daily balance, 197 Fasting, see also starvation, 72ff., 393ff. alanine oxidation, 73 amino acid entry, 229 amino acid turnover, 22 DNA, 406 fatty acid composition, 401 fatty acid depletion, 402 fatty acid unsaturation, 402 free amino acids, 160 fructose 1,6-bisphosphatase, 73 glucagon-insulin ratio, 72 glucose turnover, 21 glycogen phosphorylase, 72, 74 growth hormone levels, 423 hormone titers, 72 hypermetabolism, 73 hypoglycemia, 73 hypometabolism, 72f, 401. XCF,424 ketone bodies, 402f. lactate turnover, 19 lipid mobilization, 56 lipogenesis, 404 lipolysis, 403 liver composition, 395 liver glycogen, 72, 394 muscle composition, 395 muscle lipid, 401 muscle protein synthesis, 56 PEPCK, 73 plasma fatty acids, 402 triacylglycerols, 401 Fat tuna red muscle, 248 sonic muscle, 283 Fat fuels, 377

484 Fat metabolism recovery, 384 Fat oxidation, 378 Fat synthesis from amino acids, 169 from glutamate, 170 Fatigue, 370 Fatigue resistance sonic fibers, 283 Fatty acid desaturase, 134f. Fatty acid binding protein transport, 23 elasmobranchs, 243 Fatty acid metabolism temperature effects, 134 Fatty acid oxidation anoxia, 52 elasmobranch muscle, 243, 377 heart, 84 pyruvate utilization recovery, 384 red muscle, 80 teleost muscle, 378 white muscle, 384 Fatty acid synthesis starvation, 404 Fatty acids, see also PUFA adipose tissue, 369 as anaerobic end products, 52f. /I-oxidation, 34 binding proteins, 86 composition in starvation, 401 composition of glycerophospholipids, 132ff. depletion in starvation, 402 desaturation, 134 elongation, 134 extracellular fluid, 369 flux, 23 from acetate, 404 from amino acids, 170 from glucose, 170 hormone effects on plama fatty acids, 25 liver, 369 membrane fluidity, 402 oxidation, 21 oxidative substrates, 243 plasma concentration, 17, 24 red muscle, 369 synthesis from acetate, 404 unsaturation in starvation, 402 white muscle, 369 Feeding amino acid pool size, 204 essential amino acids, 204 FHM cell line glycerophospholipid fatty acids, 131 Fiber length, 337 Fiber size, 6 Filters

Subject Index

luminescent species, 436 Fin alanine biosynthesis, 183 cyclooxygenase, 138 Firefly recycling of luciferin, 454 Flight muscle bat, 2 hummingbird, 2 pigeon, 3 Flooding dose technique, see Bolus injection Flux regulation, 18 Food consumption effects of growth hormone, 233 Food protein sonic muscle, 282 Foraging behaviour metabolism, 359 Foregut swimbladder, 102 Fork length vs. intestine, 230 Fractional rate of protein synthesis methodology, 195 Free amino acid pool daily balance - feeding, 197 daily balance - starving, 198 Free amino acids, 159ff. a-keto acids, 160 intracellular pool, 159 metabolic connections, 160 extracellular pool, 159 Free fatty acids, see also Fatty acids exercise, 376 from viscera, 377 muscle, 376 red muscle, 48 utilization, 376 white muscle, 369 Free radical scavenger histidine-related dipeptides, 324 Freeze clamping tissue sampling, 38 Frog distribution of histidine-related dipeptides, 312 Fructose intestinal transport, 223 Fructose 1,6-bisphosphatase binding of aldolase, 300 binding of glyceraldehyde 3-phosphate dehydrogenase, 300 brain, 87 competition with F-actin, 301 effects of cortisol, 421 fasting, 73 heart, 84 iodoacetate, 299 kidney, 78 liver, 70

Subject Index Fructose 1,6-bisphosphatase (continued) red blood cells, 90 red muscle, 82 white muscle, 81 Fructose 2,6-bisphosphate anoxia, 74 Fructose 6-phosphatase starvation, 414 Fructose 6-phosphate iodoacetate, 299 Fuels exercise, 368 mobilization, 49f. selection, 371 Fumarase gas gland tissue, 105 thermogenic tissue, 247 Furan fatty acids neutral lipids, 130 G protein, 273 phospholipase A2, 138 Galactose absorption, 223 intestinal transport, 223 y-Globulins starvation, 408 Gas diffusional loss from gasbladder, 102 partial pressure, 102 resorption in swimbladder, 102 Gas deposition acidification, 111 effects of sulfonamide, 113 gas gland, ll0f. Gas gland acid production, 106, 112 anaerobic glycolysis, 106 antioxidants, 115f. bicarbonate entry, 114 buffering capacity, 112 capillary network, 103 carbon dioxide production, 106 cells, 102 contamination with other cells, 104 counter-current system, 110 endoplasmic reticulum, 103 glucose utilization, 106 glycolytic activity, 112 Golgi apparatus, 103 hormonal control, 112 hypoxia, 112 lactate dehydrogenase, 106 lactate formation, 106, 115 lactate levels, 110 lipids, 104 metabolic control, 112 metabolism schematic, 109 microvilli, 103

485 mitochondria, 103 morphology schematic, 104 perfusion, 105 proton transfer schematic, 114 fete mirabile, 110 ribosomes, 103 vagai control, 112 Gas gland tissue enzyme activities, 105 glycogen, 106 glycolytic enzymes, 107 mass-specific oxygen uptake, 105 oxygen uptake, 105 respiratory chain, 105 Gas partial pressure increase in gas gland, 110 Gastrocnemius histidine turnover, 319 Genetic adaptations nutrient transport, 230 Gill amino acids in fasting, 160 branched-chain amino acid aminotransferase, 174 cardiolipin, 129 cyclooxygenase, 138 glycerophospholipid headgroup composition, 129 histidine-related dipeptides, 314 inositol lipid turnover, 142 6-keto prostaglandin Fla, 139 lipoxins, 139 lipoxygenase products, 139 loss of luciferin, 457 Na +/K +-ATpase pressure adapation, 136 membrane lipids, 136 phospholipid changes with seawater adaptation, 135 platelet-activating factor synthesis, 143 prostanoid synthesis, 139 scaling, 343f. surface area, 343ff. symmorphosis, 343 Gill mitochondria cardiolipin, 129 Gill perfusion hypoxia, 50 Glomerulus role in luciferin loss, 457 GLP, see Glucagon-like peptide, 68, 415ff. Glucagon, 415ff. absence from hagfish gut, 411 binding sites, 420 effects of glucocorticoids, 421 effects on plasma fatty acids, 25 gas gland lactate formation, 112 gluconeogenesis, 68 glucose turnover, 21

486

Glucagon (continued) glycogenolysis, 68, 73, 418 lipolysis, 419 liver glycogen, 418 plasma fatty acids, 419 re-feeding, 74 receptors, 72f., 419f. down-regulation, 420 lipid reserves, 419 signal transduction, 77 starvation, 412 values for fish plasma, 417 Glucagon-like peptide (GLP) gluconeogenesis, 68 glycogenolysis, 68 levels in fish plasma, 418 plasma fatty acids, 419 re-feeding, 74 seasonal response, 77 starvation, 412, 418 Glucagon/insulin ratio starvation, 416 Glucocorticoids, see also Cortisol effects on transport, 233 gluconeogenesis, 421 glucose uptake, 421 protein catabolism, 421 starvation, 421 Glucokinase, 66 Gluconeogenesis, 65ff., 397ff. amino acids, 22, 170, 178, 406, 410 catecholamines, 68 cortisol, 69, 76, 422 GLP, 68, 418 glucagon, 68, 418 glucocorticoids, 421 glycerol, 249 hagfish, 411 insulin, 69 kidney, 176, 396 liver, 371, 373, 375ff., 396 non-carbohydrate precursors, 396 postprandial, 48 rate in fish liver, 382 spawning migration, 55 starvation, 396ff., 406, 410 thermal compensation, 75 vasoactive peptides, 68 vertebrate liver, 381 Gluconeogenic flux liver, 373 Glucose absorption, 223, 225 methyltestosterone effects, 232 effects of thyroid hormones, 234 aerobic exercise, 373 intestinal uptake, 225 intestine, 223 loading, 21

Subject Index

muscle uptake rates, 379 oxidation, 20f., 162 heart, 84 limits in brain, 89 red blood cells, 162 permeability effects of thyroid hormones, 234 methyltestosterone effects, 232 red blood cells, 89 plasma concentration at rest, 17 plasma pool, 379 plasma turnover, 373f. production, 20 anoxia, 74 hypoxia, 74 liver, 21 red blood cell substrate, 162 release brain, 88 insulin, 88 liver, 373 stimulation of gas gland oxygen uptake, 105 storage, 338 substrate for heart, 84 transport, 223 a-methyl-glucosides, 223 ~-methyl-glucosides, 223 2-deoxyglucose, 223 liver, 66 methyltestosterone effects, 232 Na +/glucose transporter, 223 red blood cells, 66, 89 transporter isoforms, 225 Vmax effects, 232 uptake brain, 86 carnivores, 230 glucocorticoids, 421 herbivores, 230 kinetics, 66 stimulation, 231 Vmax, 230 utilization brain, 66 hagfish heart, 66 red muscle, 370 white muscle, 370 vs. fatty acid oxidation, 21 Glucose 6-phosphate fasting, 396 flux into glycogen, 396 gas gland metabolism, 109 Glucose 6-phosphate dehydrogenase effects of diet, 172 gas gland tissue, 108 kidney, 78 sonic muscle, 285 starvation, 404 toadfish heart, 285

Subject Index Glucose 6-phosphate dehydrogenase (continued) toadfish white muscle, 285 Glucose disappearance, 20 Glucose intolerance, 65 Glucose metabolism gas gland tissue, 108 Glucose paradox, 71 Glucose sparing elasmobranchs, 378 Glucose transporter hagfish brain, 89 intestine, 163 Glucose turnover, 372, 398 cannulation, 41 glycogen synthesis, 81 in post-absorptive teleosts, 20 tuna, 248 Glucose-clamp technique, 21 Glucuronidation coelenterazine, 442 liver, 453 GLUT-l, 89 Glutamate elasmobranch muscle mitochondrial substrate, 244 flux into lipid, 170 hepatic oxidation, 170 liver mitochondrial substrate, 167 liver, 49 mammalian kidney, 80 mitochondrial transport, 245 oxidation, 22, 251, 371 thermogenic tissue mitochondria, 251 red blood cells, 161 starvation, 409 stimulation of intestinal ion transport, 166 subcellular compartmentation, 245 turnover, 22 Glutamate dehydrogenase, 171 adaptive changes, 171 allosteric regulation, 168 brain, 87 effects of diet, 172 glutamate metabolism, 246 glutamate oxidation, 245 gut, 166 heart, 84 kidney, 78, 174 liver, 70, 166 red blood cell, 162 red muscle, 82 starvation, 171 white muscle, 81 Glutamate oxidation aminooxyacetate inhibition, 245 hepatocytes, 162 vs. ammonia production, 162 Glutamate-aspartate carrier in glutamate metabolism, 246

