Biology of Metabolism in Growing Animals (Biology of Growing Animals Series - Volume 3)

Biology of Metabolism in Growing Animals Edited by

D.G. Burrin USDA /ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA

H.J. Mersmann USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA

Technical Editor

E. Salek The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Jablonna n/Warsaw, Poland

Edinburg London New York Oxford Philadelphia St. Louis Sydney Toronto 2005

Elsevier Limited © 2005 Elsevier Limited. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without either the prior permission of the publishers or a licence permitting restricted copying in the United Kingdom issued by the Copyright Licensing Agency, 90 Tottenham Court Road, London W1T 4LP. Permissions may be sought directly from Elsevier’s Health Sciences Rights Department in Philadelphia, USA: phone: (+1) 215 238 7869, fax: (+1) 215 238 2239, e-mail: [email protected]. You may also complete your request on-line via the Elsevier homepage (http://www.elsevier.com), by selecting ‘Customer Support’ and then ‘Obtaining Permissions’. First published 2005 ISBN 0 444 510133 British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging in Publication Data A catalog record for this book is available from the Library of Congress Notice Veterinary knowledge and best practice in this field are constantly changing. As new research and experience broaden our knowledge, changes in practice, treatment and drug therapy may become necessary or appropriate. Readers are advised to check the most current information provided (i) on procedures featured or (ii) by the manufacturer of each product to be administered, to verify the recommended dose or formula, the method and duration of administration, and contraindications. It is the responsiblity of the practitioner, relying on their own experience and knowldege of the patient, to make diagnoses, to determine dosages and the best treatment for each individual patient, and to take all appropriate safety precautions. To the fullest extent of the law, neither the publisher nor the editors assumes any liability for any injury and/or damage. The Publisher Printed in China

The Publisher’s policy is to use paper manufactured from sustainable forests

Keynotes

Progress in life sciences is unbelievably quick and usually unpredictable. The amount of research results communicated each minute, every day of the week makes it impossible to be up-to-date even in a very narrow scientific field. The situation as regards the transfer of these achievements to lecture halls and their integration with current “practical” scientific knowledge is even worse. The gap between the latest developments in life sciences announced by the world’s leading labs and the possibilities of their verification in medicine, biomedicine, and animal production seems to be expanding at a geometrical rate. At the same time “more and less” is known. It appears that the professional scientific world has run into difficulties in integrating what the scientific world knows. Soon, the old Scandinavian adage “the top consultants know everything about nothing” will be a truism. This series of books prepared by leading professionals will try to fill the gap between practical and basic knowledge in life sciences. We believe that the authors and their selections of the information presented in their chapters will still leave room for young animals to grow. Stepan Pierzynowski, Prof Series Editor

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INSTITUTIONS PROVIDING PATRONAGE AND FINANCIAL SUPPORT USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA

MS Milk Specialties Company

CIL Cambridge Isotope Laboratories, Inc.

ISOTEC Member of the SIGMAALDRICH Family

Lund University, Sweden

The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Poland

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Gramineer International AB, Lund, Sweden

Preface

This book Biology of Metabolism in Growing Animals is the third volume in the Elsevier book series entitled Biology of Growing Animals. This book is intended to provide in-depth reviews of the major areas of metabolism in growing domestic animals. The authors are leading, internationally recognized experts in the fields of nutrition, metabolism, and physiology and highlight some of the most recent advances in the field of metabolism. The chapters cover important new developments in interorgan, tissue-specific, and cell-specific metabolism of protein and amino acids, lipids and fatty acids and carbohydrates in monogastric and ruminant species, including humans. The study of metabolism represents a nexus of biological phenomena that integrates the nutrition, physiology, endocrinology, immunology, biochemistry and cell biology in an organism. The development of new methodological techniques and experimental approaches has provided scientists with a greater understanding of how key nutrients or substrates are metabolized at the cellular, organ and whole animal level. The book describes the impact of specific biochemical pathways and expression of critical enzymes, routes of nutrient or substrate input and anatomical or structural influences on the rates of metabolism in a given tissue or cell type. Major substrates/fuels for oxidative metabolism, key endocrine signaling pathways and intracellular molecules that regulate the major metabolic processes are described. Also discussed is the influence of ontogeny, stage of differentiation and major changes in diet, or the environment, on metabolism of growing animals. The concepts and specific findings in each area are discussed in the context of their impact on the nutrient requirements, growth, environmental impact, health and well-being of animals. Acknowledgements The editors wish to thank all of the authors for their outstanding contributions to the book. We also thank Ewa Salek for her assistance with technical editing and Jane Schoppe for administrative support. Thanks also go to the Series Editors, Stefan Pierzynowski and Romuald Zabielski, for the invitation and opportunity to put together this book. We sincerely thank the sponsors for their financial support, including USDA/ARS, Milk Specialties Company, Cambridge Isotope Laboratories, and Sigma-Aldrich-Isotec Inc. D.G. Burrin and H.J. Mersmann Editors

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Dedication

Peter J. Reeds The editors and many contributing authors of the book wish to dedicate this book to the memory of Dr. Peter Reeds. Peter Reeds was a close colleague, friend and mentor to many of the contributing authors of this book. Peter Reeds was born in England in 1945 and completed his Ph.D. in nutritional biochemistry at the University of Southampton, in 1971. His doctoral research focused on the interactions between insulin and growth hormone in the regulation of muscle protein synthesis and demonstrated the synergy between their separate mechanisms of action. Peter Reeds went on to complete postdoctoral training at the Tropical Metabolism Research Unit in Jamaica under the mentorship of Professor John Waterlow. His early years of training provided a foundation in key areas that would be central themes in his career, namely protein metabolism, isotope kinetics and growth regulation. In 1976, Peter Reeds moved to the Rowett Research Institute in Aberdeen, Scotland, to work under the guidance of the Director, Sir Kenneth Blaxter. During his years at the Rowett, Peter Reeds established himself as a leader in the science of growth regulation, protein metabolism and the nutrient requirements of farm livestock. In 1987, Peter Reeds moved to the Children’s Nutrition Research Center in the Department of Pediatrics at Baylor College of Medicine, where he resumed his longstanding interests in human pediatric nutrition and developmental aspects of growth. In 2001, Peter Reeds left the Children’s Nutrition Research Center to assume a position as Professor of Animal Sciences in the Faculty Excellence Program at the University of Illinois at Urbana-Champaign. During his career, Peter Reeds made many seminal contributions to our understanding of protein and amino acid metabolism and the biology of growth regulation. His intellectual brilliance was evident in the breadth and volume of his work. More importantly, however,

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Dedication

Peter Reeds was a wonderful human being with an irrepressible wit and sense of humor. His sense of humor was reflected in his exuberance and excitement for science, which was infectious to those with whom he worked. Peter Reeds died on August 13, 2002, from complications of Legionnaire’s disease. His legacy to the science of nutrition and metabolism will be long remembered by his countless friends, colleagues and members of the nutrition science community.

Contributors

Ball R.O. – Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2P5; The Research Institute, The Hospital for Sick Children, Toronto, Department of Nutritional Sciences, University of Toronto, Toronto, Ontoria, Canada Baracos V.E. – Department of Oncology, University of Alberta, Edmonton, Alberta, Canada T6G1Z2 Bell A.W. – Department of Animal Science, Cornell University, Ithaca, NY 14853–4801, USA Bertolo R.F.P. – Department of Biochemistry, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada, A1B 3X9 Burrin D.G. – USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA Carstens G.E. – Department of Animal Science, Texas A&M University, College Station, TX 77483–2471, USA Damon M. – INRA, Joint Research Unit for Calf and Pig Production, 35590 Saint Gilles, France Davis T.A. – United States Department of Agriculture/Agricultural Research Service, Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA Donkin S.S. – Department of Animal Sciences, Purdue University, West Lafayette, IN 47907, USA Drackley J.K. – Department of Animal Sciences, University of Illinois, Urbana, IL 61801, USA Ehrhardt R.A. – Department of Animal Science, Cornell University, Ithaca, NY 14853–4801, USA Escobar J. – Department of Animal Sciences, University of Illinois, Urbana, IL61801, USA Fiorotto M.L. – United States Department of Agriculture/Agricultural Research Service, Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA Flynn N.E. – Department of Chemistry and Biochemistry, Angelo State University, San Angelo, TX 76909, USA Greenwood P.L. – NSW Agriculture Beef Industry Centre, University of New England, Armidale, NSW 2351, Australia xi

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Contributors

Guan X. – USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA Hammon H. – Research Institute for Biology of Farm Animals (Oskar Kellner Institute), 18196 Dummerstorf, Germany Harmon D.L. – Department of Animal Sciences, University of Kentucky, Lexington, KY 40546-0215, USA Herpin P. – INRA, Joint Research Unit for Calf and Pig Production, 35590, SaintGilles, France Huntington G.B. – Department of Animal Science, North Carolina State University, Raleigh, NC 27695-7621, USA Innis S.M. – Department of Paediatrics, University of British Columbia, Vancouver, British Columbia, Canada, V5Z 4H4 Jesse B.W. – Department of Animal Science, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901-8525, USA Johnson R.W. – Department of Animal Sciences, University of Illinois, Urbana, IL 61801, USA Knabe D.A. – Department of Animal Science and Faculty of Nutrition, Texas A & M University, College Station, TX 77843-2471, USA Kristensen N.B. – Department of Animal Nutrition and Physiology, Danish Institute of Agricultural Sciences, DK-8830 Tjele, Denmark Le Dividich J. – INRA, Joint Research Unit for Calf and Pig Production, 35590 Saint-Gilles, France Lin X. – Department of Animal Science, North Carolina State University, Raleigh, NC 27695-7621, USA Louveau I. – INRA, Joint Research Unit for Calf and Pig Production, 35590 SaintGilles, France Lyvers-Peffer P. – Department of Animal Science, North Carolina State University, Raleigh, NC 27695-7621, USA Mersmann H.J. – USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA. Odle J. – Department of Animal Science, North Carolina State University, Raleigh, NC 27695-7621, USA Pencharz P.B. – Department of Paediatrics, University of Toronto, Toronto, Ontario, Canada M5G 1X8; The Research Institute, The Hospital for Sick Children, Toronto, Department of Nutritional Sciences, University of Toronto, Toronto, Ontaria, Canada Reynolds C.K. – Department of Animal Sciences, The Ohio State University, OARDC, 1680 Madison Avenue, Wooster, OH 44691-4096, USA Smith S.B. – Department of Animal Science, Texas A & M University, College Station, TX 77843-2471, USA Stoll B. – USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA Wu G. – Department of Animal Science and Faculty of Nutrition, Texas A & M University, College Station, TX 77843-2471, USA

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Regulation of metabolism and growth during prenatal life A. W. Bella, P. L. Greenwoodb, and R. A. Ehrhardt a aDepartment

of Animal Science, Cornell University, Ithaca, NY 14853-4801, USA Agriculture Beef Industry Centre, University of New England, Armidale, NSW 2351, Australia bNSW

Fetal energy and nitrogen requirements are met mostly by placental transfer of glucose and amino acids; fatty acids may contribute additional energy in some species. Placental metabolism accounts for much of the total net consumption of oxygen and macronutrients by the conceptus, and alters the composition of nutrients delivered to the fetus. The molecular basis for the facilitated transport of glucose by the placenta is well described; molecular characterization of the more complex systems for the active transport of most amino acids is under way. Maternal and placental macronutrient supply is a powerful regulator of fetal metabolism and growth, especially in late gestation. Endocrine mediation of these responses matures as gestation advances, adding to the influences of locally expressed regulators throughout gestation. Insulin, thyroid hormones, and, near term, corticosteroids, are especially influential in the direct and indirect control of fetal nutrient disposal and tissue growth. Prenatal growth retardation does not necessarily constrain the rate of neonatal growth, but at any given postnatal body weight, low-birth-weight lambs are fatter and have smaller muscles. Experimental evidence is accumulating for longer-term influences of prenatal nutrition through fetal programming of propensity for mature-onset diseases such as hypertension and type II diabetes.

1. INTRODUCTION The coordination of nutrient supply with tissue metabolism and growth during prenatal life in placental mammals is complex due to the varying influences of maternal nutrition and metabolic adaptations to the state of pregnancy, placental function, and gestational maturation of fetal endocrine and local regulatory systems. It is important to understand the separate and interdependent mechanisms by which these factors exert their effects on fetal growth and development, for several reasons. Increased neonatal mortality and morbidity in low-birthweight offspring remain major problems in some human and livestock populations, despite decades of study on the multifaceted etiology of intrauterine growth retardation (IUGR).

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Biology of Metabolism in Growing Animals D.G. Burrin and H. Mersmann (Eds.) © 2005 Elsevier Limited. All rights reserved.

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Fetal overgrowth due to maternal nutrition or diseases, such as diabetes, also increases perinatal mortality and incidence of postnatal problems. More intriguing and, possibly, with major ramifications for long-term health and productivity of humans and other animals, is the emerging evidence that fetal metabolic disturbance can lead to “programming” of increased predisposition to various disease syndromes during later postnatal life. This chapter will summarize briefly the quite detailed state of knowledge of quantitative metabolism of macronutrients in individual tissues and whole body of the fetus, and in the placenta, with emphasis on data obtained in vivo. The current understanding of placental transport of macronutrients and its implications for fetal nutrition and growth will be treated similarly. These topics will be a prelude to the major theme of regulation and coordination of metabolism and growth in the conceptus. Finally, the influence of prenatal experience on postnatal performance will be considered, with brief reference to recent experimental evidence for the concept of “fetal programming”.

2. MAJOR FEATURES OF CONCEPTUS METABOLISM AND GROWTH 2.1. Patterns of prenatal growth Early embryonic development, including organogenesis and initiation of placentation, is beyond the scope of this review. The morphology of embryo development in domestic animal species has been described by Noden and deLahunta (1985). Patterns of fetal and placental growth in the normal and growth-retarded sheep conceptus are illustrated in fig. 1. In this species, as in

Fig. 1. Patterns of fetal and placental growth in the normal (——) and growth-retarded (---) sheep conceptus. Adapted from the data of Ehrhardt and Bell (1995) and Greenwood et al. (2000).

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other placental mammals, postembryonic growth becomes quantitatively significant only after mid-gestation. However, this is preceded by rapid hyperplastic growth of the placenta, which attains all or most of its mass of dry tissue, protein, and DNA by mid-gestation (Ehrhardt and Bell, 1995). Fetal growth then follows its familiar, flattened sigmoid pattern during the latter half of gestation as it proceeds from an early exponential phase through a rapid, linear phase, and then, as term approaches, begins to diminish in rate. In most species, there is little or no increase in placental weight during this period; the ovine placenta actually diminishes in weight, mostly due to loss of extracellular water (Ehrhardt and Bell, 1995). However, the placenta undergoes extensive tissue remodeling after mid-gestation, including major proliferative growth of the umbilical vasculature (Teasdale, 1976), which is associated with a progressive increase in its functional capacity. Relations between placental size and function, and implications for fetal growth, are discussed in the next section. 2.2. Fetal requirements and metabolism of macronutrients Numerous studies on pregnant ewes have described fetal macronutrient requirements and metabolism in terms of umbilical exchanges of oxygen, nutrients, and metabolites, and of rates of net accretion of nutrients in growing tissues (see Battaglia and Meschia, 1988; Bell, 1993). These and similar data from pregnant cows (Comline and Silver, 1976; Reynolds et al., 1986; Ferrell, 1991) are summarized in table 1. During late pregnancy in these species, 35–40% of fetal energy is taken up as glucose and its fetal-placental metabolite, lactate, and a further 55% is taken up as free amino acids. In contrast to its importance as an energy source in the maternal ruminant, umbilical uptake of acetate could account for only 5–10% of fetal energy consumption. Placental capacity for transfer of longchain, nonesterified fatty acids (NEFA) and keto-acids is even more limited (see Bell, 1993), making these maternal substrates trivial contributors to fetal metabolism. Almost all of the nitrogen acquired by the fetus is in the form of amino acids, but a small net umbilical uptake of ammonia is derived from placental deamination of amino acids during the latter half of

Table 1 Fetal sources and disposal of energy and nitrogen in ewes and cows during late pregnancy Energy (kJ/kg·d)

Sources Glucose + lactate Amino acids Acetate NH3 Total Disposal Accretion Heat Urea Glutamate + serine efflux Total a Chung

Nitrogen (g/kg·d)

Ewe

Cow

Ewe

Cow

217a 177a 20b — 414

114f 156g 30h — 300

— 1.19a — 0.05e 1.24

— 1.09g — ND 1.09

133c 240a 16d 16a 405

72i 192g 15g ND 279

0.79c — 0.36d 0.11a 1.26

0.34i — 0.66g ND 1.00

et al. (1998), b Char and Creasy (1976), c McNeill et al. (1997), d Lemons and Schreiner (1983), et al. (1977), f Reynolds et al. (1986), g Ferrell (1991), h Comline and Silver (1976), i Ferrell et al. (1976).

e Holzman

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gestation (Holzman et al., 1977; Bell et al., 1989). About 60% of these amino acids are used for tissue protein synthesis, which accounts for ~18% of fetal energy expenditure (Kennaugh et al., 1987). The remaining 40% are rapidly catabolized, accounting for at least 30% of the oxidative requirements in the well-nourished sheep fetus (Faichney and White, 1987), or, in the case of glutamate and serine, taken up and metabolized by the placenta (Battaglia and Regnault, 2001). Less comprehensive studies of the fetal pig (Fowden et al., 1997) and horse (Fowden and Silver, 1995) suggest that in these species during late pregnancy, glucose is an even more important energy substrate than in fetal ruminants. The fetal horse, at least, appears to make less extensive use of amino acids as a source of energy (Silver et al., 1994; Fowden et al., 2000a). In all species studied, the fetal liver and, to a lesser extent, kidneys, develop the enzymatic capacity for gluconeogenesis during late gestation (see Fowden, 1997). In the well-fed, unstressed sheep fetus, endogenous glucose synthesis is negligible (Hay et al., 1984; Leury et al., 1990a). However, significant endogenous synthesis of glucose can be induced by maternal starvation or chronic undernutrition, presumably due to hepatic gluconeogenesis from amino acids (Hay et al., 1984; Leury et al., 1990a). Acute hypoxia and other stressors also increase net hepatic release of glucose due to increased rates of gluconeogenesis and/or glycogenolysis in fetal sheep (Rudolph et al., 1989; Townsend et al., 1991). 2.3. Metabolism of nonfetal conceptus tissues 2.3.1. Glucose metabolism The major contribution of the nonfetal components of the gravid uterus, especially the placenta, to oxygen and nutrient requirements of the conceptus is sometimes ignored. However, these requirements greatly affect the partitioning of nutrients within the gravid uterus and add substantially to the nutrient demands upon the dam. In late-pregnant ewes and cows, the aggregate weight of placentomes, consisting of fetal (cotyledonary) and maternal (caruncular) tissues, is less than 15% that of the attached fetus. However, the weight-specific metabolic rate of the placenta is so great that the uteroplacental tissues (placentomes, endometrium, myometrium) consume 35–50% of the oxygen and 60–70% of the glucose taken up by the uterus in ewes (Meschia et al., 1980) and cows (Reynolds et al., 1986). The weight-specific consumption of glucose by the diffuse placental tissues of the horse and pig is even greater than that of the epitheliochorial ruminant placenta, accounting for 80–90% of uterine glucose uptake during late gestation (Fowden, 1997). In all species, a considerable fraction of the glucose consumed by uteroplacental tissues is converted to lactate. Rates of lactate production and disposal into maternal and fetal circulations vary with species and gestational age. For example, production is relatively high and distributed mostly into the uterine circulation during late pregnancy in the mare, whereas the lower production in ruminants is mostly released into the umbilical circulation (Fowden, 1997). In ruminants, horse, and pig, a further, smaller fraction of glucose consumed by uteroplacental tissues is converted to fructose which is released into the fetal circulation and slowly metabolized by fetal tissues (Meznarich et al., 1987). 2.3.2 Amino acid metabolism Net uteroplacental consumption of amino acids, as a fraction of uterine uptake, is lower than that of glucose, presumably related to the negligible or small growth of the placenta

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and uterine tissues in sheep (Ehrhardt and Bell, 1995) and cattle (Bell et al., 1995) during late pregnancy. Nevertheless, net removal by the uteroplacental tissues has been estimated to account for 24% of uterine uptake of amino acid nitrogen in well-fed ewes during late pregnancy (Chung et al., 1998).

2.4. Gestational development of conceptus metabolism The many-fold increase in fetal mass from mid- to late gestation is, not unexpectedly, accompanied by increased absolute rates of uterine and umbilical uptake of oxygen and nutrients and of urea export by conceptus tissues, and of fetal whole-body protein synthesis in sheep and cattle (Bell et al., 1986, 1989; Reynolds et al., 1986; Kennaugh et al., 1987; Ferrell, 1991). However, when expressed on a weight-specific basis these rates are considerably greater in mid than in late gestation, concomitant with greater rates of relative growth in the immature fetus. More recent studies of fetal and uteroplacental metabolic ontogeny in the horse have shown a qualitatively similar pattern (Fowden et al., 2000a,b). The apparent absence of a decrease in weight-specific fetal oxygen consumption between mid- and late gestation in this species (Fowden et al., 2000a) may be related to its slower relative rates of fetal growth and the failure to account for the greater tissue hydration of the mid-gestation fetus. In sheep, the gestational decline in weight-specific fetal whole-body metabolic rates is associated with changes in the allometric growth of metabolically active vital organs, such as the liver, versus that of less active skeletal tissues (Bell et al., 1987a), as well as a decline in the weight-specific rate of fetal hepatic oxygen consumption (Vatnick and Bell, 1992).

3. PLACENTAL TRANSPORT OF MACRONUTRIENTS 3.1. Molecular and physiological mechanisms 3.1.1. Glucose Glucose is transported from the maternal to the fetal circulation by carrier-mediated, facilitated diffusion (Widdas, 1952; Simmons et al., 1979). This process is strongly dependent on the maternal–fetal plasma glucose concentration gradient (Simmons et al., 1979; DiGiacomo and Hay, 1990a). The predominant glucose transporter protein isoforms in the sheep placenta are GLUT-1 and GLUT-3 (Ehrhardt and Bell, 1997; Das et al., 1998), the mRNA and protein abundance of which increase with gestational age, especially for GLUT-3 (Currie et al., 1997; Ehrhardt and Bell, 1997). This, together with its low Km and localization at the apical, maternalfacing layer of the trophoblastic cell layer (Das et al., 2000), suggests that ontogenic changes in GLUT-3 expression and activity may account for much of the 5-fold increase in glucose transport capacity of the sheep placenta in vivo between mid- and late gestation (Molina et al., 1991). Other factors must include remodeling and expansion of the placenta’s effective exchange surface (Stegeman, 1974) and the increasing maternal–fetal plasma concentration gradient (Molina et al., 1991). Similar developmental patterns in placental GLUT expression have been observed in the rat (Zhou and Bondy, 1993) but not in the human (see Illsley, 2000) or horse placenta (Wooding et al., 2000), in which gestational changes were small or absent. These species differences may be due to the considerably slower rates of fetal growth and glucose demand in humans and horses, and, possibly, their greater dependence on changes in placental morphology to permit increased fetal access to glucose during late pregnancy.

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3.1.2. Amino acids Most amino acids taken up by the placenta are transported against a fetal–maternal concentration gradient, implying the use of energy-dependent, active transport processes (Young and McFadyen, 1973). Studies of isolated human and rodent placental vesicles have confirmed that the transport systems in the placenta are similar to those described for plasma membranes of other tissues (see Battaglia and Regnault, 2001). These include at least six sodium-dependent and five sodium-independent systems that have been classified systematically on the basis of their affinity for neutral, acidic, or basic amino acids, and their intracellular location (Battaglia and Regnault, 2001). Recent results from in vivo studies on sheep suggest that rapid placental transport of neutral amino acids requires both sodium-dependent transport at the maternal epithelial surface and affinity for highly reversible, sodium-independent transporters located at the fetal surface (Jozwik et al., 1998; Paolini et al., 2001). These researchers also demonstrated major differences in placental clearance among the essential amino acids, with the more rapidly transported branched-chain acids, plus methionine and phenylalanine, apparently sharing the same rate-limiting transport system (Paolini et al., 2001). 3.1.3. Fatty acids Placental capacity for maternal–fetal transport of short- and long-chain fatty acids and their keto-acid derivatives varies widely among species, associated with variations in placental structure (see Bell and Ehrhardt, 2002). Thus, the epitheliochorial placentae of ruminants and the diffuse placentae of pigs and horses allow only meager fetal access to maternal fatty acids and ketones, whereas the hemochorial placentae of rodents, lagomorphs, and, by inference, humans, are more permeable to these substrates. Molecular mechanisms for placental transport of fatty acids have yet to be defined but may involve a placenta-specific fatty-acid binding protein that has been identified in sheep (Campbell et al., 1994) and humans (Campbell et al., 1995). 3.2. Influence of placental metabolism on maternal–fetal nutrient transfer 3.2.1. Glucose metabolism Glucose entry into the gravid uterus and its component tissues is determined by maternal arterial glucose concentration (Hay and Meznarich, 1988; Leury et al., 1990b), while glucose transport to the fetus is determined by the transplacental (maternal–fetal) concentration gradient (Hay et al., 1984). In turn, the transplacental gradient is directly related to both placental and fetal glucose consumption, which are dependent on fetal arterial glucose concentration (Hay et al., 1990). Thus, as fetal glucose concentration changes relative to that of the mother, thereby changing the transplacental gradient, placental transfer of glucose to the fetus varies reciprocally with placental glucose consumption. In addition to its quantitative impact on placental transfer of glucose, placental glucose metabolism has a major qualitative influence on the pattern of carbohydrate metabolites delivered to the fetus. Rapid metabolism to lactate (~35%), fructose (~4%), and CO2 (~17%) accounted for about 56% of uteroplacental glucose consumption in late-pregnant ewes, and was directly related to placental glucose supply (Aldoretta and Hay, 1999). The fate of the remaining 44% of glucose metabolized by the placenta must include synthesis of alanine and other nonessential amino acids (Timmerman et al., 1998), directly or via lactate (Carter et al., 1995).

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Umbilical uptake and fetal oxidation of placentally derived lactate (Sparks et al., 1982; Hay et al., 1983) and fructose (Meznarich et al., 1987) are estimated to contribute approximately 20% and 5%, respectively, to fetal CO2 production, in addition to the 30% contributed by the rapid oxidation of glucose (Hay et al., 1983). 3.2.2. Amino acid metabolism Placental metabolism also affects the quantity and composition of amino acids delivered to the fetus. The significant net consumption by uteroplacental tissues of glutamate, serine, and the branched-chain amino acids (Liechty et al., 1991b; Chung et al., 1998) implies catabolism or transamination of these acids. An additional, small fraction of this net loss of amino acids will be in the form of secreted peptides. The ovine placenta has very little enzymatic capacity for urea synthesis, but produces considerable amounts of ammonia, much of which is released into maternal and, to a lesser extent, fetal circulations (Holzman et al., 1977; Bell et al., 1989). This is consistent with extensive placental deamination of branched-chain amino acids to their respective keto-acids, which are released into fetal and maternal bloodstreams (Smeaton et al., 1989; Loy et al., 1990), and with rapid rates of glutamate oxidation in the placenta (Moores et al., 1994). Transamination of branched-chain amino acids accounts for some of the net glutamate acquisition by the placenta, the remainder of which is taken up from the umbilical circulation (Moores et al., 1994). That which is not quickly oxidized combines with ammonia to synthesize glutamine, which is then released back into the umbilical bloodstream (Chung et al., 1998). Some of this glutamine is converted back to glutamate by the fetal liver, which produces most of the glutamate consumed by the placenta (Vaughn et al., 1995). This establishes a glutamate–glutamine shuttle which promotes placental oxidation of glutamate and fetal hepatic utilization of the amide group of glutamine. Similarly, the placenta almost quantitatively converts serine, mostly taken up from maternal blood, to glycine (Chung et al., 1998), reconciling the discrepancy between the negligible net uptake of glycine by the uterus and substantial net release of this amino acid into the umbilical circulation (see Hay, 1998). The complexity of interrelations among placental uptake, metabolism, and transport of amino acids was further illustrated by a study of alanine metabolism in ewes during late pregnancy (Timmerman et al., 1998). Application of tracer methodology showed that negligible net placental consumption or production of alanine masks an appreciable metabolism of maternal alanine entering the placenta which exchanges with endogenously produced alanine. Thus, most of the alanine delivered to the fetus is of placental origin, derived from placental protein turnover and transamination. 3.2.3. Fatty acid metabolism The relative abundance of polyunsaturated C20 and C22 derivatives of the essential C18 fatty acids in fetal tissues has been attributed largely to the placenta’s capacity for hydrolyzing esterified lipids (Clegg, 1981) and for desaturation and chain elongation of the resulting free polyunsaturated C18 acids (Noble et al., 1985). Thus, placental metabolism ensures an adequate fetal supply of the longer-chain ω6 and ω3 metabolites of the C18 essential fatty acids, which are the forms ultimately required by tissues, despite the poor placental transport of the parent molecules

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3.3. Factors affecting placental transport capacity 3.3.1. Placental size Placental capacity for glucose transport was substantially reduced, as were uteroplacental glucose consumption rate and fetal glycemia, in carunclectomized (Owens et al., 1987a) and heat-treated ewes (Bell et al., 1987b; Thureen et al., 1992). At least part of the absolute reduction in glucose transport capacity is presumed to be due to reduction in exchange surface area of the trophoblastic membrane, as shown in carunclectomized ewes (Robinson et al., 1995). In previously heat-treated ewes (Thureen et al., 1992), placental weight-specific glucose transport capacity was also reduced. This implies that chronic heat stress, which reduces average weight but not total number of placentomes, additionally reduces the number and/or activity of specific glucose transport proteins at maternal and/or fetal exchange surfaces. In contrast, carunclectomy, which reduces placentome number but may stimulate a compensatory increase in average weight of individual placentomes, caused a modest increase in the placental weight-specific clearance of the nonmetabolizable glucose analog, 3-O-methyl glucose (Owens et al., 1987b). This implies that glucose transporter expression was preserved or increased in the remaining placentomes. Placental insufficiency in heat-treated ewes also extends to impaired capacity for amino acid transport, including major reductions in placental uptake and fetal transfer of leucine (Ross et al., 1996) and threonine (Anderson et al., 1997). The normally extensive placental catabolism of leucine was also greatly reduced (Ross et al., 1996). 3.3.2. Maternal nutrition Recent evidence indicates that the activity of placental transport mechanisms can be modulated by maternal nutrition, independent of more chronic effects on placental size. For example, moderate undernutrition of ditocous ewes during late pregnancy caused a 50% increase in capacity for maternal–fetal glucose transport in vivo (Ehrhardt et al., 1996) which was at least partly explained by a 20% increase in total GLUT abundance, associated with a similar increase in GLUT-3 protein abundance (Ehrhardt et al., 1998). These responses help explain how placental glucose transfer remained sufficient to sustain normal fetal growth, despite chronic maternal hypoglycemia and a 26% decrease in the maternal–fetal gradient in arterial plasma glucose concentration (Bell et al., 1999). During more severe, chronic undernutrition or starvation for several days, the development of profound fetal hypoglycemia helps to sustain the maternal–fetal gradient in glucose concentration by restricting the reverse transfer of glucose to the placenta, and reducing placental glucose consumption (see Hay, 1995). Under these more stringent conditions, fetal gluconeogenesis is induced (Leury et al., 1990a), with amino acids being the presumed major substrate, consistent with increased fetal urea synthesis (Lemons and Schreiner, 1983; Faichney and White, 1987). The ultimate consequence is reduced fetal tissue protein synthesis (Krishnamurti and Schaefer, 1984) and slowing of fetal growth to a rate that can be sustained by the reduced placental nutrient supply. Effects of energy and/or protein supply on placental capacity for amino acid transport have been little studied. Fasting late-pregnant ewes for 5 days had an insignificant effect on umbilical net uptake of amino acids despite appreciable decreases in maternal arterial plasma concentrations of many amino acids (Lemons and Schreiner, 1983). This suggests that during short-term energy/ protein deprivation, placental mechanisms for active transport of amino acids are unimpaired and may even be upregulated. Under similar fasting conditions, the uteroplacental deamination of

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branched-chain amino acids appeared to be increased, judging from a 3-fold increase in the efflux of α-ketoisocaproate, the keto-acid derivative of leucine, into uterine and umbilical circulations (Liechty et al., 1991a). This suggests that increased amino acid catabolism may partly compensate for the likely reduction in placental glucose oxidation under these conditions. Placental transport and metabolism of amino acids have not been studied during more prolonged restriction of energy or protein. However, in ewes fed adequate energy but insufficient protein during the last month of pregnancy, fetal growth and protein deposition over this period were decreased by 18% (McNeill et al., 1997). It is also notable that in chronically hyperglycemic ewes with secondary hyperinsulinemia and hypoaminoacidemia, placental and fetal uptakes of several amino acids were reduced, and fetal total nitrogen uptake declined by 60% (Thureen et al., 2001). 3.4. Consequences of placental insufficiency Placental weight and associated capacity for maternal–fetal nutrient transfer are powerful determinants of fetal growth during late gestation in all species studied. This has been most persuasively demonstrated by controlled manipulation of placental size and/or functional capacity using premating carunclectomy (Alexander, 1964), heat-induced placental stunting (Alexander and Williams, 1971), or uteroplacental vascular embolization (Creasy et al., 1972). Natural variations in fetal weight due to varying litter size in prolific ewes are strongly correlated with placental mass per fetus (Rhind et al., 1980; Greenwood et al., 2000). Recently, the quite profound growth retardation of fetuses in overfed, primiparous ewes also has been attributed to a primary reduction in placental growth (Wallace et al., 2000). The probably common etiology of IUGR in experimentally induced and natural cases of placental insufficiency is illustrated by the similar patterns of association between fetal and placental weights in pregnant ewes with varying conceptus weights due to carunclectomy, heat stress, litter size, and overfeeding of primiparous dams (fig. 2). In each case, severe growth retardation was associated with chronic fetal hypoxemia and hypoglycemia during late gestation (Creasy et al., 1972; Harding et al., 1985; Bell et al., 1987b; Wallace et al., 2002).