487 Glutaminase elasmobranch kidney, 176 elasmobranch red muscle, 244f. glutamate metabolism, 246 hagfish red muscle, 245 liver, 169 muscle, 178 red muscle mitochondria, 245 teleost red muscle, 245 thermogenic tissue, 247 Glutamine elasmobranch muscle mitochondrial substrate, 244 elasmobranch plasma, 244 intestinal substrate, 166 liver mitochondrial substrate, 167 oxidation elasmobranch mitochondria, 22 muscle, 248 red blood cells, 162 thermogenic tissue mitochondria, 251 red blood cell substrate, 162 starvation, 409 stimulation of intestinal ion transport, 166 subcellular compartmentation, 245 Olutamine carrier glutamate metabolism, 246 thermogenic tissue, 247 Glutamine synthetase elasmobranch kidney, 176 elasmobranch red muscle, 245 hagfish red muscle, 245 kidney, 176 liver, 176 muscle, 176, 178, 245 teleost red muscle, 245 Olutathione peroxidase gas gland, 115 Glutathione reductase gas gland, 115 Glycemia, 398 elasmobranch starvation, 412 starvation, 396, 407 Glyceraldehyde 3-phosphate effects on luminescence, 453 gas gland metabolism, 109f. Glyceraldehyde 3-phosphate dehydrogenase association with F-actin, 303 binding in turtle brain, 297 binding to subcellular muscle structures, 292 effects of metabolites on enzyme binding, 300 effects of methodology on enzyme binding, 295 gas gland tissue, 107 glycolytic control, 81 kinetic effects of F-actin, 302 release from muscle particulate matter, 294 Glycerol gluconeogenesis, 249, 421

488 Glycerol (continued) in glycerophospholipids, 120 re-esterification, 249 structure, 120 Glycerol kinase brown adipose tissue, 249 effects of cortisol, 421 thermogenic tissue, 252 Glycerol-3-phosphate acyltransferase liver, 121 Glycerolphosphate dehydrogenase gas gland tissue, 107 Glycerophospholipid metabolism temperature effects, 134 Glycerophospholipids, 120ft. absorption, 123 basic structures, 120 biosynthesis, 121, 123 deacylation/reacylation, 122 dietary effects, 133 digestion, 123 3,5-dinitrobenzoyl derivatives, 132 embryonic development, 144 fatty acyl composition, 130 functions, 136ff. growth, 145 head group composition, 129f. larval diet, 144 lipovitellin, 127 metabolic roles, 137f. terminology, 121 transport, 123 vitellogenin, 127 Glycine brush border uptake, 227 fasting, 407 from serine, 168 muscle, 407 oxidation, 21, 371 Glycogen adipose tissue, 369 body mass, 369 brain, 46, 66, 86 burst swimming, 80 catecholamine, 373 content of tissues, 67 depletion in liver, 394 extracellular fluid, 369 GLP, 418 glucagon, 418 hagfish heart, 66 hagfish liver, 411 hypoxia, 46 in boutons, 281 indirect calorimetry, 41 insulin, 411 liver, 46, 67, 369, 394ff. liver, starvation, 400 migration, 400

Subject Index mobilization, 373 hypoxia, 51 muscle, 46, 248, 251, 283, 369, 395f. reappearance following exercise, 381 recovery, 74, 369 muscle, 359 red muscle, 248, 283, 369 repletion, 383 sonic muscle, 283 synthesis from lactate, 382 thermogenic tissue, 251 tuna red muscle, 248 white muscle, 369 Glycogen metabolism heart, 83 Glycogen phosphorylase allosteric regulation, 291 anoxia, 74 exhausting exercise, 54 fasting, 72, 74 gas gland, 106 glycolytic control, 81 hypoxia, 74 kidney, 78 propranolol, 74 role in glycogenolysis, 68 Glycogen production indirect pathway, 71 Glycogen synthase brain, 87 heart, 84 liver, 70 Glycogen synthesis brain, 88 effects of cortisol, 422 exogenous glucose, 81 heart, 85 insulin, 88 lactate, 88 recovery, 385 Glycogenesis from lactate, 82 role of enzymes, 82 with fasting, 396 Glycogenolysis, 371 Ca z+, 77 catecholamines, 68 epinephrine, 73, 420 exhausting exercise, 54 GLP, 68, 420 glucagon, 68, 73, 420 hormones, 68f, 73, 77, 420 hypoxic liver, 52 insulin, 69 liver, 52, 373 seasonality, 77 starvation, 55, 420 vasoactive peptides, 68 Glycolysis, 34

Subject Index Glycolysis (continued) activation by histidine-related dipeptides, 324 activation by calcium, 39 ATP/ADP ratio, 385 during tissue extraction, 39 Glycolytic complex, 299, 301 definition, 293 Glycolytic control muscle, 81 recovery, 81 Glycolytic enzyme binding effect of methodology, 295 Glycolytic enzymes binding to particulates, 292 multienzyme complex, 292 regulation. 291 starvation, 407 Glycolytic flux regulation through binding, 302 F-actin binding, 301 Glycolytic metabolon, 293 Glyconeogenesis, 66 Glycyl-phenylalanine brush border uptake, 227 Golgi apparatus exocytosis, 273 gas gland cells, 103f. Gonadal steroids influence on electric organ discharge, 266 Gonadectomy sex differences in electric organ discharge, 266 Growth capacity, 346 glycerophospholipids, 145 histidine, 318 intestinal nutrient absorption, 235 3H-leucine, 196 protein synthesis, 191 stunting, 346 vs. metabolism, 191 Growth hormone amino acid uptake, 233 dynamics, 424 effects on food consumption, 233 effects on plasma fatty acids, 25 lipid mobilization, 424 proline uptake, 233 regulation of nutrient transport, 23 I, 233 starvation, 423 vs. thyroid hormones, 234 Growth performance chronic stress, 202 genotype, 201 immunocompetence, 202f. social rank, 203f. Growth rate, 213f., 344, 346 scaling with body weight, 212 GTP

489 purine nucleotide cycle, 386 regulation of glutamate dehydrogenase, 174 GTP binding protein electric organ, 273 Guanine crystals reflectors, 438 swimbladder submucosa, 102 Gut amino acids in fasting, 160 nutrient transport, 221 Gymnotiform electric fish, 26 If. Hagfish glucagon, 411 glycemia, 397 liver glycogen, 411 role of insulin, 411 Hagfish heart glucose utilization, 66 Handling stress, 36f. Hatching phospholipids, 144 lipid catabolism, 144 HDL (high density lipoproteins) average composition, 25 plasma, 124 Head kidney lipoxins, 139 macrophages, 139 Heart N-acetylated histidine, 329 amino acids in fasting, 160 anserine, 329 aspartate aminotransferase, 285 ATP turnover, 379 t-receptors, 85 buffering capacity, 85 carbohydrate metabolism, 83ff. carnosine, 329 citrate synthase, 285 cyclooxygenase, 138 fatty acid oxidation, 84f. glucose 6-phosphate dehydrogenase, 285 glucose oxidation, 84 glycogen content, 67, 84 glycogen synthesis, 85 3-hydroxybutyrate dehydrogenase, 403 hypoxic stress, 85 lactate dehydrogenase, 285 lactate oxidation, 85 lactate transport, 85 lactate utilization, 381 malate dehydrogenase, 285 malic enzyme, 285 myoglobin, 85 prostanoid synthesis, 139 Heart size, 340 scaling, 344f.

490 Heat exchangers, 1, 11, 242 heat dissipation, 242 Heat flux indirect calorimetry, 41 Heat production ATPase, 248 hormonal regulation, 255 intracellular processes, 243 myosin ATPase, 248 protein synthesis, 207 proton leak, 248 rate in thermogenic tissue, 252 resting, 207 thermogenic tissue, 242 vs. substrate, 252 Heat transfer muscle, 11 Hematocrit body size, 341 (figure) physical activity, 340 scaling, 340, 345 stress, 340 Hemoglobin, 340 acidification, 111 concentration, 340 hypoxia, 50 multiple forms in fish, 342 oxygen affinity, 341 Root effects, 111, 114 spleen, 342 (figure) Henry's law, 101 Hepatectomy blood glucose, 68 glycogen balance, 72 glycogen levels, 72 plasma amino acids, 167 Hepatic portal vein amino acids, 181 postprandial ammonia, 166 Hepatocytes alanine uptake, 410 amino acid utilization, 167, 169f. ammoniogenesis, 162, 167, 169 CDP-choline phosphotransferase, 121 CDP-ethanolamine phosphotransferase, 121 cost of protein synthesis, 209 glucagon binding, 420 gluconeogenesis, 167 glutamine oxidation, 162 glycerophospholipid biosynthesis, 121 glycogenolysis, 418 ketogenesis from amino acids, 244 Na +/K+-ATPase vs. oxygen consumption, 209 oxygen consumption, 209 vs. protein synthesis, 209 phosphatidyl ethanolamine methyltransferase, 122 phosphatidyl serine decarboxylase, 122 protein synthesis, 76, 207

Subject Index vs. oxygen consumption, 207 seasonal response, "/7 thermal compensation, 75 thyroid hormone, 76 Hepatopancreas phospholipase A2, 123 Herbivores, 211 glucose uptake, 230 intestinal nutrient transport, 230 Heterogeneity liver cells, 69 Hexokinase, 52 brain, 87 gas gland tissue, 107 glycolytic control, 81 heart, 84 in tissues, 66 kidney, 78 liver, 70 red blood cells, 90 red muscle, 82, 248 regulation, 291 thermogenic tissue, 251 tuna red muscle, 248 white muscle, 81 Hexosephosphate shunt elasmobranch kidney, 79 red blood cells, 90 High-energy phosphates as endogenous fuel, 45 Histidase liver, 168 Histidine absorption, 319 N-acetylation, 329 degrading enzymes, 322 growth, 318 heart, 329 hepatic metabolism, 168 imidazole dipeptides, 309 in carnosine synthesis, 320 into muscle anserine, 320 muscle, 49, 313 osmoregulation, 315f. pK value, 325 red blood cell transport, 162 red muscle, 313 starvation, 316ff., 409 turnover, 319 uptake by tissues, 319 washout, 319 white muscle, 313 Histidine aminotransferase liver, 168 Histidine-related dipeptides as a-adrenergic antagonists, 329 buffering, 324 calcium sensitivity, 329 contractile proteins, 329

Subject Index Histidine-related dipeptides (continued) copper transport, 324 degradation, 322 enzyme activation, 324 free radical scavenger, 324 glycolysis, 324 interorgan transport, 322 Mg2+-ATPase, 324 non-muscular tissues, 314 osmoregulation, 315 oxygen transport, 324 physiological roles, 324ff. proton buffering, 324 red muscle, 313 structures, 309 white muscle, 313 Holocephalan plasma fatty acid concentration, 24 Homeostasis amino acids, 162, 199 Homeothermy, 337 cost, 337 Homeoviscous adaptation membranes, 134 Homocarnosine imidazole dipeptides, 309 imidazole pK value, 325 vertebrate muscle, 309 Hormonal control fasting, 74, 410ft., 418, 424 sonic muscle parameters, 287 Hormone-sensitive lipase in intertissue lipid exchange, 23 HSI starvation, 397 Hummingbird muscle characteristics, 2 muscle morphometric data, 6 Hydrodynamics, 344 Hydrogen shuttles a-glycerophosphate shuttle, 247, 249, 255 malate-aspartate shuttle, 178, 247, 249, 255 thermogenic tissue, 255 tuna red muscle, 249 Hydroperoxy fatty acids eicosanoid metabolism, 138f. Hydroxy fatty acids eicosanoid metabolism, 138f. 12-Hydroxy fatty acids distribution, 139 Hydroxyacyl-Coenzyme A dehydrogenase brain, 87 heart, 84 kidney, 78 liver, 70 red muscle, 82 thermogenic tissue, 251 white muscle, 81 12-Hydroxyeicosapentaenoate (HEPE)