4. REGULATION OF CONCEPTUS METABOLISM 4.1. General features The extracellular and local regulation of fetal metabolism and its relation to tissue growth has several distinctive characteristics. First, placental nutrient supply has a powerful, limiting influence on nutrient disposal, especially in late gestation when fetal demands are greatest. Second, the fetal endocrine system is largely independent from the direct influence of maternal hormones because the placenta is impermeable to most of the important metabolic regulatory peptide and steroid hormones. Thus, reported effects of maternal hormones on fetal growth must be mediated indirectly by changes in maternal metabolism and/or uteroplacental tissue growth and resulting changes in fetal nutrient supply. Examples include the effects of maternal treatment with growth hormone (GH) during early pregnancy on fetal growth in pigs (Sterle et al., 1995; Rehfeldt et al., 2001) and of maternal immunization against placental lactogen (PL) on fetal growth in sheep (Leibovich et al., 2000). Third, while most fetal endocrine organs develop the capacity to synthesize and secrete hormones early in gestation, target tissue receptor and neuroendocrine feedback systems are variably immature until late pregnancy. As a result, there is a much greater reliance on paracrine and autocrine regulation by locally expressed factors, especially in early and mid-pregnancy.

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Fig. 2. Relation between fetal and placental weights in ewes representing different models of placental insufficiency during late pregnancy. Variation in placental weight was achieved by premating carunclectomy (●; Owens et al., 1986), chronic heat treatment (䊊; Bell et al., 1987b), natural variation in litter size (▲; Greenwood et al., 2000), and overfeeding of adolescent ewes (䉭; Wallace et al., 2000). Reproduced with permission from the Society for Reproduction and Fertility (Greenwood and Bell, 2003).

4.2. Nutrient supply 4.2.1. Glucose The Km for saturable glucose transport by the sheep placenta is ~3.9 mM (Simmons et al., 1979), which is within the physiological range of glycemia in well-fed, adult sheep. Thus, uterine uptake, placental metabolism and transfer, and fetal metabolism of glucose are very sensitive to maternal arterial glucose concentration in sheep (fig. 3; Hay and Meznarich, 1988). In sheep, cows, and horses fetal utilization of glucose is highly correlated with fetal plasma glucose concentration, which, in turn is correlated with maternal glycemia (see Fowden, 1997). In contrast, fetal glucose metabolism was not related to fetal glycemia in pigs, possibly because in this species, fetal glycemia is influenced by individual relative to total fetal mass, as well as maternal nutrition (Fowden et al., 1997). It is well established that in sheep, the maternal and fetal hypoglycemia caused by starvation or chronic undernutrition is associated with increased fetal urea synthesis (Hodgson et al., 1982; Lemons and Schreiner, 1983; Faichney and White, 1987) due to increased amino acid deamination (Krishnamurti and Schaefer, 1984; Van Veen et al., 1987). Conversely, fetal hyperglycemia appears to cause substitution of glucose for amino acids as an oxidative fuel because under these conditions, increased glucose oxidation (Hay and Meznarich, 1986) is associated with decreased leucine oxidation (Liechty et al., 1991a). Interestingly, the latter response occurred independently of glucose-induced changes in fetal insulin concentration (Liechty et al., 1993). Fetal glucose supply also influences fetal endogenous glucose production, presumably due to hepatic gluconeogenesis. In addition to the association of increased endogenous production

Regulation of metabolism and growth during prenatal life

Fig. 3. Relations between maternal arterial blood glucose concentration and (A) uterine, (B) fetal, and (C) uteroplacental net uptakes of glucose in ewes during late pregnancy. Reproduced with permission from the Society for Experimental Biology and Medicine (Hay and Meznarich, 1988).

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with fetal hypoglycemia in starved or undernourished ewes (Hay et al., 1984; Leury et al., 1990a), progressive fetal hypoglycemia induced by different levels of maternal insulin infusion caused fetal endogenous glucose production to increase linearly (DiGiacomo and Hay, 1990b). A mediating role for fetal insulin was suggested by the incomplete suppression of endogenous glucogenesis by fetal infusion with insulin while maintaining basal fetal glycemia (DiGiacomo and Hay, 1990b).

4.2.2. Amino acids Effects of amino acid supply on fetal metabolism have not been studied systematically. Decreased maternal plasma concentrations of essential amino acids in fasted ewes were not associated with a significant decrease in umbilical uptake of these acids (Lemons and Schreiner, 1983). In contrast, maternal hyperglycemia with secondary hyperinsulinemia and hypoaminoacidemia caused substantial reductions in uterine, uteroplacental, and fetal uptakes of several amino acids, particularly the branched-chain acids, and a 60% reduction in total fetal uptake of nitrogen (Thureen et al., 2000, 2001). Correction of maternal amino acid concentrations by appropriate exogenous infusion restored uterine and umbilical exchanges to normal levels (Thureen et al., 2000). Maternal hyperaminoacidemia, caused by infusion of amino acids, had little effect on the umbilical uptake of most amino acids, except for increased uptake of the branched-chain acids, and did not affect fetal total nitrogen supply (Jozwik et al., 1999). However, uteroplacental production and fetal concentrations of ammonia increased moderately, implying some increase in placental deamination of amino acids.

4.3. Fetal hormones and growth factors 4.3.1. Pancreatic hormones Insulin protein and preproinsulin mRNA are detectable from early gestation in the fetal pancreas of all species studied (D’Agostino et al., 1985). In the sheep fetus, gestational increases in pancreatic and basal plasma concentrations of insulin (Alexander et al., 1968) are accompanied by a steady increase in glucose- and arginine-stimulated insulin secretion during the latter half of gestation (Aldoretta et al., 1998). Euglycemic, hyperinsulinemic clamp studies have demonstrated that fetal insulin and glucose have independent, positive effects on fetal whole-body glucose utilization (Hay et al., 1988). These observations are consistent with tissue-specific responses that vary between insulin-responsive tissues, such as skeletal muscle (Wilkening et al., 1987; Anderson et al., 2001b), and tissues unresponsive to insulin, such as the brain (Anderson et al., 2001a). Neither fetal (Jodarski et al., 1985) nor maternal (Rankin et al., 1986) plasma insulin concentration has a direct effect on placental transport of glucose, consistent with our failure to detect significant concentrations of the insulin-responsive glucose transport protein, GLUT-4, in the ovine placenta (Ehrhardt and Bell, 1997). However, fetal hyperinsulinemia indirectly promotes placental transfer and umbilical uptake of glucose through its influence on fetal glycemia and the maternal–fetal glucose concentration gradient (see Hay, 1995). In addition to its promotion of glucose uptake and metabolism in fetal tissues, a physiological increase in fetal plasma insulin stimulated umbilical uptake and whole-body utilization of amino acids when fetal glycemia and aminoacidemia were carefully controlled (Thureen et al., 2000). The specific metabolic fates of amino acids were not measured, but it is likely that protein anabolism was increased by both reduction of proteolysis (Milley, 1994)

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and stimulation of protein synthesis (Horn et al., 1983). This anabolic effect may have been reinforced indirectly by the effect of increased glucose utilization in reducing amino acid deamination (Liechty et al., 1993). Independently of its metabolic effects, insulin may influence fetal tissue growth through modulation of the expression and activity of other growth regulators such as the insulin-like growth factor (IGF) system. For example, when fetal plasma glucose and amino acid concentrations were clamped, fetal insulin infusion caused an increase in plasma concentration of IGF binding protein (BP)-3 and a decrease in hepatic expression of IGFBP-1 (Shen et al., 2001). The latter response is consistent with the opposite effects of hypoinsulinemia in the undernourished sheep fetus (Osborn et al., 1992). Ovine fetal hyperinsulinemia also increased the farnesylation of p21 Ras in ovine fetal liver, skeletal muscle, adipose tissue, and white blood cells (Stephens et al., 2001). This is significant because the Ras pathway is an important intracellular signaling element in the mitogenic actions of insulin and other growth factors, including the IGFs, and greater availability of farnesylated Ras augments mitogenic cellular responsiveness to IGF-1 and other growth factors in isolated systems (Goalstone et al., 1998). The fetal pancreas synthesizes glucagon from early in gestation, but the regulation and metabolic role of this peptide in fetal life remain unclear. Secretory responses to hypoglycemia and other metabolic stimuli in fetal sheep are small and sluggish during late gestation (Alexander et al., 1976), but birth is accompanied by a major surge in secretion of glucagon (Grajwer et al., 1977). Exogenous administration of glucagon to mimic fetal plasma concentrations observed during maternal fasting (Schreiner et al., 1980) caused hyperglycemia in the fetal sheep (Philipps et al., 1983), implying a possible role in regulation of hepatic glycogenolysis and/or gluconeogenesis. 4.3.2. Growth hormone and the IGF system During postnatal life, growth hormone (GH) is a powerful homeorhetic regulator of metabolism and growth through its direct actions on some tissues, such as adipose tissue, and its indirect actions on most lean tissues, mediated by the IGF system (see Etherton and Bauman, 1998). Notable among its pleiotropic effects are inhibition of lipogenesis and enhancement of responses to lipolytic stimuli in adipose tissue, and potent effects on cell cycle activity and protein turnover in muscle and other tissues via regulation of the expression of IGF-1 and its binding proteins in multiple tissues, including the liver. In general, these effects are greatly muted during fetal life, which is characterized by persistently high plasma levels of GH (Bassett et al., 1970; Gluckman et al., 1979) and low plasma levels of IGF-1 (Van Vliet et al., 1983). The apparent uncoupling of the GH/IGF-1 axis is consistent with low hepatic expression of the GH receptor, IGF-1, IGFBP-3, and the acid-labile subunit (ALS) (Klempt et al., 1993; Rhoads et al., 2000a). Thus, although pituitary secretion of GH is active through much of gestation (de Zegher et al., 1989), maturation of the endocrine IGF-1 system is retarded by hepatic unresponsiveness to GH, which, in postnatal life, strongly regulates expression of all three components of the ternary binding complex (IGF-1, IGFBP-3, ALS) that accounts for most circulating IGF-1 (Boisclair et al., 2001). Therefore, it is not surprising that infusion of normal sheep fetuses with GH for 10 days had no effect on fetal plasma IGF-1 levels (Bauer et al., 2000). It is possible that some direct metabolic effects of GH develop before engagement of the GH/IGF-1 system. For example, Bauer et al. (2000) reported a decrease in glucose uptake and, presumably, utilization, with no change in plasma insulin in GH-infused fetal sheep, consistent with an earlier report of apparent insulin resistance in GH-treated fetuses (Parkes and Bassett, 1985). Also, hypophysectomy of fetal lambs causes increased fat deposition that

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can be reversed by GH administration (Stevens and Alexander, 1986), implying the existence of functional GH receptors in adipose tissue during late gestation. This could account for the substantial decline in capacity for adipose tissue lipogenesis in fetal sheep during the last month of gestation (Vernon et al., 1981). Immaturity of the fetal GH/IGF-1 system raises the possibility that fetal protein anabolism and tissue growth may be limited by low levels of circulating IGF-1, despite the generally accepted notion that, during fetal life, the metabolic and trophic influences of locally expressed IGF are more important than those of systemic IGF (see Jones and Clemmons, 1995). It is therefore notable that infusion of IGF-1 into fetal sheep decreased proteolysis and amino acid catabolism (Harding et al., 1994; Liechty et al., 1996). Conversely, increased amino acid catabolism in the undernourished sheep fetus (Hodgson et al., 1982; Lemons and Schreiner, 1983) is associated with decreased plasma IGF-1 levels, whether due to maternal nutrient deprivation (Bassett et al., 1990) or placental insufficiency (Owens et al., 1994). In all species studied, fetal tissue expression and plasma levels of IGF-2 exceed those of IGF-1 (Han et al., 1988; Mesiano et al., 1989; Lee et al., 1991; Delhanty and Han, 1993). A special role for IGF-2 in the regulation of prenatal growth was demonstrated by initial gene knockout studies in the mouse (DeChiara et al., 1991). Recently, tissue-specific gene inactivation has been used to show that the IGF-2 gene is paternally imprinted in the placenta and acts to promote placental growth and functional capacity, thereby influencing fetal nutrient supply and growth in late gestation (fig. 4; Constancia et al., 2002). Lack of IGF-2 also reduced fetal hepatic glycogen storage and glycemia, associated with decreased activity but not mRNA abundance of glycogen synthase, and impaired the ability of newborn IGF-2 knockout mice to survive fasting for 12h (Lopez et al., 1999). 4.3.3. Placental lactogen Placental lactogen (PL; also known as chorionic somatomammotropin) is a major, unique protein product of the placentae of ruminants, humans, rodents, and some other species. The molecular identity and interspecies homology of these molecules, as well as their lactogenic and somatogenic effects through their ability to bind to both GH and prolactin receptors, has been reviewed recently (Gertler and Djiane, 2002). Ovine and bovine fetal plasma contains PL throughout gestation (Anthony et al., 1995) and the effective half-life of circulating PL in fetal sheep is similar to that of GH (Schoknecht et al., 1992). The physiological roles of this putative regulator of fetal metabolism and growth remain to be established definitively. Glycogen synthesis in isolated fetal hepatocytes was promoted by PL treatment in sheep (Freemark and Handwerger, 1986) and rats (Freemark and Handwerger, 1984), and we observed a 56% increase in hepatic glycogen accumulation in fetal sheep infused i.v. with native ovine PL for 14 days (table 2; Schoknecht et al., 1996). In the latter study, PL treatment caused modest increases in fetal plasma IGF-1 concentration and the relative weights of some visceral organs but did not significantly affect fetal weight. 4.3.4. Glucocorticoids In all species studied, there is a major increase in the circulating glucocorticoid concentration in the fetus toward term, mostly due to a pronounced surge in fetal adrenal cortisol secretion. The vital, pleiotropic influences of fetal cortisol on the structural and biochemical maturation of multiple fetal tissues to prepare them for postnatal functions have been reviewed by Fowden et al. (1998). Less is known about the effects of glucocorticoids on the regulation

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Fig. 4. Placental and fetal growth in mutant mice lacking paternal expression of the IGF-2 gene in labyrinthine trophoblastic tissue of the placenta, and in their wild-type littermates. Significant differences between wild-type and mutant mice are indicated: * P< 0.05; *** P<0.001. Adapted from the data of Constancia et al. (2002).

of fetal metabolism in relation to growth and development. In general, fetal cortisol appears to promote the availability of glucose to the neonatal animal by stimulating both hepatic glycogen synthesis (Barnes et al., 1978) and maturation of the capacity for hepatic glucose production (Townsend et al., 1991; Barbera et al., 1997) in the near-term sheep fetus. During late gestation, treatment with glucocorticoids reduced umbilical glucose uptake (Milley, 1996; Table 2 Effect of i.v. infusion of ovine placental lactogen for 14 days on liver glycogen concentration, content in fetal sheep at day 136 of gestation (from Schoknecht et al., 1996)a Parameter Liver weight, g Glycogen concentration, mg/g Glycogen content, g a Values