491 fatty acid precursors, 140 12-Hydroxyeicosatetraenoate (HETE) fatty acid precursors, 140 3-Hydroxybutyrate dehydrogenase elasmobranchs, 403 teleosts, 403 p-Hydroxyphenyipyruvate recycling of coelenterazine, 456 Hydroxyproline fasting, 407 muscle, 407 Hyperamino acidemia cortisol, 422 Hyperglycemia hypoxia, 52 thyroid hormones, 425 Hypermetabolism fasting, 73 Hyperoxia cell proliferation, 115 contribution of pentose shunt in gas gland, 109 edema, 115 gas gland, 115 Hyperventilation hypoxia, 50 Hypoglycemia fasting, 73 role of insulin, 411 Hypoglycemic stress brain, 87 Hypometabolism fasting, 72f. starvation, 401 Hypothyroidism, 426 Hypoxia, 374 acid secretion, 112 aerobic allometry, 352 carbohydrate metabolism, 74f. catecholamines, 91 enzyme activities, 74 glucose flux, 21 glycogen mobilization, 51 heart glycogen, 47 heart, 85 hyperglycemia, 52 hypoglycemia, 74 lactate flux, 18 lactate turnover, 19, 74 liver glycogen, 47 metabolic depression, 52 metabolic rate, 291 muscle glycogen, 46 normoglycemia, 74 physiological effects, 50 thermal acclimation, 83 Icefish hemoglobin, 340 oxygen carrying capacity, 340

492 IDL, 124f. Imidazole pK value, 325 Imidazolopyrazine autoxidation, 461 light production, 439 origin of luminescence, 459 Imino acid transporter brush border, 227 Immunocompetence feeding, 204 social rank, 203f. IMP, see Inosine 5'-monophosphate Induction amino acid transport, 235 Infusion metabolite turnover, 16f. Ingestion scaling, 344 luciferin, 446 Ingestion rate scaling with body weight, 212 Inosine 5'-monophosphate purine nucleotide cycle, 386 muscle, 380, 385 myo-lnositol in glycerophospholipids, 120 Inositol diet, 145 Inositol phosphates phosphoinositide cycle, 142 Inositol phospholipid metabolism electrocytes, 143 Insects effects on glycerophospholipid composition, 133 endothermy, 242 luminescence, 444 Insulin brain glucose release, 88 brain glycogen synthesis, 88 carbohydrate metabolism, 73 crossreactivity, 410 effects of glucocorticoids, 421 effects on liver enzymes, 412, 414 effects on plasma fatty acids, 25 gluconeogenesis, 69 glucose turnover, 21 glycogenolysis, 69 gonadotropic action, 414 hypoglycemia, 86, 411 lipogenesis, 411 liver glycogen, 411 membrane at~nity, 72 plasma dynamics, 41 lff. protein synthesis, 411 re-feeding, 74, 412 RIA, 410 spawning migration, 414

Subject Index

starvation, 410ff. values for fish plasma, 413 Insulin binding re-feeding, 73 Insulin receptor liver, 415 muscle, 415f. Insulin-like growth factors (IGFs) starvation, 424 Interference reflector guanine crystals, 438 photogenic tissue, 438 Interorgan transport anserine, 323 Interrenal steroids effects on intestinal transport, 233 metabolism, 423 Intestinal absorption luciferin, 450ff. Intestinal mucosa re-esterification of lysophospholipid, 123 Intestine amino acid oxidation, 165 amino acid requirement, 166 amino acid transport, 222 ammoniogenesis, 166 chylomicron-like particles, 124 connection to light organ, 439, 451 cyclooxygenase, 138 dipeptide transport, 163f. glucose transport, 222 glycerophospholipid headgroup composition, 129 glycerophospholipid storage, 128 length, 230 lipoproteins, 124 Na+-pumps, 222 Na +/K+-ATPase, 222 nutrient absorption, 221 passive nutrient permeability, 230 phospholipases, 123 phospholipid changes with seawater adaptation, 135f. prostanoid synthesis, 139 protein turnover, 165f. proteolysis in starvation, 406 storage for triacylglycerols, 400f. transport processes, 221 VLDL, 124 Intracellular pH calculation from NMR, 43 glycolytic flux, 292 muscle recovery, 381 Invertebrates distribution of histidine-related dipeptides, 312 Iodoacetate fructose 6-phosphate, 299 glycolytic enzyme binding, 299 heart performance, 84f.

Subject Index Ionic strength dilution method for enzyme binding, 294 IP3 phosphoinositide cycle, 142 Ischemia brain, 87 Islet hagfish, 411 Isocitrate regulation of glutamate dehydrogenase, 174 lsocitrate dehydrogenase (NAD) control by calcium, 252 thermogenic tissue, 247 Isocitrate dehydrogenase (NADP) brain, 87 heart, 84 liver, 70 red blood cells, 90 red muscle, 82 starvation, 404 white muscle, 81 Isoleucine metabolic fate, 175 red blood cell transport, 162 role in alanine biosynthesis, 183 starvation, 409 Isometric scaling gill surface area, 343 respiration, 339 lsoproterenol red cell 3-O-methyiglucose uptake, 91 red cell metabolism, 91 red cell Na+-pump, 91 Isotocin gluconeogenesis, 68 glycogenolysis, 68 signal transduction, 77 Juvenile fish cost of protein synthesis, 209 protein synthesis, 211 vs. oxygen consumption, 207 K+ current electrocytes, 268 K+-channels intestinal amino acid transport, 163 K+-gradient amino acid transport, 164 basolateral membrane, 229 Keel muscle light organ, 439 Keratin-like protein electrocytes, 272 6-Ketoprostaglandin Fla distribution, 139 Keto-testosterone sonic muscle, 287 Ketogenesis

493 elasmobranch hepatocytes, 244 Ketone bodies, 71, 378 anoxia, 88 competition with glutamine in thermogenic tissue, 247 elasmobranch fuel, 243, 411 elasmobranch red muscle, 377 oxidation heater organ, 251 brown adipose tissue, 251 oxidation in teleosts, 248 starvation, 244, 402f. substrate for heart, 85 teleost brain, 88 thermogenic tissue, 247 Kidney aglomerular, 457 alanine oxidation, 79 amino acid absorption, 173 catabolism, 177 fasting, 160 levels, 173 ammoniogenesis, 174, 176 anserinase, 322 arginase, 175 asparagine metabolism, 176 blood ammonia, 176 branched-chain amino acid metabolism, 175 brush-border membranse vesicles amino acid uptake, 173 carbohydrate metabolism, 78ff. catabolism of dipeptides, 324 cyclooxygenase, 138 enzyme activities, 78 ethanolamine plasmalogens, 129 fasting, 80 free amino acids, 49 gluconeogenesis, 79f., 176 glucose 6-phosphate dehydrogenase fasting, 80 glucose production, 20 glutamate dehdydrogenase, 174 glutamate utilization, 80 glutaminase, 176, 244 glutamine metabolism, 176 glycogen content, 67, 78 hexosephosphate shunt, 79 histidine degrading enzymes, 322 histidine-related dipeptides, 314 3-hydroxybutyrate dehydrogenase, 403 hypoxia, 80 lactate oxidation, 79 loss of luciferin, 457 PEPCK fasting, 80 phosphatidylcholine/seawater adaptation, 135 prostanoid synthesis, 139 proteolysis in starvation, 405

494 Kidney (continued) purine nucleotide cycle, 174 seawater adaptation, 135 serine metabolism, 79, 175 taurine secretion, 173 Killifish amino acid transporter, 226 intestinal transport, 226 Kleiber's rule, 336 Krebs cycle, 34 gas gland metabolism, 109 in glutamate metabolism, 246 proton pumps, 242 red blood cells, 91 Lactase intestine, 223 Lactate, 369 accumulation in white muscle, 249 adipose tissue, 369 brain, 88 burst swimming, 80 disappearance from white muscle, 384 disappearance, 82 disappearence from muscle, 381 dynamics, 375 effect of sampling methodology, 36 exchange between muscle & circulation, 19 extracellular fluid, 369 fate, 381 flux to C6-products, 71 formation gas gland, 105, 109f. acetylcholine-stimulated, 105 catecholamine-stimulated, 105 fuel for red muscle, 375 gas gland metabolism, 109 gluconeogenesis effects of cortisol, 422 gluconeogenesis, 412 hypoxia. 52 liver substrate, 69 liver, 369 load exercise, 81f. metabolism, 19, 82, 109, 371, 379 body ma~s, 19 muscle, 380 transport, 382 non-ionic diffusion, 115 oxidation, 371, 375 heart, 85 kidney, 79 red blood cells, 162 substrate, 71 plasma concentration at rest, 17 production exercise, 379 red blood cells, 91

Subject Index red muscle, 370 white muscle, 370 red muscle, 249, 369 release gas gland, 107 white muscle, 374 rest, 36, 380 retention, 19, 82 storage sites, 383 sustained swimming, 80 swimbladder blood, 107 transport, 20 heart, 85 liver, 66 red blood cells, 66 tuna white muscle, 249, 315 turnover, 82, 372, 382 body mass, 19 cannulation, 41 exercise, 19 fasting, 19 fish species, 69 hypoxia, 19, 74 in postabsortive teleosts, 19 red muscle, 1 stress, 19 vs. oxygen consumption, 19 uptake kinetics, 66 utilization, 357 vs. flux, 18 vs. lactate dehydrogenase, 356 white muscle, 249, 369, 384 Lactate dehydrogenase association with F-actin, 303 binding in turtle brain, 297 binding to subceUular muscle structures, 292 brain, 87, 355 correlation with buffering capacity, 327 enzyme binding, 295, 300 gas gland, 106f. heart, 84 isozymes, 71 kidney, 78 kinetic effects of F-actin, 302 liver, 70f. loss during muscle preparation, 296 muscle, 81, 106, 285, 314, 354ff. muscle type in gas gland, 106 raven heart, 357 red blood cells red muscle, 81 scaling in white muscle, 354f. scaling, 355 sonic muscle, 285f., 357 starvation, 396 toadfish heart, 285 toadfish white muscle, 285f. tuna white muscle, 314 vs. a-glycerophosphate dehydrogenase, 249

Subject Index Lactate dehydrogenase (continued) vs. lactate concentration, 356 white muscle, 81, 285f. Lactate pathway, 354f. Lake Baikal luminescent species, 435 Laminar flow power requirements for swimming, 356 Lamnid sharks red muscle, 243 thermogenic tissue, 242 Larval fish cost of protein synthesis, 209 glycerophospholipid diet, 144f. protein synthesis, 211 PUFA, 144 LDL (low density lipoproteins) average composition, 25 plasma, 124 Lecithin: alcohol acyltransferase plasma, 125 Lecithin: cholesterol acyltransferase plasma, 125 Lens aperture, 438 light organs, 438 Leucine activation of gluconeogenesis, 172 brush border uptake, 227 effect on branched-chain amino acid aminotransferase, 177 glutamate dehydrogenase, 168 growth, 196 metabolic fate, 175 oxidation, 22, 205, 371 protein synthesis, 196, 205 red blood cell transport, 162 regulation of glutamate dehydrogenase, 174 role in alanine biosynthesis, 183 starvation, 409 stereospecific intestinal uptake, 226 transport regulation by estradiol, 232 regulation by methyl-testosterone, 232 3H-Leucine growth, 196 protein synthesis, 196 Leukocytes chemotactic activity of leukotriene, 142 cyclooxygenase, 138 lipoxygenase products, 139 prostacyclin synthesis, 139 prostanoid synthesis, 139 Leukotrienes chemotactic activity, 142 eicosanoid metabolism, 138f. fatty acid precursors, 140 Lidocaine, 38 Life history

495

protein turnover, 211f. Light emission, 438 counterillumination, 437 photophores, 436 spectral shift, 445f. temporal variation, 446 Light filtering, 439 Light organ anatomy, 439 coelenterazine, 442 glucuronidation, 453 guanine crystals, 438 keel muscle, 439 luciferase, 442 luciferin recycling, 454 luciferin storage, 453 luminescent species, 436 peroxidase, 442 relation to intestine, 439 relation to pyloric caeca, 439 storage of luciferin, 452 Lindane brain metabolism, 88 Lipase adipose tissue, 403 bile-salt activated, 123 red muscle, 403 Lipid as endogenous fuel, 47 availability, 374 depletion cortisol, 422 starvation, 399f., 405 droplets tuna red muscle, 248 from acetate, 404 gas gland, 104f. lipoproteins, 124 liver composition, 395f. liver, 25 metabolism hatching, 144 embryonic development, 144 mobilization, 400 growth hormone, 424 muscle content, 25, 395f., 401 oxidation elasmobranch liver, 244 elasmobranch tissues, 243 embryonic development, 144 transport, 124 albumin-like protein, 125 lipoproteins, 124f. Lipid reserves effects of thyroid hormones, 425 glucagon, 419 starvation, 419 Lipogenesis anoxia, 52

496 Lipogenesis (continued) from amino acids in muscle, 178 insulin,411 perivenous cells,69 starvation,404 Lipogenic enzymes effectsof diet, 172 Lipolysis, 123, 378 cortisol,422 effectsof growth hormone, 424 starvation,419 Lipoprotein lipase,125 activationby apoproteins, 126 in intertissuelipidexchange, 23 Lipoproteins apoproteins, 126 average composition, 25 contribution to plasma fraction,25 lipidtransport, 124 phosphatidylcholine, 126 plasma, 25 PUFA, 126 remodelling, 125 uptake receptor-mediated, 126f. pinocytosis, 127 vitellogenin, 127 Lipovitellin lipid composition, 127 Lipoxins eicosanoid metabolism, 138f. gill, 139 macrophages, 139f. Lipoxygenase eicosanoid metabolism, 138f. fatty acid specificity, 142 tissue products, 139 12-Lipoxygenase, 139 15-Lipoxygenase, 139 Lithium-sensitive phosphatase electrocytes, 143 Liver, 68ff. amino acids, 369 effects of salinity, 171 exercise, 53 fasting, 160 ammoniogenesis, 49 anserinase, 322 apoprotein receptors, 126 arginase, 175 as energy store, 71 branched-chain a -ketoacid dehydrogenase, 175 branched-chain amino acid aminotransferase, 174 carbon cycling, 71 cardiolipin, 129 carnosinase, 322

Subject Index CDP-choline-l,2diacylglycerol choline phosphotransferase, 121 composition change in starvation, 395 cortisol, 76 binding, 423 cyclooxygenase, 138 depletion of lipids, 400 dipeptide degradation, 322 DNA content in starvation, 406 enzymes, 70 activation by cortisol, 69 ethanolamine plasmalogens, 129 fasting, 72 fat synthesis, 170 fatty acid synthesis, 404 fatty acids, 369 fractional rate of protein synthesis, 204f. free amino acids, 49 fructose 6-phosphatase, 412 gluconeogenesis, 170, 371, 375, 406, 412 from amino acids, 170 gluconeogenic flux, 373 glucose, 369 production, 20, 372 release, 373 transport, 66 glutamate dehydrogenase, 172 glutamate production from amino acids, 168 glycerol-3-phosphate acyltransferase, 121 glycerophospholipid headgroup composition, 129 glycerophospholipid storage, 128 glycogen, 46, 75, 338, 358, 369, 394, 405 anoxia survival, 75 anoxia tolerance, 74 content, 46, 67 fasting, 72 mobilization, 394 re-feeding, 73 seasonality, 76 starvation, 55 glycogenolysis, 52, 373 heterogeneity, 69 histidine degrading enzymes, 322 histidine metabolism, 168 histidine-related dipeptides, 314 3-hydroxybutyrate dehydrogenase, 403 insulin binding, 72 insulin receptor, 415 lactate, 369 dehydrogenase, 71 transport, 66 iipase, 125, 422 lipids, 405 content, 25, 47 oxidation, 244 lipogenic enzymes, 172 lobes metabolic activity, 69

497

Subject Index

Liver (continued) mass in starvation, 406 microsomal lipid synthesis, 121 mitochondrial ammoniogenesis, 167 mitochondrial phospholipids/temperature, 136 ornithine decarboxylase, 172 periportal cells, 69 perivenous cells, 69 phosphofructokinase- 1, 414 phospholipid changes with seawater adaptation, 135 platelet-activating factor synthesis, 143 postprandial amino acids, 167 prostanoid synthesis, 139 protein, 369, 405f. synthesis, 76, 167 synthesis vs. amino acid pool, 204f. proteolysis in starvation, 405 purine nucleotide cycle, 167 pyruvate kinase, 412 serine metabolism, 168 signal transduction, 77 starvation, 405 storage for triacylglycerols, 399 thyroid hormone binding, 426 transdeamination, 371 triacylglycerols, 369, 399 lipase, 419 mobilization, 375 VLDL synthesis, 125 Locomotion, 337 Luciferase, 440ff. amino acid sequence, 444 chemiluminescence, 444 coelenterazine recycling, 457 digestive tract, 442 effects of glyceraldehyde 3-phosphate, 453 light organs, 442 molecular weight, 443 monooxygenase, 443 Luciferin, 436, 440ff. autoxidation, 451, 461 binding protein, 452 blood, 457 counterillumination, 456 de novo synthesis, 454 diet, 446 diffusion, 458 effects of glyceraldehyde 3-phosphate, 453 eggs, 458 exogenous sources, 447ff. from oxyluciferin, 454 ingestion, 446 intestinal absorption, 450 losses from body, 457 maternal transfer, 450 metabolism, 447 origins, 459 oxidation, 443

peroxide formation, 443 plasma binding, 452 quantum yields, 441 receptors, 458 recycling, 447, 454 red cell binding, 452 retention, 456 signalling, 456 spectra, 441 stomach, 451 storage, 447, 452 structures, 440 transport, 452 trophic transfer, 446 uptake, 458 Luciferin-binding protein, 452 Luciferinol, 442 Luciferyl-sulfate coelenterazine, 442 Luciferyl-sulfokinase luciferin regeneration, 442 Luminescence, see also bioluminescence effects of cyanide effects of epinephrine, 454 inhibition by cyanide, 442 phylogenetic distribution, 461 quantum yield, 455 starvation, 461 Luminescent organisms fish diet, 448 Luminescent species light organs, 436 Luminescent system distribution in fishes, 441 Lymphatic system transport of lipoproteins, 124 Lysine brush border uptake, 227f. intestinal transport, 163 osmoregulation, 316 Lysosomes lipase, 403 Macrophages cost of protein synthesis, 208f. fractional protein synthesis rate vs. oxygen consumption, 208 12-hydroxy fatty acids, 139 leukotrienes, 139 lipoxins, 139f. lipoxygenase products, 139 Maintenance cost protein turnover, 214 Maintenance synthesis, 210 Malate regulation of glutamate dehydrogenase, 174 Malate aspartate shuttle, 178, 247, 249, 255 thermogenic tissue, 255 Malate dehydrogenase

498 Malate dehydrogenase (continued) brain, 87 gas gland tissue, 105 heart, 84, 285 in glutamate metabolism, 246 kidney, 78 liver, 70 red blood cells, 90 red muscle, 82, 285f. sonic muscle, 285f. thermogenic tissue, 247 toadfish heart, 285 toadfish white muscle, 285f. white muscle, 81, 285L Malate/a-ketoglutarate carrier in glutamate metabolism, 246 Malformation role of glycerophospholipids, 145 Malic enzyme (NADP) brain, 87 glutamine oxidation, 244 glycogenesis, 82 heart, 84, 285 in glutamate metabolism, 246 in muscular glycogen synthesis, 383 kidney, 78 liver, 70 red muscle, 82, 285 sonic muscle, 285 starvation, 404 thermogenic tissue, 247 toadfish heart, 285 toadfish white muscle, 285. white muscle, 81, 285 Maltase intestine, 223 Mammals distribution of histidine-related dipeptides, 312 endothermy, 242 muscle buffering capacity, 326 Manometry gas gland metabolism, 105 Marine mammals distribution of histidine-related dipeptides, 312 muscle buffering capacity, 326 Mate call toadfish, 281 Maternal transfer luciferin, 450, 458ff. Membranes composition, 134 electric organs, 269f. temperature, 134 transporter, 222 vesicles, see also basolateral membrane & brush border membranes preparations, 223 Mesenteric fat, 47 starvation, 56

Subject Index

Mesentery lipase, 422 Metabolic acid load exercise, 81f. Metabolic allometry, 336L Metabolic cost branchial pump, 345 cardiac pump, 345 Metabolic depression, 74E hypoxia, 52 low temperature, 72 Metabolic intermediates extraction, 39 flux, 15 measurements, 40 sampling, 38 storage, 39f. turnover studies, 41 Metabolic pathway efficiency, 33f. regulation, 291 Metabolic rate, 359 aerobic scaling exponent, 359 anoxia, 291 burst swimming, 291 effects of thyroid hormones, 425 hypoxia, 291 large animals, 359 scaling, 345 slow swimming, 291 Metabolic substrates systemic circulation, 15ft. Metabolism aerobic, 335ff. allometry, 344 basal, 336 down-regulation, 401 exercise, 368 fatty acid contribution, 24 muscle, 337, 359 respiration rate, 338 routine, 371 scaling, 336ff. standard metabolic rate, 336 Methionine brush border uptake, 227 protein consumption, 206 protein synthesis, 206 3-O-Methylglucose brain glucose transport, 89 uptake red cell, 91 isoproterenol, 91 Methyl-histidine excretion, 322 histidine-related dipeptides, 309ff. imidazole pK value, 325 N-Methylglycine intestinal transport, 163

Subject lndex

Mg2+-ATPase, 324 role of histidine-related dipeptides, 324 Microsome membranes composition of phosphatidylcholine, 132 composition of phosphatidylethanolamine, 132 Microsomes CDP-choline-l,2diacylglycerol choline phosphotransferase, 121 lipid synthesis, 121 phospholipase A2 Microtubules electric organ, 273 Microvilli gas gland cells, 103 Midshipman sonic muscle, 279ff. sound production, 279ff. Migration, see also Spawning migration body weight, 399 lipid loss, 399 lipid utilization, 376 vertical, 101, 342 vitellogenin, 408 Milt cyclooxygenase, 138 Mitochondria amino acid oxidation, 371 /5-hydroxybutyrate dehydrogenase, 246 carnitine palmitoyl-Coenzyme A transferase, 377 flux capacities, 378 gas gland cells, 103 glutaminase, 169, 176, 178 glutamine oxidation, 22 in boutons, 281 interfibrillar, 9 membranes, 242 composition of phosphatidylcholine, 132 composition of phosphatidylethanolamine, 132 temperature effects on lipid composition, 135 mobilization in starvation, 407 muscle glutaminase, 178 muscle, 371 oxidative substrates, 244 phosphorylation, 242 proton leak, 241 quantity in red muscle, 377 renal glutaminase, 176 respiration, 242 acidosis, 386 thermogenic tissue, 252 sonic muscle, 284 subsarcolemmal, 9 Mitochondrial density sonic muscle, 284 Mitochondrial volume, 3 density, 3