Controlb

Placental lactogenc

115.8 ± 9.2 79.3 ± 6.9 8.4 ± 0.7

124.8 ± 9.9 105.0 ± 5.6* 13.1 ± 1.7*

are means ± SEM, n = 5. with saline containing ovine plasma (15 ml/l), days 122 to 136 of gestation. c Infused with ovine placental lactogen (1.2 mg/d), days 122 to 136 of gestation; caused a 4-fold increase in fetal plasma concentration of placental lactogen. * Treatment effect was significant at P<0.05. b Infused

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Barbera et al., 1997) and placental uptake of fetal glutamate (Barbera et al., 1997; Timmerman et al., 2000). The latter response was associated with decreased hepatic output of glutamate apparently due to decreased fetal hepatic uptake of glucogenic amino acids, including glutamine, and diversion of hepatic glutamine to metabolism in the TCA cycle rather than glutamate synthesis (Timmerman et al., 2000). Other growth-related effects of the prepartum increase in fetal cortisol include induction of the hepatic GH receptor and hepatic synthesis of IGF-1 (Li et al., 1996) but suppression of IGF-1 expression in muscle independently of any change in GH receptor gene expression (Li et al., 2002). Cortisol also suppresses IGF-2 expression in liver, muscle, and adrenal glands (Li et al., 1993), stimulates the deiodination of thyroxine (T4) to triiodothyronine (T3) (Fowden et al., 1998), and appears to downregulate the production of PL by binucleate cells in the ovine placenta (Ward et al., 2002). 4.3.5. Thyroid hormones The fetal thyroid secretes T4 from early in gestation, and thyroidectomy of fetal sheep in midgestation causes generalized growth retardation and delayed maturation of the skin, skeleton, and pulmonary and neuromuscular systems (Hopkins and Thorburn, 1972). Fetal sheep made hypothyroid by thyroidectomy or hypophysectomy suffered a 20–30% decrease in umbilical oxygen uptake that was restored to normal by exogenous T4 administration (Fowden and Silver, 1995). This reduction in oxygen consumption was accompanied by abnormal blood-gas status and reductions in rate of glucose oxidation and the fraction of oxygen consumption used for glucose oxidation, all of which also were normalized by T4 replacement (Fowden and Silver, 1995). Interestingly, plasma T3 levels remained low and were unchanged by thyroid or pituitary ablation or exogenous T4, suggesting that at least before maturation of the enzymatic capacity for T4 deiodination near term (Polk et al., 1988), thyroid hormone effects may be mediated directly by T4. However, it should be noted that administration of T3 alone, albeit in supraphysiological doses, caused an increase in oxygen consumption of fetal sheep (Lorijn et al., 1980). In addition to its negative effects on glucose utilization, thyroid deficiency impaired the ability of fetal sheep to increase hepatic glucogenesis in response to fasting (Fowden et al., 2001). Recent evidence also suggests that the cortisol-induced increase in deiodination of T4 to T3, and the consequent prenatal surge in fetal plasma T3, at least partly mediates the maturational effects of cortisol on the hepatic somatotropic axis (Forhead et al., 2000). 4.3.6. Catecholamines Prenatal maturation of the sympathoadrenomedullary system is vital to enable the perinatal animal to respond to the stresses of parturition and adaptation to the extrauterine environment. In precocial species such as the sheep, central nervous (splanchnic) control of the adrenal medulla develops relatively early; in other species, functional innervation is not apparent until after birth (see Slotkin and Seidler, 1988). During late gestation, the fetal sheep responds to acute hypoxia (Cohen et al., 1982) and hypoglycemia (Harwell et al., 1990) with pronounced increases in adrenomedullary secretion of epinephrine and norepinephrine. Metabolic consequences include rapid stimulation of hepatic glucose production, presumably through increased glycogenolysis (Jones et al., 1983), and mobilization of NEFA (Harwell et al., 1990), associated with reduced pancreatic secretion and plasma concentrations of insulin (Bassett and Hanson, 1998), and attenuated action of IGF-1 (Hooper et al., 1994). Restoration of normal insulinemia

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by insulin infusion abolished most of the metabolic and growth-inhibitory effects of prolonged catecholamine infusion in the sheep fetus (Bassett and Hanson, 2000). This suggests that establishment and maintenance of hypoinsulinemia is a necessary mediating factor for the adverse effects of elevated circulating catecholamines on fetal growth. 4.3.7. Leptin The peptide hormone leptin is synthesized and secreted primarily by adipose tissue in postnatal animals and is considered to play an important role in the regulation of energy balance (Ahima and Flier, 2000). Leptin has been detected in ovine fetal plasma as early as day 40 of gestation (Ehrhardt et al., 2002) and its concentration increases moderately throughout gestation, especially during the last 2 weeks (Ehrhardt et al., 2002; Forhead et al., 2002). The gestational increase in plasma concentration is accompanied by increased abundance of leptin mRNA in perirenal adipose tissue in late pregnancy (Yuen et al., 1999). Our results indicate that before 100 days of gestation, tissues other than adipose tissue, such as brain and liver, are the primary source of circulating leptin, and that this role is assumed by brown adipose tissue only after this tissue develops appreciably during the last one-third of gestation (fig. 5; Ehrhardt et al., 2002). Regulation of tissue expression and biological actions of leptin during fetal life have yet to be studied systematically. The increase in plasma leptin during late gestation in fetal sheep was associated with the prepartum surge in fetal cortisol and abolished by fetal adrenalectomy (Forhead et al., 2002). It also appears that expression of leptin mRNA in perirenal brown adipose tissue in the sheep fetus responds positively to hyperinsulinemia but not hyperglycemia (Devaskar et al., 2002). The functional significance of fetal leptin is unclear. Leptin signaling apparently is not essential during prenatal life because leptin-deficient ob/ob mice are born relatively normal (Mounzih et al., 1998). Also, fetal plasma leptin was unaffected by changes in maternal nutrition sufficient to change fetal glycemia and insulinemia in late-pregnant ewes (Ehrhardt et al., 2002; Mühlhäusler et al., 2002; Yuen et al., 2002). Fetal plasma leptin was correlated with body fatness as represented by the relative mass of unilocular cells in perirenal and interscapular brown adipose tissue (Mühlhäusler et al., 2002). Infusion of leptin for several days into the sheep fetus caused decreases in relative abundance of leptin mRNA and the proportion of unilocular cells in perirenal adipose tissue, suggesting a feedback effect on adipose tissue function (Yuen et al., 2003). However, the relevance of this observation is unclear because of the unphysiologically high levels of plasma leptin in treated fetuses. A potential role for fetal and/or maternal leptin in the regulation of placental function is suggested by the abundant expression of the physiologically relevant long (Ob-Rb) form of the leptin receptor by the ovine placenta (Ehrhardt et al., 1999; Thomas et al., 2001). 4.4. Coordination of fetal metabolism and growth The mechanisms relating nutrient supply to expression of endocrine and local regulatory factors and, thence, tissue metabolism and growth, can be illustrated by synthesis of the present knowledge on IUGR, whether caused by placental insufficiency, maternal undernutrition, or insulin-induced maternal hypoglycemia. Effects on the local expression of trophic factors and the cellular growth of skeletal muscle will serve as an example of tissue responses to an altered extracellular milieu. The putative relationships discussed below are schematically represented in fig. 6. Placental insufficiency during late gestation is generally characterized by fetal hypoxemia and hypoglycemia, whether caused by surgical reduction (carunclectomy; Harding et al., 1985),

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Fig. 5. Relative abundance of leptin mRNA in ovine fetal tissues at different stages of gestation. Adapted from the data of Ehrhardt et al. (2002).

placental embolization (Creasy et al., 1972), maternal heat stress (Bell et al., 1987b), or overfeeding of adolescent ewes (Wallace et al., 2002). Associated endocrine changes include decreased fetal plasma concentrations of insulin (Robinson et al., 1980) and IGF-1 and -2 (Owens et al., 1994), and increased concentrations of cortisol (Phillips et al., 1996). All of these changes can be elicited by maternal undernutrition or insulin-induced hypoglycemia, implicating fetal glycemia as an important primary signal (Mellor et al., 1977; Osgerby et al., 2002). However, it must be recognized that hypoxemia may reinforce these responses through its stimulation of fetal adrenal secretion of cortisol and catecholamines, and the inhibitory influence of the latter on fetal insulin secretion. It seems likely that hypoinsulinemia is a primary, coordinating mediator of the numerous metabolic and trophic consequences of reduced fetal nutrient supply. Disruption of fetal pancreatic insulin secretion has a potent, negative effect on fetal growth (Fowden et al., 1995),

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Fig. 6. Schematic outline of some important factors linking maternal undernutrition and placental insufficiency to intrauterine growth retardation.

associated with decreased fetal tissue uptake and metabolism of glucose (Fowden and Hay, 1988), decreased uptake of amino acids and increased proteolysis (Carver et al., 1997), and reduced circulating levels of IGF-1 (Gluckman et al., 1987). However, although circulating IGF-1 may be of increasing importance during late gestation, it is likely that local tissue expression and actions of this and other growth factors are more significant mediators of tissue growth responses to altered nutrient supply. For example, fetal muscle strongly expresses IGF-1 throughout gestation (Dickson et al., 1991; Lee et al., 1993) and disruption of the IGF-1 gene causes lethal abnormalities in muscle development (Liu et al., 1993), consistent with the extensive evidence for the role of IGF-1 in regulation of myogenesis (Florini et al., 1996). It therefore seems likely that the reduced mitotic activity of myosatellite cells and growth of skeletal muscle in acutely undernourished or placentally growth-retarded sheep fetuses (Greenwood et al., 1999) was mediated, at least partly, by reduced local expression of IGF-1, possibly caused by elevated plasma levels of cortisol (Li et al., 2002). Finally, although this section has focused on IUGR to illustrate aspects of the coordination of nutrient supply with growth in the fetus, it should be recognized that even in optimally fed, healthy animals, fetal growth is constrained by placental capacity for nutrient transfer during late pregnancy. This phenomenon ensures that the unborn animal’s demands upon its dam’s nutrient reserves are not excessive, and reduces the possibility of birth injury to itself

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and its mother. The capacity for increased growth in response to increased nutrient supply was demonstrated by the almost 20% increase in birth weight of singleton lambs that had been infused directly with glucose for the last 30 days of gestation, in ewes that were extremely well fed (Stevens et al., 1990).

5. INFLUENCE OF PRENATAL METABOLISM AND GROWTH ON POSTNATAL PERFORMANCE AND HEALTH 5.1. Postpartum metabolism and growth We recently have reported some of the metabolic characteristics of naturally growth-retarded lambs from prolific ewes immediately after birth and during neonatal growth to a nominal live weight (LW) of 20 kg (Greenwood et al., 2002; Greenwood and Bell, 2003). At birth, these lambs tended to be hypoglycemic and had elevated plasma urea nitrogen levels. More striking was the apparent immaturity of their hepatic GH/IGF system as represented by greatly elevated plasma concentrations of GH and low concentrations of IGF-1. This blood picture was associated with reduced hepatic expression of the GH receptor and the GH-dependent ALS necessary to form the ternary binding complex which contains most circulating IGF-1 in postnatal life (Rhoads et al., 2000a,b). Postnatal changes in superficial indices of carbohydrate and protein metabolism were little affected by birth weight in small and normal lambs that were artificially reared with ad libitum access to milk replacer. The very high concentrations of plasma GH in small, newborn lambs decreased markedly within 2 days of birth but remained significantly higher than concentrations in normal lambs for about 2 weeks. During the same period, plasma IGF-1 increased steadily in both groups but remained significantly lower in the small lambs (Greenwood et al., 2002). These observations suggest that the apparent immaturity of the GH/IGF axis in growthretarded newborn lambs persists for several weeks after birth. Interestingly, only during this early postnatal phase did the absolute growth rates of low-birth-weight lambs (248 g/d) lag significantly behind those of normal birth weight lambs (353 g/d) (Greenwood et al., 1998). Thereafter, during rapid growth from about 2 weeks of age to slaughter at 20 kg (attained at 6.5–8 weeks of age), plasma IGF-1 concentrations were persistently higher but GH concentrations were not different in low-versus normal-birth-weight lambs, perhaps related to the higher relative energy intakes and plasma insulin concentrations (see below) of the small lambs. This study did not examine the consequences of low birth weight after weaning. However, plasma GH concentrations tended to be higher during adolescence (~132 days of age) and adulthood (~378 days of age) in low-birth-weight male lambs from carunclectomized ewes compared to lambs of normal birth weight and were negatively correlated with indices of birth size (Gatford et al., 2002). Plasma insulin concentrations increased rapidly during the early postnatal period in small lambs feeding ad libitum, consistent with their very high levels of energy intake. Then, from about 2 weeks of age until slaughter at 20 kg, plasma insulin concentrations were persistently higher in low-compared with normal-birth-weight lambs. We speculate that this relative hyperinsulinemia may be due to the predisposition of growth-retarded neonates to develop insulin resistance (Hales et al., 1996). Plasma leptin concentrations were somewhat higher in rapidly fattening, low-birth-weight lambs during the first week post partum, but not thereafter (Ehrhardt et al., 2003), despite the fact that at any subsequent body weight up to 20 kg LW, these lambs were significantly fatter than their normal-birth-weight counterparts (Greenwood et al., 1998).

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Additional aspects of whole-body and tissue growth and development in lambs suffering IUGR are discussed in another, recent review (Greenwood and Bell, 2003).

5.2. Fetal programming of postnatal pathophysiology The human epidemiological evidence for fetal programming has implicated IUGR as an important risk factor for mature onset of diseases including hypertension and type II diabetes (Barker, 1998). Although the methodology and interpretation of aspects of this work recently have been challenged (Huxley et al., 2002), these epidemiological associations have been replicated experimentally in rodents (Langley-Evans, 2001) and various models of IUGR in sheep (McMillen et al., 2001; Greenwood and Bell, 2003). For example, low-birth-weight offspring born to protein-deprived rats (Langley and Jackson, 1994) and placentally insufficient ewes (McMillen et al., 2001) display hypertension during postweaning growth and adulthood. The rat model also has been used to demonstrate a relation between prenatal nutrition and the later development of insulin resistance (Langley et al., 1994), and similar evidence is emerging from sheep studies (Greenwood and Bell, 2003). Mechanisms linking prenatal nutrition, organ and tissue development, and the programming of later pathophysiology are unclear. However, excessive fetal exposure to glucocorticoids is a consistent feature of most animal studies involving prenatal nutrient deprivation, especially during late gestation. Also, treatment of pregnant rats and sheep with glucocorticoids during late pregnancy can replicate some of the programming effects of fetal undernutrition on later development of hypertension and insulin resistance (Langley-Evans, 2001; Greenwood and Bell, 2003). Growing evidence from studies on sheep and other species indicates that fetal programming can involve long-term sequelae to changes in the early prenatal environment that do not necessarily cause changes in fetal gross morphology. For example, modest undernutrition of ewes during the first half of pregnancy had no effect on growth of lambs during fetal or postnatal life but caused relative hypertension and increased activity of the hypothalamic–pituitary–adrenal (HPA) axis in lambs aged 12–13 weeks (Hawkins et al., 2000). Consistent with these responses, maternal undernutrition between early and mid-gestation caused increased expression of the glucocorticoid receptor in adrenals, kidney, liver, lungs, and perirenal adipose tissue of the fetus at term (~145 days) (Whorwood et al., 2001). At the same time, there were marked changes in the enzymatic capacity of several fetal tissues to deactivate cortisol, which may have led to excessive fetal exposure to this hormone during late gestation. Some of these tissue-specific fetal responses were evident as early as day 77 of gestation. A central role for corticosteroids in the mediation of fetal programming was further implicated by the remarkable finding that exposure of ewes to high doses of dexamethasone for only 2 days in early pregnancy resulted in hypertensive offspring at 3–4 months of age (Dodic et al., 1998). This hypertension amplified with age to beyond 3 years and was associated with increased cardiac output (Dodic et al., 1999) but no change in responsiveness of the HPA axis (Dodic et al., 2002). Glucose metabolic responses to insulin were unaltered, but the ability of insulin to suppress net fatty acid release from adipose tissue (plasma nonesterified fatty acid concentration) was moderately enhanced (Gatford et al., 2000).