499 muscle fiber, 10 sonic fibers, 286 Mitochondrial volume density tuna red muscle, 250 vertebrate muscles, 7 Monocarboxylate carrier /~-hydroxybutyrate, 246 pyruvate transport, 246 Monooxygenase luciferase, 443 Mormyriform electric fish, 26 If. Morphometry red muscle, 1 Mosquitofish metabolic rate, 344 Motoneuron cholinergic, 281 innervation of electric organ, 262 Motor nerve sonic muscle, 280 MS222, 38 Mucopolysaccharides amino acid gluconeogenesis, 22 Mucus nitrogen loss,198 Mucus production amino acid gluconeogenesis, 22 Muscle, see also Red muscle, White muscle, Sonic muscle & Heart 31P-spectra, 44 A C h R subunits,269 adenylates, 380 A D P pool, 385 alanine biosynthesis,183 allometry, 346 amino acid metabolism, 11, 177ff. amino acid pool in starvation,160, 178 aminotransfcrases, 178 ammoniogcnesis, 179 A M P deaminase, 380, 385 AMP, 380 anaerobic capacity,327 anserinase, 322 ATP/ADP ratio,385 branched-chain amino acid aminotransferase, 174, 178 buffering capacity,325ff. starvation,316ff. capillarymanifolds, II catheptic enzymes, 405 composition change in starvation,395 contraction, 337 conversion into electricorgan, 260 creatine phosphate, 45 cyclooxygenase, 138 cytosol pH, 325 differentiation,270 fate of lactate,381

Subject Index

500

Muscle (continued) fibers, 337 cross-sectional area, 4 diffusion distance, 9 ultrastructure, 4 volume of mitochondria, 3 fiber size, 3 vs. subsarcolemmal mitochondria, 9 free fatty acids, 376 gluconeogenesis from amino acids, 178 glucose uptake, 383 glutamate dehydrogenase, 178 glutathione reductase, 115 glycerophospholipid headgroup composition, 129 glycerophospholipid storage, 128 glycogen, 46, 338, 358, 371, 380, 405 mobilization in starvation, 398 starvation, 55 elasmobranchs, 399 glycogen synthesis, 381, 383 glycogenolysis, 398 glycolytic enzyme binding, 292 heat transfer, 11 histidine, 178 histidine degrading enzymes, 322 IMP, 179, 380 insulin receptor, 415f. lactate, 380 dehydrogenase, 327 exchange, 19 release/retention, 19, 82, 382 lipases, 122 lipid, 25, 377, 405 content, 25 starvation, 56, 401 iipogenesis from amino acids, 178 malate-aspartate shuttle, 178 metabolism, 337, 359 mitochondria cardiolipin, 129 ethanolamine plasmalogens, 129 modifed as heater organ, 250 morphometric data, 6 myoglobin, 327 NMR, 381 non-bicarbonate buffering, 325 oxidative fibers, 377 phosphagens, 379 phosphate, 380 polyribosomes, 193 protein, 405 degradation, 178 metabolism, 11 percent of whole animal protein, 213 proportion of amino acids, 407 retention, 178 starvation, 406ff. synthesis, 178, 193

utilization in starvation, 400 proteolysis, 170, 398, 410 purine nucleotide cycle, 179 pyruvate, 383 respiration, 380 resting lactate, 36 starvation, 55, 160, 178, 398, 405f. steady-state swimming, 367 storage for triacylglycerols, 399 substrate transfer, 11 triacylglycerol mobilization, 375 trimethylamine, 49 twitch contraction time, 337 utilization of ketone bodies, 377 weight vs. glycogen, 358 Muscular dystrophy dystrophin, 270 Mussel protein synthesis, 196 stable isotopes, 196 my5 expression in electrocytes, 270

myo-lnositol in glycerophospholipids, 120f., 129 metabolism in electrocytes, 143 Myoeardium oxygen consumption, 375 work, 375 Myocytes ~-adrenergic stimulation, 86 MyoD expression in electrocytes, 270 Myofibrillar protein anoxia, 53 percent of whole animal protein, 213 Myofibrils ADP pool, 385 electrocytes, 271 fasting, 407 free ADP, 386 Mg2+-ATPase, 324 role of histidine-related dipeptides, 324 sonic muscle, 282 space in white muscle, 379 synthesis, 407 Myofilament volume density, 358 Myogenic electric organ, 265 Myoglobin, 342 correlation with buffering capacity, 327 heart, 85, 342 oxygen storage, 338 red muscle, 311, 342 scaling, 345 Myosin in electrocytes, 270, 272 protein synthesis, 192 Myosin ATPase

Subject Index

Myosin ATPase (continued) activation during tissue extraction, 39 creatine phosphate, 45 elasmobranch muscle, 243 heater organ heat production, 248 phosphofructokinase, 304 thermogenesis, 242, 248, 250 Myosin light chains sonic muscle, 283 Na + ions electric organ discharge, 263 Na+-amino acid coupling ratios intestinal transport, 228 Na+-channel, 259, 265 Na+-current electrocytes, 268 Na+-dependent transport amino acids, 164, 226 coupling ratio, 223 intestine, 222 stimulation by growth hormone, 233 Na+-efflux heater mitochondria, 254 Na+-gradient amino acid transport, 164 basolateral membrane, 229 Na+-influx intestinal amino acid transport, 163 Na+-pump intestinal amino acid transport, 163 intestine, 163, 222 isoproterenol, 91 red blood cells, 91 Na +/glucose coupling effects of thyroid hormones, 234 methyltestosterone effects, 232 Na+/glucose transporter amino acid sequence, 224 brush border, 223 cDNA, 223 evolution, 224 gene, 224 pyloric caeca, 224 site density, 224 subunits, 224 Na +/H +-exchanger red blood cells, 91 Na +/K+-ATpase amino acid transport, 164 contribution to hepatocyte oxygen consumption, 209 effects of thyroid hormones, 234 elasmobranch muscle, 243 gill membrane lipids, 136 heater organ heat production, 248 in autoradiogram, 269 in electric organs, 269, 273 intestinal amino acid transport, 163

501 intestine, 163, 222 methyltestosterone effects, 232 thermogenesis, 242, 248 Na +/proline transporter E. coli, 224 intestine, 227 NAD(P) role in luminescence, 443 Nasal photophores, 443 Negative allometry, 337, 357 standard metabolism, 359 Neuromuscular junction presence of dystrophin, 270 sonic muscle, 281 Neutral amino acids intestinal transport, 163 Neutral lipase, 403 Neutral lipids furan fatty acids, 130 seawater adaptation, 135 Neutral protease starvation, 56 Neutrophils eicosanoid biosynthesis, 141 leukotrienes, 142 lipoxygenase products, 139 Niche shift, 359 Nicotinic cholinergic neurons, 262 Nitrogen excretion, see also Ammonia amino acid imbalance, 206 protein metabolism, 22 scaling with body weight, 212 Nitrogen flux protein synthesis, 192 Nitrogen loss amino acid oxidation, 198 ammonia production, 198 daily balance - feeding, 197 daily balance - starving, 198 digestive enzymes, 198 mucus, 198 scales, 198 urea production, 198 Nitrogen retention efficiency, 211 Nodes of Ranvier electric organ axon, 266 Non-bicarbonate buffering muscle, 325, 327 Non-essential amino acids, see also amino acids fasting, 409 oxidation, 22 plasma, 161 post-prandial, 161 uptake from portal blood, 181 Non-mediated transport, 222 Non-steady state conditions flux measurements, 17 Norepinephrine anoxia, 52

502 Normoxia contribution of pentose shunt in gas gland, 109 Nuclear magnetic resonance, 37, 42ff. muscle metabolism, 42 Nuclei thyroid hormone binding, 426 Nutrient absorption adaptive changes, 222 growth, 235 intestine, 221 Nutrient transport adaptations to diet, 229f. genetic adaptations, 230 intestine permeability, 230 regulation, 229 stimulation by growth hormone, 233 Nutrient transporter sequence homology, 235 Nutrition effects on hormones, 420 protein synthesis, 191 protein turnover, 192 Ocular muscle anserinase, 322 electric organ, 261 Olfactory cilia phospholipase C, 122 Olfactory epithelium phospholipase C, 122, 143 Olfactory nerve plasmalogens, 129 alkylacylphospholipids, 129 Omnivores, 211 glucose uptake, 230 Oocytes injection of AChR mRNA, 270 Oogenesis, 127 Ophidine HPLC, 311 imidazole dipeptides, 309 vertebrate muscle, 309 Optical structures photogenic tissue, 438 Organ protein synthesis, 192 Orientation muscle capillaries, 7 Omithine decarboxylase starvation, 172 Osmoregulation carnosine, 315 histidine-related dipeptides, 315 muscle amino acids, 315f. Ostracods coelenterazine, 436 luciferin, 436 Ouabain

SubjectIndex Na+/K+-ATPase in brain heater, 254 Ovarian fluid cyclooxygenase, 138 Ovarian follicles protein kinase C, 143 steroidogenesis, 143 Ovary cyclooxygenase, 138 prostanoid synthesis, 139 histidine-related dipeptides, 314 Ovulation eicosanoids, 142 Oxaloacetate heart performance, 85 Oxamate gas deposition, 112 gas gland lactate production, 112 Oxidative capacity white muscle, 384 3-Oxoacid CoA transferase thermogenic tissue, 247 Oxygen affinity, 341f. carrying capacity, 339ff. convective transport, 339 delivery, 338 in luminescence, 443 reaction with imidazolopyrazines, 461 storage, 338 tension in gas gland, 113 Oxygen consumption amino acid oxidation, 22 basal, 371 brain, 89 burst swimming, 359 gas gland tissue, 106 indirect calorimetry, 41 post-exercise, 383 postprandial increase, 205f. protein synthesis, 191 red blood cells, 91 red muscle, 370, 373 vs. lactate turnover, 19 white muscle, 370 Oxygen debt recovery, 75 Oxygen delivery, 339, 341, 343, 345 Oxygen flux capillary to muscle, If. muscle, 9 Oxygen minimum zone, 355 Oxygen radicals gas gland, 115 removal, 115 Oxygen transfer, 340 Oxygen transport, 91 histidine-related dipeptides, 324 Oxygen uptake gas gland tissue, 105

Subject Index

Oxygen uptake (continued) symmorphosis, 343 Oxyluciferin, 443, 445, 454 conversion to luciferin, 454 P450 enzymes epoxide formation, 139 Pacemaker nucleus electric organ, 262 nicotinic cholinergic neurons, 262 Palmitate cardiac performance, 85 oxidation in heart, 84f. substrate for heart, 84f. Palmitoyl-L-carnitine elasmobranch muscle mitochondrial substrate, 244 oxidation thermogenic tissue mitochondria, 251 tuna red muscle, 248 Pancreas, 123 Paracellular pathway intestinal transport, 223 Parasympathicomimetic drugs gas deposition, 112 Parr-smolt transformation cortisol, 233 Parvalbumin sonic muscle, 282f. Pasteur effect, 51 anoxia, 74 gas gland, 107, 113 Pentose phosphate shunt, see also hexose phosphate shunt gas gland enzymes, 108 gas gland metabolism, 109 gas gland tissue, 108ff. heart, 83 liver, 108 fete mirabile, 115 starvation, 404 PEPCK effects of cortisol, 422 fasting, 73 glycogenesis, 82 heart, 83 in muscular glycogen synthesis, 383 insulin, 69 kidney, 78f. starvation, 396, 414 subcellular distribution, 71 transcription, 397 Peptido-leukotrienes gill function, 142 Periportal cells glucose release, 69 Perivenous cells glycogenesis, 69 lipogenesis, 69

503 Peroxidase light organs, 442 Peroxisomes metabolism of alkylglycerophosphate, 122 Pesticides brain metabolism, 88 pH ATP/ADP ratio, 385 effects on glycolytic enzyme binding, 295 Phase polarity electric organ discharge, 263 Phenotypie adaptations intestinal transport, 230 Phenylalanine brush border uptake, 227f. free pool, 195 intraperitoneal injection, 195 intravenous injection, 195 microinjection, 195 oxidation, 22 protein-bound pool, 195 red blood cell transport, 162 white muscle, 195 3H-Phenylalanine protein synthesis, 193 specific activity, 193 L-2,6-3H-Phenylalanine protein synthesis, 195 specific activity, 195 time course, 195 Phenylephrine glycogenolysis, 77 Phloretin glucose transport, 225 Phlorizin glucose transport, 223f. Phorbol ester protein kinase C, 143 Phosphagens exercise, 385 Phosphatase lithium-sensitive, 143 Phosphate buffering, 325, 328 effects on glycolytic enzyme binding, 295 limitations, 386 muscle, 380 homogenate buffering, 328 signal in 31P-NMR, 44 stimulation of respiration, 386 utilization in recovery, 385 Phosphatidic acid structure, 120 Phosphatidyl ethanolamine methyltransferase hepatocytes, 122 Phosphatidyl serine decarboxylase hepatocytes, 122 Phosphatidylcholine, 120f. fatty acid composition of membranes, 132

504 Phosphatidylcholine (continued) fish tissues, 129 growth promotion, 145 in lipovitellin, 127 lipoproteins, 126 temperature acclimation, 134f. Phosphatidylethanolamine, 120f. changes with seawater adaptation, 135 fatty acid composition of membranes, 132 in fish tissues, 129 in lipovitellin, 127 liver mitochondria, 136 temperature acclimation, 134f. Phosphatidylglycerol, 120f. Phosphatidylinositol, 120f. fatty acid composition, 131 growth promotion, 145 in fish tissues, 129 in lipovitellin, 127 Phosphatidylserine, 120f. in fish tissues, 129 in lipovitellin, 127 Phosphatidylserine decarboxylase temperature effects, 135 Phosphoadenosine luciferin regeneration, 442 Phosphocreatine anaerobic glycolysis, 385 exercise, 385 muscle recovery, 381 resynthesis, 385 Phosphocreatine/creatine ratio NMR, 43 Phosphoenolpyruvate binding of lactate dehydrogenase, 300 binding of pyruvate kinase, 300 in muscle recovery, 383 Phosphoenolpyruvate carboxykinase, see PEPCK Phosphofructokinase-1 association with F-actin, 303 binding in turtle brain, 297 binding to F-actin, 299 binding to subcellular muscle structures, 292 binding to troponin C, 299 brain, 87 enzyme binding, 295, 300 gas gland tissue, 107, 113 glycolytic control, 81 heart, 83f. insulin, 69 kidney, 78 kinetic effects of F-actin, 302 liver, 70 myosin ATPase, 304 phosphorylation, 68, 300 polymerization, 300 red blood cells, 90 red muscle, 82 regulation, 291

Subject Index

release from muscle particulate matter, 295 role in gluconeogenesis, 68 starvation, 412 temperature effects, 75 white muscle, 81, 113 6-Phosphogluconate dehydrogenase carbon dioxide formation, 109 effects of diet, 172 gas gland tissue, 108, 110 kidney, 79 starvation, 404 Phosphoglucose isomerase gas gland tissue, 107 3-Phosphoglycerate kinase, 34 binding to subcellular muscle structures, 292 binding in turtle brain, 297 glycolytic control, 81 Phosphoglycerides, see glycerophospholipids teminology, 121 Phosphoinositide cycle, 122, 142f., 146 phospholipase C, 138 Phospholipase A2 bile salts, 123 calcium, 138 eicosanoid metabolism, 138 hepatopancreas, 123 microsomes, 122 Phospholipase C, 146 eicosanoid metabolism, 138 molecular forms, 143 olfactory cilia, 122 olfactory epithelium, 143 phosphoinositide cycle, 142 Phospholipase D, 122 Phospholipases intestine, 123 microsomes, 122 muscle, 122 olfactory cilia, 122 Phospholipids in intertissue lipid exchange, 23 mobilization in starvation, 402 plasma concentration at rest, 17 seawater adaptation, 135 starvation, 56 terminology, 121 Phosphorylation/dephosphorylation acetyl-coenzyme A carboxylase, 404 branched-chain alpha-ketoacid dehydrogenase, 175 glycogen phosphorylase, 68 glycolytic flux, 292 phosphofructokinase-1, 68, 300 pyruvate kinase, 68 triacylglycerol lipase, 403, 419 Phosvitin, 127 Photocytes luminescence, 436, 438 Photogenic cells, 453

Subject Index Photogenic oxidation, 436 Photogenic tissue capillary network, 438 filter, 438 innervation, 438 lens, 438 reflectors, 438 Photophores, 436, 442 light emission, 436 luciferin storage, 453 nasal, 443 secretions, 438 spectral characteristics, 436 Photoprotein binding of calcium, 443 Photoreceptor membrane glycerophospholipids, 137 Phycobiliprotein photoprotein, 445 Physical activity hematocrit, 340 Physoclists, 102 Physostomes, 102 Pigeon pectoralis muscle, 3 muscle morphometric data, 6 Pinocytosis lipoprotein uptake, 127 Pituitary growth hormone, 424 Plasma amino acids, 23 elasmobranch starvation, 412 fasting, 409 essential amino acids, 161 fatty acids effects of ACTH, 422 effects of hormones, 419 starvation, 419 glucose elasmobranch, 247 pool, 379 lecithin: alcohol acyltransferase, 125 lecithin: cholesterol acyltransferase, 125 lyso-glycerophospholipids, 123 non-essential amino acids, 161 platelet-activating factor acetylhydrolase, 143 postprandial amino acid pool, 167, 204 protein percent of whole animal protein, 213 starvation, 408 substrates list of resting concentrations, 17 wax esters, 125 Plasmalemma in electrocytes, 272 Plasmalogens, 120 brain, 129 olfactory nerve, 129

505 white muscle, 129 Plasmologenase, 122 Platelet-activating factor synthesis in tissues, 143 Platelet-activating factor acetylhydrolase plasma, 143 Polyethylene glycol pyruvate kinase kinetics, 301 Polyribosomes muscle protein synthesis, 193 Polyunsaturated fatty acids, 24 Portal system transport of intestinal lipoproteins, 124 Positive allometry spleen weight, 340 Posterior intestine immunological role, 163 protein absorption, 163 Power output weight specific, 357 Preluciferin in luciferin recylcing, 454 Pressure adaptation gill Na+/K+-ATPase, 136 Prey luminescence, 447 Prey immobilization electric organ, 261 Progesterone metabolism, 423 Prolactin effects on plasma fatty acids, 25 Proline brush border uptake, 227 elasmobranch muscle mitochondrial substrate, 244 fasting, 407 muscle, 407 oxidation in thermogenic tissue mitochondria, 251 transporter correlation with diet, 165 uptake, 231 brush border membrane vesicles, 231 dependence on diet, 230 effects of growth hormone, 233 effects of steroids, 233 effects of thyroid hormones, 234 stimulation, 231 Propranolol glycogen phosphorylase, 74 pyruvate kinase activity, 74 Prostacyclin (PGI) blood clotting, 142 eicosanoid metabolism, 138f. elasmobranch leukocytes, 139 fatty acid precursors, 140 thrombocyte aggregation, 142 Prostaglandins

506 Prostaglandins (continued) D series distribution, 139 fatty acid precursors, 140 E-series distribution, 139 fatty acid precursors, 140 eicosanoid metabolism, 138f. F-series fatty acid precursors, 140 distribution, 139 ovulation, 142 spawning activity, 142 vasoactive properties, 142 Prostanoids, 137 vasoactive properties, 142 synthesis, 139 tissue distribution, 139 Protein adipose tissue, 369 as endogenous fuel, 48 catabolism nitrogen excretion, 22 consumption rate definition, 193 content of muscles, 50 degradation rate definition, 193 resynthesis, 192 degradation, 192f., 371 anoxia, 53 cortisol, 202 muscle, 178 pathways, 196 protein recycling, 199 role of genetics, 201 scaling with body weight, 212 starvation, 199 extracellular fluid, 369 growth daily balance- feeding, 197 daily balance - starving, 198 growth rate definition, 193 imidazole pK value, 325 liver, 369 composition, 395f. metabolism effects of cortisol, 422 muscle, 11 mobilization glucocorticoids, 421 starvation, 405 muscle content, 395L nitrogen daily flux - schematic, 197 daily absorption, 197 oxidation exercise, 53

Subject Index plasma concentration at rest, 17 pool red muscle, 369 retention muscle, 178 retention efficiency definition, 193 sparing dietary lipid, 206 white muscle, 369 Protein kinase enzyme binding, 299 exhausting exercise, 54 Protein kinase C phosphoinositide cycle, 143 Protein loss, 192 Protein synthesis, 191ft., 197ff. cortisol, 202 cost, 191,206ff., 214 direct approach, 206f. indirect approach, 207 daily balance - feeding, 197 daily balance - starving, 198 dietary amino acids, 199 diurnal variation, 196 effects of thyroid hormones, 425 essential amino acids, 205 fasting, 407 muscle, 56 heat production, 207 hormonal stimulation, 199 insulin, 411 3H.leucine, 196 limits through amino acid transport, 167 liver, 48 mass synthesized per day, 192 methionine-deficient diet, 206 muscle, 178 nutrition, 191 oxygen consumption, 191, 207 postprandial, 48, 205f. rate definition, 193 retention efficiency, 192 definition, 193 growth, 198 protein synthesis, 198 ribosomal activity, 199 role of environment, 201 role of nutrition, 201 scaling with body weight, 212 sewage sludge diet, 212 stable isotopes, 196 starvation, 199, 407 thyroid hormones, 76, 425 tritiated amino acids, 195 vs. amino acid transport, 165 Protein synthetic scope, 210 Protein turnover

Subject Index Protein turnover (continued) definition, 192f. environmental influence, 196 fasting, 407 formulae, 193 general model, 197 genotype, 201 measurements, 193 nutritional status, 192 parameters, 193 rates in fish species, 210 sewage sludge diet, 212 starvation, 199 Protein-nitrogen absorption rate definition, 193 Protein-nitrogen retention efficiency growth, 201 Proteolysis muscle, 410 starvation, 396, 405, 410 Proton gas gland metabolism, 109 hemoglobin oxygen affinity, 111 Proton buffering histidine-related dipeptides, 324 Proton extrusion gas gland, 112 Proton gradient, 34 brown adipose tissue, 241 gas gland, 112 heater mitochondria, 254 proton release, 112 Proton influx heater mitochondria, 254 Proton leak elasmobranch thermal tissue, 250 heater organ heat production, 248 Proton transfer gas gland schematic, 114 PUFA (poly-unsaturated fatty acids) dioxygenase, 137 eicosanoid metabolism, 137f. embryonic development, 144 in diet, 133 lipoproteins, 124, 126 phospholipids, 130f. starvation, 56 temperature acclimation, 134 Puget Sound, 450 Pulse-type fish electric organ discharge, 262 Pupfish metabolic rate, 343 Purine nucleotide cycle, 386 kidney, 174 liver, 167 muscle, 179 Putter-yon Bertalanffy model, 191 Pyloric caeca