6. FUTURE PERSPECTIVES The development almost four decades ago of novel techniques for studying fetal and placental physiology and metabolism in utero has led to considerable understanding of the regulation

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of the metabolic and developmental processes that culminate in the birth of a healthy, wellgrown neonate. Nevertheless, unexplained dysfunctions of conceptus growth remain, associated with unacceptable incidence of perinatal morbidity and mortality in many human and domestic animal populations. There also is a new awareness of the possible longer-term effects of nutritional and other environmental insults during fetal life, some of which may be quite subtle and without influence on gross morphology. Unraveling the mechanisms underlying such effects will be the major challenge of prenatal biology for the foreseeable future and should lead to a greater understanding of both human mature-onset pathologies and unexplained variation in the productivity and disease resistance of domestic animals.

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Lee, C.Y., Bazer, F.W., Etherton, T.D., Simmen, F.H., 1991. Ontogeny of insulin-like growth factors (IGF-I and IGF-II) and IGF-binding proteins in porcine serum during fetal and postnatal development. Endocrinology 128, 2336–2344. Lee, C.Y., Chung, C.S., Simmen, F.A., 1993. Ontogeny of the porcine insulin-like growth factor system. Mol. Cell. Endocrinol. 93, 71–80. Leibovich, H., Gertler, A., Bazer, F.W., Gootwine, E., 2000. Active immunization of ewes against ovine placental lactogen increases birth weight of lambs and milk production with no adverse effect on conception rate. Anim. Reprod. Sci. 64, 33–47. Lemons, J.A., Schreiner, R.L., 1983. Amino acid metabolismin the ovine fetus. Amer. J. Physiol. 244, E459–E466. Leury, B.J., Bird, A.R., Chandler, K.D., Bell, A.W., 1990a. Effects of maternal undernutrition and exercise on glucose kinetics in fetal sheep. Brit. J. Nutr. 64, 463–472. Leury, B.J., Bird, A.R., Chandler, K.D., Bell, A.W., 1990b. Glucose partitioning in the pregnant ewe: effects of undernutrition and exercise. Brit. J. Nutr. 64, 449–462. Li, J., Forhead, A.J., Dauncey, M.J., Gilmour, R.S., Fowden, A.L., 2002. Control of growth hormone receptor and insulin-like growth factor-I expression by cortisol in ovine fetal skeletal muscle. J. Physiol. 541, 581–589. Li, J., Owens, J.A., Owens, P.C., Saunders, J.C., Fowden, A.L., Gilmour, R.S., 1996. The ontogeny of hepatic growth hormone receptor and insulin-like growth factor I gene expression in the sheep fetus during late gestation: developmental regulation by cortisol. Endocrinology 137, 1650–1657. Li, J., Saunders, J.C., Gilmour, R.S., Silver, M., Fowden, A.L., 1993. Insulin-like growth factor-II messenger ribonucleic acid expression in fetal tissues of the sheep during late gestation: effects of cortisol. Endocrinology 132, 2083–2089. Liechty, E.A., Boyle, D.W., Moorehead, H., Lee, W.-H., Bowsher, R.R., Denne, S.C., 1996. Effects of circulating IGF-I on glucose and amino acid kinetics in the ovine fetus. Amer. J. Physiol. 271, E177–E185. Liechty, E.A., Boyle, D.W., Moorehead, H., Liu, Y.M., Denne, S.C., 1993. Increased fetal glucose concentration decreases ovine fetal leucine oxidation independent of insulin. Amer. J. Physiol. 265, E617–E623. Liechty, E.A., Denne, S., Lemons, J.A., Klein, C.L., 1991a. Effects of glucose infusion on leucine transamination and oxidation in the ovine fetus. Pediat. Res. 30, 423–429. Liechty, E.A., Kelley, J., Lemons, J.A., 1991b. Effect of fasting on uteroplacental amino acid metabolism in the pregnant sheep. Biol. Neonate 60, 207–214. Liu, J.-L., Baker, J., Perkins, A.S., Roberson, E.J., Efstratiadis, A., 1993. Mice carrying null mutations of the genes encoding insulin-like growth factor-I and type 1 receptor. Cell 75, 59–72. Lopez, M.F., Dikkes, P., Zurakowski, D., Villa-Komaroff, L., Majzoub, J.A., 1999. Regulation of hepatic glycogen in the insulin-like growth factor II-deficient mouse. Endocrinology 140, 1442–1448. Lorijn, R.H.W., Nelson, J.C., Longo, L.D., 1980. Induced fetal hyperthyroidism: cardiac output and oxygen consumption. Amer. J. Physiol. 235, H302–H307. Loy, G.L., Quick, A.N. Jr., Hay, W.W. Jr., Meschia, G., Battaglia, F.W., Fennessey, P.V., 1990. Fetoplacental deamination and decarboxylation of leucine. Amer. J. Physiol. 259, E492–E497. McMillen, I.C., Adams, M.B., Ross, J.T., Coulter, C.L., Simonetta, G., Owens, J.A., Robinson, J.S., Edwards, L.J., 2001. Fetal growth restriction: adaptations and consequences. Reproduction 122, 195–204. McNeill, D.M., Slepetis, R., Ehrhardt, R.A., Smith, D.M., Bell, A.W., 1997. Protein requirements of sheep in late pregnancy: partitioning of nitrogen between gravid uterus and maternal tissues. J. Anim. Sci. 75, 809–816. Mellor, D.J., Matheson, I.C., Small, J., 1977. Some changes in the composition of maternal and fetal plasma from chronically catheterised sheep during short periods of reduced feed intake in late pregnancy. Res. Vet. Sci. 23, 119–121.

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Polk, D.H., Wu, S.Y., Wright, C., Reviczky, A.L., Fisher, D.A., 1988. Ontogeny of thyroid hormone effect on tissue 5′-monoiodinase activity in fetal sheep. Amer. J. Physiol. 254, E337–E341. Rankin, J.H.G., Jodarski, G., Shanahan, M.R., 1986. Maternal insulin and placental 3-O-methyl glucose transport. J. Dev. Physiol. 8, 247–253. Rehfeldt, C., Kuhn, G., Nürnberg, G., Kanitz, E., Schneiders, F., Beyer, M., Nürnberg, K., Ender, K., 2001. Effects of exogenous somatotropin during early gestation on maternal performance, fetal growth, and composition traits in sheep. J. Anim. Sci. 79, 1789–1799. Reynolds, L.P., Ferrell, C.L., Robertson, D.A., Ford, S.P., 1986. Metabolism of the gravid uterus, foetus and utero-placenta at several stages of gestation in cows. J. Agr. Sci. 106, 437–444. Rhind, S.M., Robinson, J.J., McDonald, I., 1980. Relationships among uterine and placental factors in prolific ewes and their relevance to variations in foetal weight. Anim. Prod. 30, 115–124. Rhoads, R.P., Greenwood, P.L., Bell, A.W., Boisclair, Y.R., 2000a. Organization and regulation of the gene encoding the sheep acid-labile subunit of the 150-kilodalton insulin-like growth factor-binding protein complex. Endocrinology 141, 1425–1433. Rhoads, R.P., Greenwood, P.L., Bell, A.W., Boisclair, Y.R., 2000b. Nutritional regulation of the genes encoding the acid-labile subunit and other components of the circulating insulin-like growth factor system in the sheep. J. Anim. Sci. 78, 2681–2689. Robinson, J.S., Chidzanja, S., Kind, K., Lok, F., Owens, P., Owens, J., 1995. Placental control of fetal growth. Reprod. Fertil. Dev. 7, 333–344. Robinson, J.S., Hart, I.C., Kingston, E.J., Jones, C.T., Thorburn, G.D., 1980. Studies on the growth of the fetal sheep: the effects of reduction of placental size on hormone concentration in fetal plasma. J. Dev. Physiol. 2, 239–248. Ross, J.C., Fennessey, P.V., Wilkening, R.B., Battaglia, F.C., Meschia, G., 1996. Placental transport and fetal utilization of leucine in a model of fetal growth retardation. Amer. J. Physiol. 270, E491–E503. Rudolph, C.D., Roman, C., Rudolph, A.M., 1989. Effect of cortisol on hepatic gluconeogenesis in the fetal sheep. J. Dev. Physiol. 11, 219–223. Schoknecht, P.A., Currie, W.B., Bell, A.W., 1992. Kinetics of placental lactogen in mid- and late-gestation ovine fetuses. J. Endocrinol. 188, 95–100. Schoknecht, P.A., McGuire, M.A., Cohick, W.S., Currie, W.B., Bell, A.W., 1996. Effect of chronic infusion of placental lactogen on ovine fetal growth in late gestation. Domest. Anim. Endocrinol. 13, 519–528. Schreiner, R.L., Nolen, P.A., Bonderman, P.W., Moorehead, H.C., Gresham, E.L., Lemons, J.A., Escobedo, M.B., 1980. Fetal and maternal hormonal response to starvation in the ewe. Pediat. Res. 14, 103–108. Shen, W.-H., Yang, X., Boyle, D.W., Lee, W.H., Liechty, E.A., 2001. Effects of intravenous insulin-like growth factor-I and insulin administration on insulin-like growth factor binding proteins in the ovine fetus. J. Endocrinol. 171, 143–151. Silver, M., Fowden, A.L., Taylor, P.M., Knox, J., Hill, C.M., 1994. Blood amino acids in the pregnant mare and fetus: the effects of maternal fasting and intrafetal insulin. Exp. Physiol. 79, 423–433. Simmons, M.A., Battaglia, F.C., Meschia, G., 1979. Placental transfer of glucose. J. Dev. Physiol. 1, 227–243. Slotkin, T.A., Seidler, F.J., 1988. Adrenomedullary catecholamine release in the fetus and newborn: secretory mechanisms and their role in stress and survival. J. Dev. Physiol. 10, 1–16. Smeaton, T.C., Owens, J.A., Kind, K.L., Robinson, J.S., 1989. The placenta releases branched-chain amino acids into the umbilical and uterine circulations in the pregnant sheep. J. Dev. Physiol. 12, 95–99. Sparks, J.W., Hay, W.W. Jr., Bonds, D., Meschia, G., Battaglia, F.C., 1982. Simultaneous measurements of lactate turnover rate and umbilical lactate uptake in the fetal lamb. J. Clin. Invest. 70, 179–192. Stegeman, J.H.G., 1974. Placental development in the sheep and its relation to fetal development. Bjd. Dierk. 44, 1–72.

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Stephens, E., Thureen, P.J., Goalstone, M.L., Serson, M., Leitner, J.W., Hay, W.W. Jr., Draznin, B., 2001. Fetal hyperinsulinemia increases farnesylation of p21 Ras in fetal tissues. Amer. J. Physiol. 281, E217–E223. Sterle, J.A., Cantley, T.C., Lamberson, W.R., Lucey, M.C., Gerrard, D.E., Matteri, R.L., Day, B.N., 1995. Effects of recombinant porcine somatotropin on placental size, fetal growth, and IGF-I and IGF-II concentrations in pigs. J. Anim. Sci. 73, 2980–2985. Stevens, D., Alexander, G., 1986. Lipid deposition after hypophysectomy and growth hormone treatment in the sheep fetus. J. Dev. Physiol. 8, 139–145. Stevens, D., Alexander, G., Bell, A.W., 1990. Effect of prolonged glucose infusion into fetal sheep on body growth, fat deposition and gestation length. J. Dev. Physiol. 13, 277–281. Teasdale, F., 1976. Numerical density of nuclei in the sheep placenta. Anat. Rec. 185, 187–196. Thomas, L., Wallace, J.M., Aitken, R.P., Mercer, J.G., Trayhurn, P., 2001. Circulating leptin during ovine pregnancy in relation to maternal nutrition, body composition and pregnancy outcome. J. Endocrinol. 169, 465–476. Thureen, P.J., Merson, S., Hay, W.W. Jr., 2000. Regulation of uterine and umbilical amino acid uptakes by maternal amino acid concentrations. Amer. J. Physiol. 279, R849–R859. Thureen, P.J., Padbury, J.F., Hay, W.W. Jr., 2001. The effect of maternal hypoaminoacidaemia on placental uptake and transport of amino acids in pregnant sheep. Placenta 22, 162–170. Thureen, P., Trembler, K.A., Meschia, G., Makowski, E.L., Wilkening, R.B., 1992. Placental glucose transport in heat-induced fetal growth retardation. Amer. J. Physiol. 263, R578–R585. Timmerman, M., Chung, M., Wilkening, R.B., Fennessey, P.V., Battaglia, F.C., Meschia, G., 1998. Relationship of fetal alanine uptake and placental alanine metabolism to maternal plasma alanine concentration. Amer. J. Physiol. 275, E942–E950. Timmerman, M., Teng, C., Wilkening, R.B., Fennessey, P., Battaglia, F.C., Meschia, G., 2000. Effect of dexamethasone on fetal hepatic glutamine-glutamate exchange. Amer. J. Physiol. 278, E839–E845. Townsend, S.F., Rudolph, C.D., Rudolph, A.M., 1991. Cortisol induces perinatal hepatic gluconeogenesis in the lamb. J. Dev. Physiol. 16, 71–79. Van Veen, L.C.P., Teng, C., Hay, W.W., Jr., Meschia, G., Battaglia, F.C., 1987. Leucine disposal and oxidation rates in the fetal lamb. Metabolism 36, 48–53. Van Vliet, G., Styne, D.M., Kaplan, S.L., Grumbach, M.M., 1983. Hormone ontogeny in the ovine fetus. XVI. Plasma immunoreactive somatomedin C/insulin-like growth factor I in the fetal and neonatal lamb and in the pregnant ewe. Endocrinology 113, 1716–1720. Vatnick, I., Bell, A.W., 1992. Ontogeny of fetal hepatic and placental growth and metabolism in sheep. Amer. J. Physiol. 263, R619–R623. Vaughn, P.R., Lobo, C., Battaglia, F.C., Fennessey, P.V., Wilkening, R.B., Meschia, G., 1995. Glutamineglutamate exchange between placenta and fetal liver. Amer. J. Physiol. 268, E705–E711. Vernon, R.G., Robertson, J.P., Clegg, R.A., Flint, D.J., 1981. Aspects of adipose-tissue metabolism in foetal lambs. Biochem. J. 196, 819–824. Wallace, J.M., Bourke D.A., Aitken, R.P., Leitch, N., Hay, W.W. Jr., 2002. Blood flows and nutrient uptakes in growth-restricted pregnancies induced by overnourishing adolescent sheep. Amer. J. Physiol. 282, R1027–R1036. Wallace, J.M., Bourke, D.A., Aitken, R.P., Palmer, R.M., Da Silva, P., Cruickshank, M.A., 2000. Relationship between nutritionally-mediated placental growth restriction and fetal growth, body composition and endocrine status during late gestation in adolescent sheep. Placenta 21, 100–108. Ward, J.W., Wooding, F.B.P., Fowden, A.L., 2002. The effects of cortisol on the binucleate cell population in the ovine placenta during late gestation. Placenta 23, 451–458. Whorwood, C.B., Firth, K.M., Budge, H., Symonds, M.E., 2001. Maternal undernutrition during early to midgestation programs tissue-specific alterations in the expression of the glucocorticoid receptor,

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11β-hydroxysteroid dehydrogenase isoforms, and type 1 angiotensin II receptor in neonatal sheep. Endocrinology 142, 2854–2864. Widdas, W.F., 1952. Inability of diffusion to account for placental glucose transfer in the sheep and consideration of the kinetics of a possible carrier transfer. J. Physiol. 118, 23–39. Wilkening, R.B., Molina, R.D., Battaglia, F.C., Meschia, G., 1987. Effect of insulin on glucose/oxygen and lactate/oxygen quotients across the hindlimb of fetal lambs. Biol. Neonate 51, 18–23. Wooding, F.B.P., Morgan, G., Fowden, A.L., Allen, W.R., 2000. Separate sites and mechanisms for placental transport of calcium, iron and glucose in the equine placenta. Placenta 21, 635–645. Young, M., McFadyen, I.R., 1973. Placental transfer and fetal uptake of amino acids in the pregnant ewe. J. Perinatal Med. 1, 174–182. Yuen, B.S.J., McMillen, I.C., Symonds, M.E., Owens, P.C., 1999. Abundance of leptin mRNA in fetal adipose tissue is related to fetal body weight. J. Endocrinol. 163, R11–R14. Yuen, B.S.J., Owens, P.C., McFarlane, J.R., Symonds, M.E., Edwards, L.J., Kauter, K.G., McMillen, I.C., 2002. Circulating leptin concentrations are positively related to leptin messenger RNA expression in adipose tissue of fetal sheep in the pregnant ewe fed at or below maintenance energy requirements during late gestation. Biol. Reprod. 67, 911–916. Yuen, B.S.J., Owens, P.C., Mühlhäusler, B.S., Roberts, C.T., Symonds, M.E., Keisler, D.H., McFarlane, J.R., Kauter, K.G., Evens, Y., McMillen, I.C., 2003. Leptin alters the structural and functional characteristics of adipose tissue before birth. FASEB J. Express article 10.1096/fj.02–0756fje. Published online, April 22, 2003. Zhou, J., Bondy, C.A., 1993. Placental glucose transporter gene expression and metabolism in the rat. J. Clin. Invest. 91, 845–852.

PART II Protein metabolism

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Regulation of skeletal muscle protein metabolism in growing animals T. A. Davis and M. L. Fiorotto United States Department of Agriculture/Agricultural Research Service, Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA

The skeletal musculature is not only of great significance to the physiological function and long-term well-being of the growing animal, but, by virtue of its large mass, has tremendous impact on the overall rate of protein metabolism in the whole body. Protein deposition is very rapid during early life and this is largely driven by the high fractional rate of protein synthesis in skeletal muscle. A number of factors regulate the growth, development, and metabolic activity of the skeletal musculature, and these include the intrinsic or genetic factors that influence muscle differentiation as well as the extrinsic factors such as nutrients, hormones, and activity that influence muscle hypertrophy.

1. INTRODUCTION In the mature adult, the skeletal musculature constitutes the largest single protein pool in the body, and comprises approximately 60% of the body’s metabolically active mass. Thus, despite its relatively low basal rate of metabolism, skeletal muscle mass is such that changes to its composition and/or its size have implications for the overall metabolism of the body. Until relatively recently, the primary interests in skeletal muscle metabolism were related to its functional role in dictating locomotor performance, specifically speed, strength, and endurance, and its influence on the quantity and quality of meat products that constitute a primary source of protein and micronutrients in the human diet. More recently, however, a renewed interest in the contribution of the muscle metabolism to the overall health of the human individual has emerged. This interest, together with the developments in our understanding of the regulation of gene expression and cellular signalling, have spurred substantial amounts of research to advance our understanding of how muscle mass, metabolism, and function respond to nutrients, hormones, growth factors, activity, and other anabolic agents. The fully differentiated skeletal muscle is made up of multinucleated myofibres. The postnatal growth rate of muscle mass is a function of the total number of fibres, and the growth

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rate of each fibre. Thus, understanding the regulation of myofibre formation during prenatal life, and the rate of muscle protein accretion in postnatal life, are critical for evaluating the animal’s maximal capacity for muscle growth. The mature myofibre is composed primarily of the three basic systems required for muscle contraction: the myofibrillar proteins composed primarily of the contractile elements and the associated structural proteins; an extensive membrane system that regulates the release and uptake of ions in response to the neural inputs; and the mitochondrial and cytoplasmic system of enzymes involved in the generation of the ATP required to drive functional processes. These components vary in their relative abundance, as well as in their level of activity, thereby giving rise to substantial diversity in fibre function and size. The combination of functional and metabolic properties of fibres is used as the basis for the standard classification of muscle fibres in the adult. Muscle fibres can be divided into two main categories on the basis of their twitch characteristics, that is, slow-twitch (S) or fast-twitch (F), which correspond with the Type I and Type II nomenclature, respectively. The contractile property of the myofibrils is largely determined by the ATPase activity of the myosin heavy chain (MHC) isoform expressed within each fibre (Schiaffino and Reggiani, 1996). Fibres are also identified in a variety of ways by their metabolic properties: slow-twitch fibres, and a subcategory of fast-twitch fibres, generate ATP predominantly by oxidative metabolism (O). All fast-twitch fibres also can generate limited amounts of ATP by glycolysis and, hence, they are categorized as glycolytic (FG or Type IIB) or fast-twitch, oxidativeglycolytic (FOG or Type IIA). The capacity for oxidative metabolism is supported by a relatively high abundance of mitochondria, enzymes for fatty acid oxidation, and oxygen delivery and uptake. The latter is effected by a high capillary density, and the presence of large amounts of myoglobin in the sarcoplasm, both of which impart a red tint to the muscle, so that SO and FOG fibres also are referred to as red muscle. Because these metabolic properties render to the fibres a greater degree of fatigue-resistance, they constitute primarily those muscles that undergo prolonged periods of sustained slow isometric contraction, such as postural muscles (e.g. soleus and rhomboides), or muscles that are required for continuous episodes of isotonic contraction (e.g. diaphragm and jaw muscles). In contrast, FG fibres have a paucity of these components and thus, muscles where these fibres predominate (e.g. longissimus dorsi) are much lighter in colour and are referred to as white muscles. The origin of myofibre heterogeneity is complex and not entirely understood. There is evidence that the various fibre types are derived from distinct myoblast lineages. However, within an organism, the relative abundance of different fibre types varies between muscles according to their physiological function. There is also variability between individuals of the same breed, between different breeds of the same species, and between different species (Rehfeldt et al., 1999). Fibre-type proportions are of relevance with respect to livestock, as they are a key determinant of meat quality (Koohmaraie, 2003). Fibre diversity has its origins in the embryo, is amplified during the process of differentiation during fetal and early postnatal life, and thereafter is fine-tuned in response to the functional demands placed on the muscle. By and large, however, fibre-type composition is a genetically determined, inherited trait. Muscle fibre hypertrophy, on the other hand, occurs after the fibres have differentiated. Hypertrophy is largely a postnatal event and metabolically is dominated by the accretion of muscle-specific proteins. Unlike the early processes of determination, commitment, and differentiation that are normally orchestrated by signalling molecules and intracellular pathways inherent to the developing organism and are primarily under genetic control, muscle hypertrophy is highly responsive to external cues, such as nutrient availability, muscle use, and various hormones. The aim of this review is to consider the factors that influence muscle protein metabolism in the growing organism. We shall address the “heritable/congenital” component, i.e. the

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developmental aspects of muscle growth that principally determine fibre number and fibretype diversity, and the role of external influences that modulate muscle hypertrophy. Owing to space limitations, this review cannot cover all aspects of muscle metabolism in equal depth. Rather, we have selected those areas in which substantial progress has been made recently, and have focused largely on early life, when the most marked changes occur.

2. MUSCLE DIFFERENTIATION 2.1. From myoblast to myofibre Much of our understanding of early myogenesis is derived from studies on the chick. However, the overall pattern is similar in mammals as indicated by the extensive study of the mouse in which the advent of transgenic technology has permitted the function of individual genes to be identified. Myogenesis begins in early embryonic life and, with the exception of craniofacial muscles, the skeletal musculature develops from mesodermal progenitor cells in the somites (Perry and Rudnicki, 2000; Buckingham, 2001). The somites develop in pairs from aggregates of epithelial cells on either side of the neural tube and notochord and mature in a rostro-caudal direction under the regulation of positive signals and negative regulators, in the form of diffusible molecules, produced by the tissues adjacent to the somites (Buckingham, 2001; Buckingham et al., 2003; Francis-West et al., 2003) (fig. 1). Cells of the dorsal surface of the somite are compartmentalized into the dermomyotome and are specified to form myogenic and dermal progenitors, whereas signals to cells on the ventral aspect of the somite specify the formation of the sclerotome which gives rise to the ribs and axial skeleton (Buckingham, 2001). Myogenic precursor cells originate from the dorso-medial (epaxial) and ventro-lateral (hypaxial) edges of the dermomyotome. The epaxial precursors delaminate and translocate ventrolaterally to form the myotome, under the dermomyotome, and eventually they expand and differentiate to form the deep back muscles. The hypaxial myogenic precursors of the dermomyotome migrate ventrally to form the ventral body wall muscles, tongue, and diaphragm, or delaminate and migrate into the limb buds to give rise to the limb musculature. Specification of myogenic cells occurs upon the activation of myogenic regulatory factors (MRF), specifically those that encode the basic helix–loop–helix transcription factors Myf5, MyoD, MRF4, and myogenin (Molkentin and Olson, 1996; Perry and Rudwicki, 2000).

Fig. 1. Schematic representation of somite structure and key molecules responsible for myogenic specification, determination, delamination, and migration.

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Myf5 and MyoD are the earliest MRFs to be expressed in the dermomyotome, and their patterns of expression appear to be spatially and temporally regulated. Wnt-1 produced in the dorsal neural tubes induces Myf5 expression in the epaxial region, whilst Wnts produced by the dorsal ectoderm induce MyoD expression by the dermomyotome. Sonic hedgehog (Shh), produced from the notochord and floor plate of the neural tube, also appears to play a role in the early activation of Myf5 in the determination of the epaxial dermomyotome (Buckingham et al., 2003). Cells committed to the muscle lineage express the homeobox gene, Pax-3, and its expression is most likely activated by Wnts and Shh. During early myogenesis, however, the differentiation of cells that express Pax-3 and are committed to the muscle lineage is blocked by the expression of inhibitory factors. These include the growth factor, bone morphogenic protein-4 (BMP-4), and fibroblast growth factors, both of which are produced by the lateral plate mesoderm. Inhibition of differentiation is critical as it enables the cells to continue proliferation, delaminate, and migrate. In the regions of the limbs, migrating cells do not express MRFs until they reach the limbs where, after some delay during which they undergo several rounds of replication, they give rise to the appendicular muscles (Buckingham, 2001; Buckingham et al., 2003; Francis-West et al., 2003). Delamination and migration of muscle precursor cells are crucial steps in myogenesis (Birchmeier and Brohmann, 2000; Francis-West et al., 2003) and require the activation of the c-met receptor by hepatocyte growth factor (HGF, also known as scatter factor) (Scaal et al., 1999; Birchmeier and Brohmann, 2000). Transcription of the c-met gene is activated by Pax-3, and in its absence no limb muscles form, whereas when a constitutively active form is expressed, there is an overproduction of hypaxial muscles (Epstein et al., 1996). Lbx1 is another transcription factor required for migration of the somitic cells, with particular importance for those cells that give rise to the dorsal muscle masses of the limbs (Brohmann et al., 2000). Once commitment of cells to the myogenic lineage has occurred, the cells are prevented from differentiating further by a variety of gene products produced by the cells themselves, as well as the cells’ matrix, and a variety of mitogens that promote proliferation (Perry and Rudnicki, 2000; Fuchtbauer, 2002). The maintenance of the committed, but not fully differentiated, state is a critical determinant of myofibre number and size because it permits myoblast proliferation to continue and, hence, to expand the population of cells that can give rise to myofibres (Fuchtbauer, 2002). Indeed, all instances of muscle hypertrophy that are associated with increased myofibre number have their origin in embryonic and fetal life, and by birth, fibre number is fixed (Rehfeldt et al., 1999). The degree of myoblast proliferation in vivo is the product of the balance between activities of stimulatory and inhibitory factors (Fuchtbauer, 2002). The former include Msx1, basic fibroblast growth factor (bFGF), HGF, and the insulin-like growth factors (IGF)-I and -II. The transforming growth factor (TGF-β) super-family of peptides exert a variety of effects which appear to be species-dependent, but by and large, they inhibit terminal differentiation with either little effect or even inhibition of proliferation (Fuchtbauer, 2002). Members of the TGF-β family that have been shown to regulate myogenesis include TGF-β itself, activin, BMPs, and growth differentiation factor 8, also known as myostatin (McPherron et al., 1997; Fuchtbauer, 2002). Their precise role and the incurred responses vary between muscle beds and according to the net balance between positive and negative regulators. The in vivo significance of these factors in regulating myoblast hyperplasia and fibre formation has been demonstrated in studies with an extensive variety of transgenic animals. Notable among these has been the development of transgenic mice in which the myostatin gene is inactivated (McPherron et al., 1997). Myostatin is expressed beginning early in embryonic life and inhibits cell cycle progression, thereby limiting myoblast proliferation

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(Lee and McPherron, 1999). Myostatin also inhibits differentiation by down-regulating MyoD and myogenin expression and activity (Langley et al., 2002). Hence, in its absence, there is greater myoblast proliferation and this ultimately results in the formation of a larger number of muscle fibres. The characterization of myostatin and its mechanism of action pointed to the identification of the genetic basis for the muscle hypertrophy in some breeds of cattle, commonly known as “double-muscling” (Grobet et al., 1997). In vivo, myostatin is regulated by follistatin, and follistatin overproduction increases muscle mass, whereas impaired production reduces muscle mass. The IGFs are unique in their actions in that they can stimulate both proliferation and differentiation by activating the type 1 IGF receptor; these effects, however, are temporally separated. The switch from proliferative to myogenic effects is associated with changes in the intracellular signalling pathways from primary activation of the mitogen-activated protein kinase (MAPK) pathway during the proliferative phase, to signalling through the phosphatidylinositol 3-kinase (PI 3-kinase) pathway for differentiation (Coolican et al., 1997). In cell culture, the proliferative phase is associated with IGF-I-stimulated expression of cell cycle markers and cell proliferation and reduced expression of myogenic markers, whereas during differentiation IGF-I promotes expression of myogenin and muscle-specific gene expression (Engert et al., 1996). The exact trigger responsible for switching the response from proliferation to differentiation is unclear, especially in vivo. Studies in the chick embryo have demonstrated that increased local expression of IGF-I in the limb at an early stage of development before cells have differentiated increases myoblast number with the formation of larger muscles containing an increased number of myofibres (Mitchell et al., 2002), in much the same way as, although independently of, decreased myostatin expression. Similarly, administration of growth hormone (GH) to pregnant sows in early gestation (10–24 days) indirectly stimulates fetal IGF secretion and results in greater myofibre number at birth (Rehfeldt et al., 1993). This response contrasts with the response to over-expression of IGF-I in the differentiated muscle, where muscle hypertrophy is not associated with increased fibre number (Musaro et al., 2001; Fiorotto et al., 2003). The stimulation of muscle differentiation by the MRFs entails not only up-regulation of their expression within presumptive muscle cells, but also the co-ordinated orchestration of a series of events that enables them to be transcriptionally active. These include: the downregulation of Id proteins which mitigate the binding of MRFs to their E-box consensus sequence (Benezra et al., 1990); the association with the myogenic enhancer factor 2 (MEF2) family of transcription factors that bind to both their own DNA site and form protein–protein interactions with the MRFs (Molkentin and Olson, 1996); and the dissociation of histone deacetylases from the transcription factors and subsequent recruitment of histone acetylases to E-boxes associated with muscle-specific genes. The resulting acetylation of histones produces the chromatin relaxation necessary to permit transcription of the underlying gene (McKinsey et al., 2001, 2002). This differentiation step is associated concomitantly with inhibition of cell cycling, and the alignment and subsequent fusion of the adhered myoblasts to form myotubes. Fibres form in two waves: the first wave occurs during early embryogenesis and results in the formation of primary myotubes that shape and position the orientation of individual muscles (Ontell and Dunn, 1978; Ontell, 1982). These primary fibres are originally in clusters, but progressively become separated by the basal lamina. Subsequently, a secondary population of myoblasts located under the basal lamina of the primary fibres begins to proliferate using the plasma membrane of the primary fibres as scaffolding, but without fusing to them. These then fuse among themselves to form secondary myotubes, and gradually separate from the