507 D-amino acid oxidase, 166 coelenterazine, 442 histidine-related dipeptides, 314 luciferin storage, 453 Na+/glucose transporter, 224 relation to light organ, 439 Pyruvate elasmobranch muscle mitochondrial substrate, 244 fatty acid oxidation, 384 in muscle, 383 oxidation in red blood cells, 162 oxidation in thermogenic tissue mitochondria, 251 Pyruvate carboxylase brain, 87 glycogenesis, 82 heart, 84 in muscular glycogen synthesis, 383 kidney, 78 liver, 70 muscle, 81f. Pyruvate dehydrogenase activation, 380 control by calcium, 252 dephosphorylation during exercise, 380 regulation in muscle, 384 thermogenic tissue, 247 Pyruvate kinase, 34 association with F-actin, 303 binding in turtle brain, 297 binding to subcellular muscle structures, 292 brain, 87 correlation with lactate disappearance, 384 effects of metabolites on enzyme binding, 300 equilibrium conditions, 383 gas gland tissue, 107 glycogenesis, 82 heart, 83f. hypoxia, 74 in muscular glycogen synthesis, 383 insulin, 69 kidney, 78 kinetic effects of F-actin, 302 liver, 70 negative allometry, 357 propranolol, 74 raven heart, 357 red blood cells, 90 red muscle, 82 regulation, 68, 291 reversal, 83, 383f. scaling, 355 starvation, 412 temperature effects, 75 white muscle, 81, 384 Pyruvate oxidation elasmobranch muscle mitochondria, 247 tuna red muscle, 248

508 Pyruvate transport monocarboxylate carrier, 246 red blood cells, 90 Quantum yield luminescence, 455 luciferins, 441 Rab6p in electric organ, 273 Rat soleus muscle morphometric data, 6 Rate of energy loss starvation, 405 Rays electric organs, 261 Re-feeding, 73f., 173, 393, 412f. anabolic effects, 73 insulin binding, 73 liver glycogen, 73 ornithine decarboxylase, 173 plasma hormones, 74 plasma insulin, 412 Receptor-mediated endocytosis apoproteins, 126 Receptors apoproteins, 126 glucagon, 72f., 419f. luciferin, 458 Recovery, 20 burst exercise, 375 fat metabolism, 384 fatty acid oxidation, 384 glycogen dynamics, 381L glycogen repletion, 82 glycogen synthesis in muscle, 385 glycolytic control, 81 lactate dynamics, 380f. lipid oxidation, 381 metabolites, 380 oxygen debt, 75 rate, 386 Rectal gland glycerophospholipid headgroup composition, 129 phosphoinositide cycle, 142 Recycling luciferin, 454 Red blood cell alanine oxidation, 162 amino acids, 161 amino acid concentration, 23 amino acid metabolism enzymes, 162 amino acid utilization, 162 band Ill anion exchanger, 114 carbohydrate metabolism, 89f. carbonic anhydrase, 114 ethanolamine plasmalogens, 129 glucose

Subject Index metabolism, 91 oxidation, 91 permeability, 89 transport, 66, 89 hexosemonophosphate shunt, 90 intracellular pH, 91 catecholamines, 91 lactate production, 91 lactate transport, 66 luciferin binding, 452 mitochondria, 66, 90 oxidative capacity, 90 oxygen consumption, 91 proton transfer schematic, 114 swelling, 37, 91 Red muscle acid lipase, 403 aerobic capacity, 250 amino acids, 369 content, 50 exercise, 53 oxidation, 178, 371 anaerobic energy production, 370 anserine in starvation, 318 anserine transport, 324 asparagine synthetase, 178 ATP turnover, 370, 374 branched-chain a-ketoacid dehydrogenase, 175 buffering capacity, 326f. burst power requirements, 357 capillary manifolds, 5 capillary surface per fiber volume, 10 carnosine in starvation, 318 citrate synthase, 250 cytochrome c oxidase, 352 depletion, 407 elasmobranch, 377 fatty acids, 369 oxidation, 80 fine structure, 4 free fatty acids, 48 glucose, 369 oxidation, 373 utilization, 370 glutaminase, 244f. glutamine oxidation, 248 glutamine synthetase, 178, 245 glycogen, 46, 67, 80, 369, 372 heat production, 243 hexokinase, 247, 373f. histidine in starvation, 318 histidine turnover, 320 histidine uptake, 320 ketone bodies, 377 lactate, 369 accumulation, 80 as substrate, 249 exchange, 20 production, 370

Subject Index Red muscle (continued) lipase, 422 lipid content, 48, 401 mass, 373 mitochondria, 371 morphometry, 1 oxidation rates, 377 oxidative capacity, 370 oxygen consumption, 179, 370, 373 power output, 357 protein, 369 content, 50 proteolysis, 405 provision of fat fuel, 376f. scaling, 342 sealing of power output, 358 serine hydroxymethyl transferase, 178 structural design, lff. sustained swimming, 80 triacylglycerol lipase, 48 triacylglycerols, 369 uptake of 2-deoxyglucose, 373 work output, 370 Red muscle (tuna) alpha-glycerophosphate dehydrogenase, 249 fat content, 248 glycogen, 248 hexokinase, 248 hydrogen shuttles, 249 lipid droplets, 248 morphometrie data, 6 palmitoylcarnitine oxidation, 248 pyruvate oxidation, 248 Redox balance elasmobranch muscle mitochondria, 245 Reflectors guanine, 438 lens, 438 luminescent species, 436 Regulation glycolytic enzymes, 291 Reptiles distribution of histidine-related dipeptides, 312 lactate oxidation, 381 muscle glycogen synthesis, 381 Resorbing bladder, 102 Respiration isometric scaling, 339 role of phosphate, 386 surface area, 339 white muscle, 384 Respiratory area scaling, 343 scaling exponents, 343 Respiratory chain gas gland tissue, 105 Respiratory control ratio elasmobranch muscle mitochondria, 247 Respiratory quotient, 42

509 exercised muscle, 384 Rest glycolytic enzyme binding, 292 Rete mirabile capillaries, 115 carbonic anhydrase, 113 gas gland, 1I0 swimbladder, 102 Retina di-PUFA, 133 rod membrane lipids, 137 Ribosomal activity protein synthesis, 199 Ribosomal protein percent of whole animal protein, 213 Ribosomes gas gland cells, 103 protein synthesis, 199 RNA Activity definition, 193 Roe glycerophospholipid headgroup composition, 129 Root effect gas gland, 111 red blood cell, 114 rRNA production protein synthesis, 209 RTG-2 cell line cost of protein synthesis, 208 fractional protein synthesis rate vs. oxygen consumption, 208 glycerophospholipid fatty acids, 131 Sach's organ, 263 Salinity lipid composition, 135 Salt gland (birds) phosphoinositide cycle, 143 Sampling artifacts, 36 freezing rate, 38 freeze clamping, 38 techniques, 35f. handling stress, 36f. tissue extraction, 35 Sand goby metabolic rate, 344 Sarcomere in electric organ, 270 in electrocytes, 261 length, 3, 7 Sarcomeric actin, 271 Sarcoplasmic protein synthesis, 407 starvation, 407 Sarcoplasmic reticulum calcium release during tissue extraction, 39 mobilization in starvation, 407

510 Sarcoplasmic reticulum (continued) sonic muscle, 282 thermogenic tissue, 252 Sarcosine, 49 Scale cells cost of protein synthesis, 208f. fractional protein synthesis rate vs. oxygen consumption, 208 Scales nitrogen loss, 198 Scaling aerobic metabolism, 336ff. anaerobic metabolism, 354ff. blood volume, 339 circulation time, 339 contraction frequency, 344 growth rate, 344 heart size, 344f. hematocrit, 340 repiration, 339 spleen, 340 stroke volume, 344 Scaling exponent, 336, 344 frequency distribution for standard metabolic rate, 336 sustained swimming, 344 weight-specific activity, 346 sprint swimming, 344 Seasonality anoxia, 76 carbohydrate metabolism, 76 hormone responses, 76 hypoxia tolerance, 74 liver glycogen, 76 Seawater adaptation cortisol, 233 hydrogen peroxide, 459 superoxide anion, 459 Secretory activity photocytes, 438 Secretory bladder, 102 Serine brush border uptake, 227 in glycerophospholipids, 120 metabolism, 175 liver, 168 oxidation, 371 kidney, 79 transport Na +-dependent, 161 red blood cells, 161 Serine dehydratase kidney, 175 liver, 168, 175 Serine-hydroxymethyl transferase kidney, 175 liver, 168 liver, 175

Subject Index red muscle, 178 Serine-pyruvate transaminase kidney, 175 liver, 175 Serum amino acids in fasting, 160 Sewage sludge diet protein growth, 212 protein turnover, 212 Sex differences electric organ discharge, 262, 266f. sonic muscle, 279ff. sound duration, 280 sound frequency, 280 Sex steroids influence on electric organ discharge, 266 Sexual dimorphism electric organ discharge frequency, 267 sonic nerve terminals, 281 Signal transduction pathways, 77 Single concentrating effect, 101, 110f. gas gland, 112 Skin cyclooxygenase, 138 glycerophospholipid headgroup composition, 129 glycerophospholipid storage, 128 keratin, 272 lipoxygenase products, 139 lipoxygenase specificity, 142 Slow muscle fibers, 337 Slyke muscle buffering, 326L Smooth muscle cells secretory bladder, 102 Snakes distribution of histidine-related dipeptides, 312 Sneaker males toadfish, 281 Social communication electric organs, 261 Social rank growth performance, 203 immunocompetence, 203 stress response, 203 Solubility swimbladder gas, 101 Somatostatin effects on plasma fatty acids, 25 Sonic motoneuron, 281 Sonic motor pathway, 281 Sonic muscle, 279ff. aspartate aminotransferase, 285 ATPase, 283 bouton, 281 Ca 2+ ATPase, 282 calcium content, 282 citrate synthase, 284f. fatigue resistance, 283

Subject Index Sonic muscle (continued) fiber typing, 283 glucose 6-phosphate dehydrogenase, 285 glycogen content, 283 lactate dehydrogenase, 285 malate dehydrogenase, 285 malic enzyme, 285 mitochondrial density, 284 mitochondrial volume, 286 motor nerve, 280 myofibrils, 282 myosin light chains, 283 NAD diaphorase, 283 neuromuscular junction, 281 relaxation, 280 sarcoplasmic reticulum, 282 T-tubule system, 282 vs. muscle mass, 284 Z-line, 282 Sonic muscle weight androgens, 287 estradiol, 287 estrogen, 287 hormonal control, 287 keto-testosterone, 287 testosterone, 287 Sonic nerve terminal (bouton) midshipman, 281 sexual dimorphism, 281 Sound production, 279ff. Spawning activity eicosanoids, 142 Spawning migration alanine flux, 22 alanine gluconeogenesis, 22, 55, 170 anserine, 315 carboxypeptidase A, 407 cathepsin D, 407 corticoids, 423 fatty acid composition, 402 histidine, 315 insulin, 414 lactate gluconeogenesis, 55 lipid changes, 402 muscle proteolysis, 170 Species difference hormonal response, 77 Spectral shifts photoproteins, 445 Sphingomyelin, 121 Spleen amino acids in fasting, 160 contraction, 340 cyclooxygenase, 138 ethanolamine plasmalogens, 129 hemoglobin, 341 histidine-related dipeptides, 314 platelet-activating factor synthesis, 143 scaling, 340