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primaries by forming their own basal lamina. There is a third population of myoblasts, the satellite cells, that remain quiescent, sandwiched between the plasma membrane and basal lamina. The expression of the homeobox protein, Pax-7, distinguishes satellite cells from other lineages of myoblast, and appears to be essential for the formation of satellite cells (Seale et al., 2000). Satellite cells proliferate to enlarge the myonuclear population present in the myofibres to which they are attached, and contribute only to myofibre hypertrophy, not new fibres (except to repair muscle damage). To this point in the process of skeletal muscle development, the primary effect of perturbations in the growth process are likely to manifest themselves as variations in the number of myofibres that form. The timing of the perturbation will determine if primary or secondary fibres are affected. It is unclear if impairment of satellite cell replication during the terminal phase of muscle formation can permanently impact the size of a muscle’s reserve of satellite cells and, hence, postnatal muscle growth potential. 2.2. Compositional development Once myoblasts have fused, the expression of muscle-specific proteins dominates muscle growth. The cells undergo a complex set of transformations to create the highly structured myofibrils which have the capacity to perform contractile work. This process of maturation is critical for the developing offspring as it is essential for postnatal survival: skeletal muscles are a prerequisite for independent breathing and suckling. Indeed, the offspring die at birth in all transgenic animals in which normal skeletal muscle development is impaired by targeted disruption of key regulatory molecules (e.g. Venuti et al., 1995). However, there is a wide variation in the level of muscle maturity at birth among species, and between muscle groups within the same individual. From these observations, it is evident that birth occurs at different stages of muscle development across species, and that within an individual, maturation proceeds in a rostral to caudal direction, and from proximal to distal in the appendages. Hence, altricial species, like rabbits, rodents, and most carnivores, are born with functional head and thoracic muscles, whereas their lower abdominal and limb muscles, especially those of the hindlimbs, are still immature. In contrast, precocial species, such as ungulates, have relatively longer gestations and the newborn muscle is at a fairly advanced stage of maturity. Indeed, locomotor function is attained very soon after birth. The maturation of myotubes into fully functional myofibres involves the co-ordinated development of the metabolic machinery of the cells, the ion transport membrane system, and the contractile elements. The complex sarcoplasmic reticulum and T-tubular system responsible for coupling excitation and contraction followed by muscle relaxation develop in a co-ordinated fashion and attain their mature configuration at approximately 2 weeks of age in the rat. At this point, the membrane system constitutes approximately 40% of the nonmyofibrillar compartment in the muscle fibre (Schiaffino and Margreth, 1969). Contractile proteins, not present in the myotube at fusion, comprise 55−65% of total muscle protein by 2 weeks of age in the rat (Yates and Greaser, 1983; Fiorotto et al., 2000a). The accretion of myofibrillar proteins, therefore, is a major determinant of whole-body protein accretion during this developmental period. The diversity in fibre-type characteristics results from the combination of the inherent properties of the myoblast lineage from which the fibres were derived and extrinsic signals from the organism. Thus, in the development of a myofibre from a myotube, in addition to the first-order general pattern of compositional development that occurs (described above),

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significant changes also occur in the isoform composition of muscle proteins and the supporting metabolic machinery. These result in a second order of compositional changes that produce the development of the adult phenotype of individual fibre types (Schiaffino and Reggiani, 1996). A significant proportion of the proteins that constitute the thick, intermediate, and thin filaments initially are expressed as immature isoforms and subsequently are replaced by the adult isoforms during maturation (table 1). In the thick filaments, all primary fibres initially express a combination of embryonic and slow MHC isoforms, or neonatal and slow MHC isoforms. The associated myosin light chains (MLC) also differ according to the MHC isoform with which they are associated and, therefore, their expression changes during maturation. Secondary fibres, in contrast, initially express the embryonic and neonatal MHC isoforms, which are replaced usually by any of the adult fast MHC isoforms according to the functional characteristics of the individual muscle. This process of terminal maturation is amenable to regulation by hormones, activity pattern, and neural inputs, and contrasts with the earlier differentiation of myoblast lineage which proceeds independently of extrinsic factors. A fundamental conundrum regarding the differentiation among fibre type relates to the mechanism that co-ordinates the appropriate expression of the plethora of proteins responsible for the metabolic and contractile characteristics of muscle. Recent studies have focused on the

Table 1 Contractile protein isoforms expressed in the developing and mature muscles of the rat Developing muscles

Adult muscles

Embryonic

Neonatal

Fast

Slow

Myosin heavy chains embryonic β-slow

neonatal (embryonic)

2B 2X 2A

β-slow

MLC-1fast (MLC-3fast)

MLC-1fast MLC-3fast

MLC-1slow/ventricular (MLC-1slow-α)

MLC-2fast

MLC-2fast

MLC-2slow

α-skeletal (α-cardiac)

α-skeletal

α-skeletal

TnC-fast

TnC-fast

TnC-slow/cardiac

TnI-fast TnT-fast, fetal isoforms

TnI-fast TnT-fast, adult isoforms

TnI-slow TnT-slow

TM-β TM-αfast

TM-αfast (TM-β)

TM-αslow TM-αfast TM-β

Myosin light chains (MLC) MLC-1embryonic MLC-1slow-α MLC-1fast MLC-2fast Actin α-cardiac α-skeletal Troponins (Tn) TnC-fast TnC-slow/cardiac TnI-slow TnT-cardiac TnT-slow Tropomyosins (TM) TM-β TM-αfast TM-αslow

Minor isoforms are indicated in parentheses. Isoform profile indicated for neonatal developing muscles is that of the majority of hindlimb muscle fibres, which are secondary generation fibres destined to become fast-type fibres. Adapted from Schiaffino and Reggiano (1996).

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role of calcineurin (CaN), a calcium and calmodulin-dependent serine/threonine phosphatase, in the regulation of expression of those genes responsible for the slow muscle phenotype (Schiaffino and Serrano, 2002; Spangenburg and Booth, 2003). CaN is activated when there are high intracellular steady-state levels of Ca2+, typical of slow fibres that are subjected to chronic, low-frequency nerve stimulation. Once activated, CaN dephosphorylates the transcription factors, nuclear factor of activated T-cells (NFAT) and MEF-2, so that they can then be translocated into the nucleus where, in conjunction with MRFs, they effect changes in gene expression (McKinsey et al., 2001, 2002). These factors bind to their respective DNA consensus sequences which form a characteristic motif, the slow upstream element (SURE), present in the promoter of a variety of slow muscle genes such as slow troponin I and myoglobin (Calvo et al., 2001). Despite the ability of CaN to sense and transduce changes in cell calcium levels into changes in gene expression, it is by no means a global regulator of the SO fibre phenotype as was initially proposed. For example, it has been observed that expression of MHC-IIa, the predominant MHC isoform in FOG muscles, is also highly responsive to CaN activation (Allen and Leinwand, 2002). Considerable progress has been made in identification of the mechanisms that co-ordinate the contractile and metabolic characteristics of muscle. Again, the sustained elevation of intracellular calcium appears to be a central factor in signalling not only the fast-to-slow shift in muscle gene expression (Allen and Leinwand, 2002), but also an increase in mitochondrial biogenesis (Ojuka et al., 2003). In addition to CaN, calcium activates calcium/calmodulindependent protein kinases (CaMK) which catalyse a series of reactions that result in the transcription of a coactivator of nuclear receptors, peroxisome proliferator-activated receptor coactivator-1α (PGC-1α) (Handschin et al., 2003). PGC-1α plays a pivotal role in glucose metabolism, mitochondrial biogenesis, and adaptive thermogenesis by activating various transcription factors. Specifically, in muscle PGC-1α has been shown to stimulate mitochondrial DNA replication, mitochondrial abundance, cytochrome c and cytochrome oxidase levels, GLUT4 expression, and uncoupling protein expression. PGC-1α also enhances its own transcription (Handschin et al., 2003). Consequently, once activated, an autoregulatory loop is set up which sustains PGC-1α expression and its downstream effects, and thereby maintains stable expression of the oxidative phenotype. In addition to calcium, PGC-1α expression is regulated by thyroid hormone and AMPactivated protein kinase, an enzyme that is activated by chronic reductions in the cellular ATP/AMP ratio, for example, with energy deprivation (Irrcher et al., 2003; Ojuka et al., 2003; Spangenburg and Booth, 2003). These mechanisms that co-ordinate the metabolic properties of a muscle with its contractile properties and pattern of use, however, are pertinent primarily to the development of slow-twitch and/or oxidative properties, presumably in muscles where these characteristics are not present. This suggests that fast-twitch, glycolytic properties are the default phenotype of skeletal muscle and there is, indeed, some evidence to support this suggestion. During terminal maturation, the loss of polyinnervation and acquisition of single innervation from a nerve with a low-frequency firing pattern is necessary for the development and maintenance of slow-twitch characteristics. Moreover, if the soleus is denervated at birth in the rat, slow myosin isoenzymes are gradually replaced by fast myosins (Gambke et al., 1983). Thus, the replacement of the immature isoforms of myosin specifically by adult slow myosin occurs only with the appropriate neural input. In the mature muscle, cross-innervation of a mature fast-twitch muscle with the nerve from a slow-twitch muscle gradually transforms the entire contractile phenotype and metabolic properties to those of a slow muscle (Barany and Close, 1971). Thyroid hormone also plays a critical role in the maturation of skeletal muscle. Moreover, because thyroid hormone is sensitive to changes in energy balance, it may serve as a signal

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to the muscle to produce adaptive changes in muscle metabolism. The importance of thyroid hormone has been studied extensively with respect to the regulation of MHC expression where it is required for down-regulation of the neonatal isoform of MHC in muscles destined to become either fast or slow (Gambke et al., 1983; Adams et al., 1999). Furthermore, in the absence of thyroid hormone, the accumulation of slow MHC is accelerated, whereas that of IIA MHC is down-regulated. Thus, variations in energy balance that produce changes in thyroid hormone might be anticipated to alter the postnatal pattern of muscle maturation. Our studies in suckling rats suggest that the changes in thyroid hormone have to be relatively severe in order to effect changes at the level of gene expression. However, changes in muscle use and protein turnover also occur as a consequence of alterations in food intake and growth rate. These changes serve to mitigate the effect of altered gene expression and, consequently, the maturation of muscle phenotype is preserved (Fiorotto and Davis, 1997; Fiorotto et al., 2000a). The suckling pig responds to mild hypothyroidism during the suckling period by increasing slow MHC expression, although the effect is somewhat mitigated by increases in nuclear thyroid hormone receptor (Harrison et al., 1996). Relatively severe energy restriction in post-weaned pigs has similar effects, increasing the abundance of slow MHC at both the protein and mRNA level, and increasing the oxidative properties of the muscles, but with substantial muscle-to-muscle variability (White et al., 2000). Although the increase in slow MHC expression is compatible with the known effects of hypothyroidism on MHC expression, the enhanced oxidative properties are the opposite of those anticipated on the basis of PGC-1α regulation by thyroid hormone. However, they are compatible with a change in AMP kinase activity, which increases with a chronic deficit in energy intake, and thereby promotes mitochondrial biogenesis and fatty acid oxidation. Overall, these responses to a chronic deficit in energy intake represent beneficial adaptations by the muscle to enhance its metabolic efficiency: a slow muscle requires less energy than a fast-twitch muscle to generate the same amount of tension, and it is able to derive more of its energy by fatty acid oxidation and oxidative phosphorylation (Henriksson, 1990). The regulation of the fast-twitch, glycolytic phenotype of muscles is much less clearly understood than that of slow-twitch muscle. Some genes expressed in fast fibres contain a characteristic binding motif, the fast intronic regulatory element (FIRE), analogous to the SURE motif in slow fibre genes (Nakayama et al., 1996). Myoblast lineage established during fetal life appears to be a primary determinant of whether a fibre matures into a fast fibre. As noted previously, gene mutations that promote secondary myoblast proliferation result primarily in fast fibre hypertrophy. During terminal maturation, thyroid hormone is required for the down-regulation of neonatal MHC and, if present at high levels, thyroid hormone tends to drive the expression of fast MHC in muscle fibres that normally would be slow (Nakayama et al., 1996). The role of innervation also appears to be less critical in the development of fast-twitch fibres than for slow fibres. In both rodents and chickens, denervation delays the elimination of immature MHC isoforms, but does not prevent the development of the fast phenotype. Inactivity also tends to promote the fast phenotype, although this is attributable in part to the preferential atrophy of slow fibres. In post-weaned pigs (Katsumata et al., 2000), but possibly not neonatal pigs (Louveau and Le Dividich, 2002), mild undernutrition up-regulates the expression of the growth hormone receptor (GHR) on FOG and FG fibres which normally express the lowest level of GHR. Together with the reduction in thyroid hormone expression, the resulting changes in hormone responsiveness may be responsible for the metabolic shift that occurs in muscle during undernutrition and which enables it to derive more of its energy from lipid oxidation. A broader implication of these findings, however, relates to the anti-insulin effects of GH in the undernourished animal which serve to divert

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nutrients from muscle towards the visceral organs. This is demonstrated by the differential response in rates of tissue growth in protein-malnourished piglets where body protein partitioned into gastrointestinal tissue is preserved, while that of skeletal muscle is reduced (Ebner et al., 1994). 2.3. Role of protein synthesis and degradation in the regulation of compositional development As the above discussion of the developmental changes in muscle composition indicates, protein synthetic rates in the immature muscle must sustain not only the de novo accretion of myofibrillar proteins and membrane structures, but also their continuous and co-ordinated replacement as the tissue develops its mature compositional and functional characteristics. The faster accumulation of myofibrillar proteins compared to sarcoplasmic proteins is explained almost entirely by their higher fractional synthesis rate compared to other protein components (Fiorotto et al., 2000a). As compositional maturity is attained, the synthesis rate of myofibrillar proteins decreases to a greater extent than sarcoplasmic proteins, and in the mature muscle, sarcoplasmic protein synthesis rates are higher than for myofibrillar proteins. However, once the mature composition is attained, the rates of degradation also differ in parallel and results in the maintenance of constant composition. In altricial mammals such as rodents and rabbits, the differential regulation of protein synthesis in the different protein pools occurs in the immediate postnatal period. In precocial animals, the full complement of myofibrillar proteins in fibres is mostly completed by birth, although they still undergo some limited, second-order compositional maturation postnatally. Nonetheless, the intrauterine pattern of development and mechanisms of regulation at comparable stages of maturity are likely to be similar across species. In the mature muscle, there are fibre-type differences in the rate of protein turnover that reflect their compositional differences; slow fibres have higher rates of protein turnover than fast-twitch fibres (Bark et al., 1998; Dardevet et al., 2002), and this diversity emerges only upon maturation (Davis et al., 1989). These phenotypic differences are attributable to the differences in the synthesis rate of muscle proteins in combination with the variation in their relative abundances among muscles. In skeletal muscles from mature pigs, the average synthesis rate of mitochondrial proteins is higher than for sarcoplasmic proteins and this, in turn, tends to be slightly higher than for the myofibrillar proteins (Balagopal et al., 1997; Fiorotto et al., 2000a). Although the ratio of myofibrillar to sarcoplasmic proteins tends to be greater in slower muscles (Hemel-Grooten et al., 1995), the difference in synthesis rates is substantially lower than that of mitochondrial proteins, which are more abundant in the slower, oxidative muscles. The greater overall protein synthetic activity of the slow muscles is supported by a higher ribosomal abundance and entails minimal changes in protein synthetic efficiency. In addition to the inherent variation in synthesis rates, the myofibre protein components can also differ in their responses to extrinsic stimuli. For example, in adult porcine muscle, stimulation of protein synthesis by insulin appears to be limited to the mitochondrial proteins (Boirie et al., 2001); the developmental decline in muscle protein synthesis rates is dominated by myofibrillar proteins (Fiorotto et al., 2000a). Clearly, these differences among muscle protein components have to be factored into our understanding of the overall regulation of skeletal muscle protein metabolism. In the newly differentiated muscle, the high ribosomal abundance is the principal factor that enables high rates of protein synthesis to be attained, and its reduction with maturation is one mechanism that may underlie the general reduction in fractional synthesis rates

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observed for all muscle proteins. However, this cannot explain the differences in composition of proteins synthesized, and some regulation must occur at the level of gene transcription. During the transition from myoblast to myotube, genes encoding non-muscle proteins are repressed, while those specific for muscle proteins are induced in a co-ordinated manner. There is then a commensurate change in the composition of proteins expressed (Devlin and Emerson, 1978, 1979; Shani et al., 1981). In vivo, it has been demonstrated that the stoichiometry of the total mRNAs encoding all isoforms within a protein family is maintained accurately, and that production of individual myofibrillar proteins in appropriate stoichiometric amounts, therefore, is regulated at the message level (Wade et al., 1990). However, such changes would not explain the differential responses of sarcoplasmic and myofibrillar proteins even if the decrease in ribosomal abundance were accompanied by a reduction in the proportion of myofibrillar mRNAs. Such a change in mRNA composition would increase the translational efficiency of sarcoplasmic proteins, but decrease the translational efficiency of myofibrillar proteins. Translational efficiency, however, increases for all proteins in the immature muscle. Clearly, therefore, although differences in mRNA abundances are involved, as we have demonstrated in newborn pigs (Fiorotto et al., 2000b), this also cannot be entirely responsible for the compositional differences in protein synthesis, suggesting that there must also be regulation of mRNA at the translational and post-translational levels.