511 storage of erythrocytes, 338 Sprint swimming scaling exponent, 344 Standard metabolic rate, 336 scaling, 344, 359 negative allometry, 359 Starvation, 393ff. actin levels, 408 alanine aminotransferaase, 406 alanine release, 410 amino acid gluconeogenesis, 170 amino acids, 370, 409 aminotransferases, 171 ammonia excretion, 167, 171 aspartate aminotransferase, 406 body protein loss, 182 elasmobranch fish, 255, 411 enzyme activities, 406 epinephrine, 420 glucagon/insulin ratio, 416f. glucocorticoids, 421 glucose synthesis from alanine, 182 glucose turnover, 374 glutamate dehydrogenase, 171 glycogen utilization, 55 glycolytic enzymes, 407 growth hormone levels, 423 histidine-related dipeptides, 317f. ketone bodies, 377 lipid depletion, 376, 400 lipoprotein composition, 125 luminescence, 461 mobilization of mitochondria, 407 mobilization of sarcoplasmic reticulum, 407 muscle amino acids, 178 muscle carnosine, 316ft. muscle histidine, 178, 316ft. myofibrillar protein, 407 nitrogen loss, 56 ornithine decarboxylase, 172 plasma branched-chain amino acids, 182 plasma proteins,. 408 protein degradation, 56, 199, 407 protein mobilization, 405 protein synthesis, 199, 406f. protein turnover, 199 renal amino acid metabolism, 177 triacylglycerol lipase, 419 Steady-state conditions, flux measurements, 17 lactate turnover, 20 Steady-state swimming, 374 red muscle, 367 Steroidogenesis A23187, 143 protein kinase C, 143 Steroids non-genomic actions, 232 regulation of nutrient transport, 231s 235

512 Stoichiometry intestinal transport, 223 Stomach cyclooxygenase, 138 luciferin, 451 Storage luciferin, 452f. Stress carbohydrate metabolism, 76 hematocrit, 340 social rank, 203 subordinate fish, 202 Stroke volume, 344 hypoxia, 50 Structural lipids lipids, 402 starvation, 400 Stunting, 346 Subordinate fish feeding, 201 growth, 201 Subsarcolemmal mitochondria vs. muscle fiber size, 9 Substrate flux, 15 Substrate transfer muscle, 11 Subunits AChR, 269 Succinate from aspartate, 53 hypoxia, 52 Succinate dehydrogenase scaling, 346 thermogenic tissue, 247 Succinyl-CoA synthetase thermogenic tissue, 247 Sucrase intestine, 223 Sulfonamide carbonic anhydrase, 113 Superoxide anion in luminescence, 459 in seawater, 459 Superoxide dismutase gas gland, 115 Sustained swimming, 80, 375 red muscle, 80 scaling exponents, 3 ~ Swim tunnel, 378 Swimbladder, see also gas gland acidification, 111 blood pH, 111 counter-current exchange, 102 gas partial pressure, 101 metabolism, 101ft. origin, 102 perfusion catecholamines, 112 resorbing bladder, 102

SubjectIndex secretory bladder, 102 sonic muscle, 279ff. sound production, 279ff. tissue layers, 103 volume, 101 Swimming metabolism, 371 Symbionts bioluminescence, 438 Symmorphosis, 339ff., 345 Sympathetic system photogenic tissue innervation, 438 Synaptic membranes electric organ, 269f. Synaptosomes enzyme association, 296 T-tubule system in electrocytes, 261 sonic muscle, 282 Tail beat frequency, 337 Taurine intestinal transport, 226 muscle buffering, 328 red blood cells, 161 renal secretion, 173 Teleosts distribution of histidine-related dipeptides, 312 muscle buffering capacity, 326 Temperature acclimation gluconeogenesis, 75 protein synthesis, 76 Q10, 75 enzyme activities, 75 fiber volume, 83 heart performance, 85 hypoxia, 83 Krebs cycle, 86 lipid metabolism, 134f. membrane composition, 134 muscle recruitment, 83 myocyte response, 86 phospholipid structure, 134 swimming performance, 83 Testes cyclooxygenase, 138 prostanoid synthesis, 139 protein kinase C, 143 steroidogenesis, 143 Testosterone effect on electric organ discharge, 267 metabolism, 423 sonic muscle, 287 TF cell line glycerophospholipid fatty acids, 131 Thermal compensation gluconeogenesis, 75 hepatocytes, 75 Thermal tolerance

Subject Index

Thermal tolerance (continued) locomotion, 83 Thermogenesis thyroid hormone, 425 Thermogenic mechanisms, 242 Thermogenic tissue, 250ff. ot-glycerophosphate oxidation, 251 amino acid oxidation, 251 carbohydrate oxidation, 251 contractile apparatus, 250 enzyme activities, 251 glycerol kinase, 252 glycogen, 251 hexokinase, 251 ketone bodies vs. glutamine, 247 ketone body oxidation, 251 metabolic organization - schematic, 254 mitochondrial respiration, 252 myosin ATPase, 250 proton leak, 251 sarcoplasmic reticulum, 252 substrate oxidation, 251 swordfish brain, 251 thermogenin, 251 Thermogenin brown adipose tissue, 241 swordfish thermogenic tissue, 251 Threshold electrocytes, 263 Thrombocytes A23187, 140f. aggregation eicosanoids, 142 cyclooxygenase, 138 lipoxygenase, 141 prostaglandin synthesis, 140 Thromboxane distribution, 139 eicosanoid metabolism, 138f. fatty acid precursors, 140 thrombocyte aggregation, 142 Thyroid hormones conversion of 3"4 to T3, 426 effects on plasma fatty acids, 25 metabolic effects, 425 mitochondrial respiration, 255 protein synthesis, 76 receptors, 426 regulation of nutrient transport, 231, 233 role in thermogenic tissues, 255 starvation, 425f. stimulation of nutrient uptake, 233 thermogenesis, 242, 425 Tissue protein synthesis, 192 Tissue extraction metabolites, 38f. temperature effects, 39 Tissue respiration, 346 Toadfish

513

sonic muscle, 279ff., 357 sound production, 279ff. Torpor, 75 Transaldolase gas gland tissue, 108, 113 Transdeamination liver, 371 Transketolase gas gland tissue, 108, 113 Transport amino acids, 162 carrier-mediated, 222 chloride-dependent, 222 diffusion, 222 epithelia, 222 glucose, 223 intestine, 221 ionic requirements, 222 luciferin, 452 monosaccharides, 223 Na+-dependent, 222 non-mediated, 222 paracellular, 223 phlorizin-inhibitable, 223 Transporter density, 231 Triacylglycerol lipase phosphorylation, 403, 419 red muscle, 48 starvation, 419 Triacylglycerols, 369 adipose tissue, 369 embryonic development, 144 extracellular fluid, 369 in intertissue lipid exchange, 23 liver, 369 mobilization during exercise, 376 plasma concentration at rest, 17 red muscle, 369 starvation, 401 stores, 375, 377 white muscle, 369 Tricarboxylic acid cycle, see also Krebs cycle gas gland metabolism, 109 3,5,Y-Triiodo-L-thyronine see thyroid hormones Trimethylamine muscle, 49 Triosephosphate isomerase association with F-actin, 303 Triton X-100 detergent solubilization technique, 296f. tRNA protein synthesis, 209 Trophic transfer luciferin, 446 Tropomyosin in electrocytes, 270 Troponin C phosphofructokinase binding, 299 Tryptophan

514

Tryptophan (continued) red blood cell transport, 162 Tubulin enzyme binding, 292 Tunas thermogenic tissue, 242 Turbulent flow power requirements for swimming, 356 Turtle glycolytic enzyme binding in brain, 297 Tween 80 leucine transport, 232 "l~vitch contraction time, 337 "l~rosine red blood cell transport, 162 "I~osine kinase insulin receptor, 415 Ucrit, 179, 370ff. lactate turnover, 375 red muscle glucose utilization, 373 Ultrastructure vertebrate muscles, 6 Urea excretion, 405 nitrogen loss, 198 Urotensin II effects on plasma fatty acids, 25

Vagus gas deposition, 112 Valine brush border uptake, 227 effect on branched-chain amino acid aminotransferase, 177 metabolic fate, 175 starvation, 409 Van Slyke, 326 Vargula luciferin, see also luciferin derivatives, 442 distribution in fishes, 441 structure, 440 Vascular heat exchangers, 242 Vasoactive peptides gluconeogenesis, 68 glycogenolysis, 68 Vasotocin gluconeogenesis, 68 glycogenolysis, 68 signai transduction, 77 Vertical migration, 101 Viscera cytochrome c oxidase, 352 triacylglycerol mobilization, 375 Visceral fat, 47 storage for triacylglycerols, 399f. supply of free fatty acids, 377 Vitellogenesis, 127 ViteUogenin

Subject Index

density, 127 lipid composition, 127 migration, 408 receptor-mediated micropinocytosis, 127 starvation, 408 transport, 127 uptake, 127 VLDL (very low density lipoproteins) average composition, 25 composition, 124 estrogen, 127 hepatic synthesis, 125 hydrolysis, 126 in intertissue lipid exchange, 23 lipid transport, 124 receptor-mediated endocytosis, 127 uptake by oocytes, 127 Vocalization frequency, 280 toadfish, 280 Voltage-clamp electrocytes, 268 Volume of mitochondria muscle fiber, 3 yon Bertalanffy growth equation, 211, 343 Warburg gas gland metabolism, 105 Water liver content, 395f. muscle content, 395f. Wave-type fish electric organ discharge, 262 Waveform electric organ discharge, 262 Wax ester plasma, 125 Weakfish sonic muscle, 281 Weight gain protein turnover, 192 Weight, see scaling Weight-specific activity scaling exponents, 346 Whales histidine-related dipeptides, 313 White muscle aerobic capacity, 379 amino acids, 315ff., 369 content, 50 exercise, 53 oxidation, 371 anaerobic energy production, 370 anserine in starvation, 318 anserine transport, 324 aspartate aminotransferase, 285 ATP utilization, 370 buffering capacity, 356 burst swimming, 80

Subject Index White muscle (continued) capillary surface per fiber volume, 10 carnosine in starvation, 318 citrate synthase, 284f., 354 cytochrome c oxidase, 352 depletion, 407 fatty acids, 369 fraction rate of protein synthesis, 204f. fractional protein synthesis rate, 213 free phenylalanine, 195 gas gland, 106 glucose 6-phosphate dehydrogenase, 285 glucose, 369 utilization, 370 glycogen content, 46, 67, 80, 369, 371f., 374 histidine, 315ff. content, 312f. starvation, 318 turnover, 320 uptake, 320 washout, 319 histidine-related dipeptides, 312f. lactate, 369, 371 accumulation, 80, 249 dehydrogenase, 285, 354 exchange, 19, 20 production, 370 retention, 19 lipid content, 401 malate dehydrogenase, 285

515 malic enzyme, 285 mitochondria, 371 density, 284 volume density, 379 myofibril space, 379 oxygen consumption, 370 phenylalanine specific activity, 195 plasmalogens, 129 protein, 369 content, 50 synthesis vs. amino acid pool, 204f. proteolysis, 405 anoxia, 53 release of lactate, 374 scaling of protein metabolism, 212 transport of deoxyglucose, 382 triacylglycerols, 369 work output, 370 White muscle- tuna morphometric data, 6 Work output red muscle, 370 white muscle, 370

Xenopus oocytes injection of AChR mRNA, 270 Z bands electrocytes, 272 sonic muscle, 282

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