3. POSTNATAL MUSCLE GROWTH 3.1. Satellite cells and hyperplasia Postnatal growth of skeletal muscle is driven by hypertrophy of the existing fibres. This requires both an increase in myonuclear content, and the accretion of muscle proteins. Myonuclei are postmitotic and, thus, satellite cells are entirely responsible for the postnatal increase in muscle fibre DNA. This is clearly demonstrated in mice in which the expression of Pax-7 is abolished and consequently no satellite cells form (Seale et al., 2000). These muscles contain both primary and secondary fibres but they fail to hypertrophy postnatally. Indeed, in a variety of circumstances normally associated with accelerated postnatal muscle growth, the inhibition of satellite cell replication will prevent the growth response (Rosenblatt and Parry, 1992). Recent evidence suggests that subpopulations of satellite cells may exist that can be distinguished by their proliferative potential (Perry and Rudnicki, 2000; Seale and Rudnicki, 2000). There is a reserve population of quiescent, non-differentiated cells that retains its mitogenic potential and has the capacity for self-renewal. Under the appropriate stimulation, these satellite cells become activated, migrate as necessary, and undergo a limited number of replications before they terminally differentiate. These cells can no longer divide and undergo fusion into the myofibre. In the rat, satellite cells comprise approximately 32% of muscle nuclei at birth and decrease to 10% at 4 weeks of age and less than 5% at sexual maturity when the cells are largely mitotically quiescent (Allbrook et al., 1971). A similar pattern is seen in the pig, in which satellite cells constitute approximately 20% of total muscle nuclei at birth, and 4% at 64 weeks of age (Campion et al., 1981; Mesires and Doumit, 2002). These values vary according to the metabolic properties of the muscle. The significance of myonuclear number in the context of muscle growth relates to the observation that in skeletal muscle, “myonuclear domain” size, i.e. the quantity of cytoplasm regulated by a single myonucleus (and reflected by the protein:DNA ratio), is tightly regulated (Allen et al., 1999). This implies that the amount of protein that can be deposited without further addition of

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myonuclei is limited. Nuclear domain size under “steady state” conditions appears to vary according to the metabolic activity of the fibre. It is smaller for oxidative than glycolytic fibres, and for any given fibre type, values increase with age. Although satellite cells become quiescent as growth rate plateaus, their proliferation can be reactivated in response to muscle injury, denervation, or increased muscle stretch and it is essential for muscle repair and hypertrophy (Bischoff, 1994). The close similarity between the developmental changes in satellite cell replication and protein synthesis strongly suggests that these processes may be linked. This is further supported by the differential response of immature and mature skeletal muscles to suboptimal nutrient intakes. Alterations in food intake (greater or less than average) in the neonatal animal, provided they are not severe, alter DNA and protein accretion proportionally as indicated by the maintenance of relatively normal, age-appropriate protein:DNA ratios despite a wide range of growth rates (Fiorotto and Davis, 1997). In transgenic animals, sustained over-expression of IGF-I in the skeletal muscle promotes satellite cell replication and transiently increases the accretion of total muscle DNA; this increase precedes the enhanced accumulation of muscle protein and results in protein:DNA ratios that temporarily are lower than normal (Fiorotto et al., 2003). These ultimately increase to age-appropriate values, but never surpass those in wild-type control animals. A potential link between satellite cell replication and the capacity for protein synthesis is through the regulation of ribosomal production, ribosomal abundance being the primary determinant of a cell’s maximal capacity for protein synthesis. Regulation of ribosome biogenesis is achieved in the majority of cells by altering the rate of rRNA synthesis by rDNA transcription (Zahradka et al., 1991), the regulation of which is coupled to cell cycling via the retinoblastoma gene product, pRb (Hannan et al., 2000). We have demonstrated that the enhanced replicative capacity of satellite cells from muscles that overexpress IGF-I is associated with increased phosphorylation of pRb upon mitogen stimulation (Chakravarthy et al., 2001). Thus, when rates of satellite cell division are high, pRB is phosphorylated, and in this form it enables a key transcription factor for rDNA transcription, UBF, to transactivate rDNA genes to promote rRNA synthesis. Thus, accretion of ribosomes is necessarily correlated to the rate of cell division. 3.2. Role of protein synthesis in the regulation of muscle growth A rapid increase in the absolute rate of growth occurs during early postnatal life and a majority of this growth is comprised of skeletal muscle protein (Young, 1970). The more rapid accretion of muscle protein than other tissue proteins results in an increase in the proportion of the body protein pool that is represented by muscle protein from ~30% in the newborn to ~50% in the adult (fig. 2). However, the fractional rate of growth, i.e. the amount of weight gained in relation to the existing mass, is extremely high at birth and decreases with development, with the most rapid change in the fractional rate of growth occurring during the neonatal period. This developmental decline in the fractional rate of growth is largely explained by a developmental decline in the fractional rate of protein deposition in skeletal muscle (Shields et al., 1983; Mitchell et al., 2001). Changes in the rate of protein deposition are driven by changes in the rates of protein synthesis or protein degradation such that a decline in the fractional rate of protein deposition can be due to a decline in the fractional rate of protein synthesis, an increase in the fractional rate of protein degradation, or both. The developmental decline in protein deposition in skeletal muscle is due to a developmental decline in the fractional rate of muscle protein synthesis

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Fig. 2. Relative changes in the proportion of whole-body protein mass attributable to skeletal muscle in the rat between birth and weaning (Fiorotto et al., unpublished observations).

(Kelly et al., 1984; Denne and Kalhan, 1987; Davis et al., 1989) (fig. 3). In fact, the fractional rate of muscle protein synthesis in the pig and rat is about 3-fold higher in the newborn than at weaning, and the rate of decline is attenuated as development proceeds (Kelly et al., 1984; Davis et al., 1989, 1996; Baillie and Garlick, 1992; Fiorotto et al., 2000a). This developmental decline in skeletal muscle protein synthesis is more profound in muscles containing predominately FG fibres than in those containing primarily SO fibres (Davis et al., 1989). By contrast, fractional protein degradation rates in skeletal muscle decline modestly with development. The rate of protein synthesis is determined by the abundance of ribosomes, the efficiency of the translational process, and potentially, the concentration of translatable mRNA (Kimball and Jefferson, 1988). Because the majority of RNA in tissues is rRNA, ribosomal abundance can be estimated from the RNA to protein ratio, or can be measured more precisely from the amount of 18S rRNA expressed per unit protein. The efficiency of the translation process can

Fig. 3. Relationship between the postnatal decline in the rate of muscle protein accretion and the fractional synthesis rate of skeletal muscle proteins in the hindlimbs of rats. (Data compiled from Davis et al., 1989; Fiorotto et al., 2000a.)

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be calculated from the amount of protein synthesized per unit RNA and reflects how well the protein synthetic machinery is functioning. Chronic changes in protein synthesis are thought to be a result of a change in ribosome number. Thus, the high rate of protein synthesis in immature muscle and its overall decline with development are driven largely by an elevated number of ribosomes at birth and a developmental decline in ribosome concentration as the musculature matures (Kelly et al., 1984; Davis et al., 2001). Rapid changes in the rate of protein synthesis, including those due to food ingestion, are generally regulated by changes in the efficiency of translation process secondary to modulation of the rate of translation initiation (Harmon et al., 1984; Kimball and Jefferson, 1988; Kimball et al., 1994), the rate-limiting step in protein synthesis. One of the best characterized steps involved in the regulation of translation initiation, depicted in fig. 4, is the binding of initiator methionyl-tRNA (met-tRNA) to the 40S ribosomal subunit to form the 43S preinitiation complex via mediation of eukaryotic initiation factor (eIF) 2 (Pain, 1996; Kimball et al., 1997; Webb and Proud, 1997). The eIF2-mediated met-tRNA binding to the 40S subunit is further regulated by the activity of eIF2B, which exchanges GDP for GTP on eIF2 (Kimball et al., 1996). A second well-characterized step in translation initiation, shown in fig. 4, is the binding of mRNA to the 43S preinitiation complex via mediation of the assembly of the eIF4F complex of proteins (Lin et al., 1994; Rhoads et al., 1994; Sonenberg, 1994). The three proteins comprising the eIF4F complex are eIF4A, an RNA helicase, eIF4E, the protein that binds to the m7GTP cap structure at the 5′-end of the mRNA, and eIF4G, a scaffolding protein that binds to the 40S ribosomal subunit. Thus, mRNA binds to the 40S ribosomal subunit through the association of eIF4E with eIF4G. The availability of eIF4E for binding to eIF4G is regulated by its association with 4E-BP1, a repressor protein that competes with eIF4G for binding to eIF4E (Pause et al., 1994). Upon stimulation by an anabolic agent, such as insulin, 4E-BP1 becomes phosphorylated, resulting in reduced affinity of eIF4E for 4E-BP1, release of eIF4E, and enhanced binding of eIF4E to eIF4G to form the active eIF4E:eIF4G complex (Gingras et al., 1999). Activation of translation initiation is mediated through a signal transduction pathway involving a protein kinase referred to as the mammalian target of rapamycin (mTOR) which,

Fig. 4. Regulation of translation initiation. Abbreviations: eIF, eukaryotic initiation factor; 4EBP1, eIF4E binding protein; Met-tRNA, initiator methionyl tRNA; S6K, 70 kDa ribosomal protein S6 kinase 1; 43S, 43S ribosomal subunit; 48S, 48S ribosomal subunit.

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in addition to phosphorylating 4E-BP1, also phosphorylates and activates the 70 kDa ribosomal protein S6 kinase, S6K1 (Jefferies et al., 1994; von Manteuffel et al., 1997). These phosphorylation events lead to an increase in the rate at which most proteins are synthesized and, in addition, the preferential increase in translation of mRNAs encoding elements of the translational apparatus, including ribosomal proteins and elongation factors. Recent studies in growing pigs suggest that the overall developmental decline in the response of skeletal muscle protein synthesis to feeding involves regulation by eIF2B (Davis et al., 2000). Availability of eIF4E for 48S ribosomal complex formation follows a similar pattern. This response is primarily modulated by the developmental change in the feedinginduced activation of the factors involved in the binding of mRNA to the 43S preinitiation complex. 3.3. Role of protein degradation in the regulation of muscle growth Less is known about the mechanisms that regulate protein degradation than those that regulate protein synthesis. It is known that there are multiple pathways in mammalian tissues for the degradation of proteins and that these pathways are highly controlled and selectively degrade specific protein substrates. These pathways include the lysosomal–autophagic system, the calpain–calpastatin system, and the ubiquitin–proteasome system (Goll et al., 1989; Attaix et al., 1999). The lysosomal–autophagic systems involve primarily cathepsins. Most evidence suggests that this pathway of degradation is unselective and may be of special importance under conditions in which cellular proteolysis is maximally activated. The calpain–calpastatin system is the major calcium-activated pathway of protein degradation. At least two main calpain isoforms, μ− calpain and m-calpain, have been identified and the system is subject to inhibition by the protein, calpastatin. The proteases play an important role in muscle myofibrillar protein turnover by catalysing initial disruption of the structure via proteolysis at the Z-disc. Released myofilaments can then be degraded into amino acids by the proteasome and/or lysosomal enzymes (Goll et al., 1992). The ubiquitin–proteasome pathway is widely distributed among tissues and has a relatively broad protein specificity. It consists of a recognition system involving the protein ubiquitin, which is responsible for targeting the protein substrates towards degradation by forming a polyubiquitin complex, and a multifunctional protease, referred to as the proteasome, which degrades the proteins. The role of these proteolytic pathways in the regulation of muscle growth and development remains to be explored.

4. REGULATORS OF PROTEIN SYNTHESIS 4.1. Feeding Dietary protein is utilized very efficiently for the deposition of whole-body protein during early postnatal life (Pellett and Kaba, 1972; McCracken et al., 1980; Fiorotto et al., 1991; Davis et al., 1993a). The accumulated evidence suggests that young animals utilize their dietary amino acids more efficiently for growth because they are capable of a greater increase in muscle protein synthesis in response to feeding than older animals (Davis et al., 1991, 1996). Feeding stimulates protein synthesis in the whole body of the newborn human (Denne et al., 1991); in skeletal muscle of the suckling lamb (Oddy et al., 1987; Wester et al., 2000); and in skeletal muscle of the post-weaned, but still growing, rat (Garlick et al., 1983). However, the stimulation of muscle protein synthesis by feeding is blunted or absent in adult mammals (Melville et al., 1989; Baillie and Garlick, 1992; Tessari et al., 1996).

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Stimulation of protein synthesis by feeding decreases with development in neonatal pigs.

In the suckling pig (Davis et al., 1996, 1997; Burrin et al., 1997a) and rat (Davis et al., 1991, 1993b), protein synthesis in skeletal muscle is maximally stimulated after eating. Figure 5 shows that the postprandial rise in protein synthesis in skeletal muscle of the neonatal pig declines sharply during the first 4 weeks of life. Although feeding stimulates protein synthesis in all tissues of the neonatal animal, the magnitude and the developmental decline in the response to feeding are most pronounced in skeletal muscle (Burrin et al., 1991, 1995, 1997a; Davis et al., 1991, 1993b, 1996). This enhanced ability of skeletal muscle protein synthesis to respond to the provision of nutrients in young growing animals should not be surprising, because the rate of protein deposition during the postprandial period must be higher than the rate of protein loss during the postabsorptive period to permit growth of skeletal muscle. Recent studies have examined the developmental changes in the expression and activation of factors that regulate the feeding-induced stimulation of protein synthesis in skeletal muscle of young, growing pigs. The results show that eIF2B activity, which regulates the binding of met-tRNA to the 40S ribosomal subunit, is unaffected by feeding but decreases with development. The stimulation of muscle protein synthesis by feeding, and the developmental decline in this response, involve regulation by the eIF4F complex (Davis et al., 2000; Kimball et al., 2002). In skeletal muscle of the neonatal pig, feeding increases the phosphorylation of 4E-BP1, resulting in dissociation of the inactive 4E-BP1 . eIF4E complex, and increased association of the active eIF4E . eIF4G complex. This response leads to a global increase in the rate of muscle protein synthesis. These feeding-induced changes in the activity of factors that regulate eIF4F formation decrease with development in parallel with the developmental change in the feeding-induced stimulation of muscle protein synthesis. A response to feeding has been observed in “teenage” rats (Yoshizawa et al., 1997). However, the magnitude of the response is smaller than that in neonatal pigs, thus further supporting a developmental decline in the feeding-induced formation of the eIF4F complex. The developmental changes in the feeding-induced eIF4F activation occur in parallel with increased phosphorylation of S6K1, which is involved in the translation of mRNAs encoding specific proteins that regulate translation initiation (Davis et al., 2000; Kimball et al., 2002). An increased phosphorylation of both 4E-BP1 and S6K1 suggests involvement of the mTOR signalling pathway in this process. Furthermore, rapamycin, a specific inhibitor of mTOR,

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strongly attenuates the feeding-induced assembly of both eIF4F and S6K1 activation (Kimball et al., 2000). Thus, the enhanced activation of the eIF4F complex following food consumption likely plays an important role in the postprandial stimulation of muscle protein synthesis in growing animals and the efficient use of dietary amino acids for muscle protein deposition in the neonate. 4.2. Insulin Studies performed in incubated muscles and in perfused hindlimbs of growing animals clearly demonstrate that insulin stimulates protein synthesis (Jefferson et al., 1977; Davis et al., 1987; Kimball et al., 1994). The infusion of physiological concentrations of insulin in fasted, weaned rats stimulates muscle protein synthesis in vivo to rates similar to those found in the fed state (Garlick et al., 1983). This response to feeding can be blocked by co-administration of anti-insulin serum (Preedy and Garlick, 1986). Furthermore, insulin has been shown to stimulate whole-body amino acid utilization and protein synthesis in the fetal sheep (Liechty et al., 1992; Thureen et al., 2000), protein synthesis in hindlimb of the young lamb (Wester et al., 2000), and skeletal muscle protein synthesis in the weaned rat (Garlick et al., 1983). In marked contrast to studies conducted in growing animals, most studies in adult animals (Baillie and Garlick, 1992; McNulty et al., 1993) and humans (Gelfand et al., 1987; Heslin et al., 1992; Louard et al., 1992) show little, if any, response of muscle protein synthesis to physiological increases in insulin. This suggests that the response of muscle protein synthesis to insulin is developmentally regulated. Insulin plays a key role in the increased response of skeletal muscle protein synthesis to feeding, and thus the increased rate of protein deposition, during the early postnatal period. In fasted and fed neonatal pigs, there is a positive curvilinear relationship between the postprandial increase in fractional muscle protein synthesis rates and circulating insulin concentrations (Davis et al., 1997). Studies using a hyperinsulinemic–euglycemic–euaminoacidemic clamp technique show that when amino acids and glucose are maintained at fasting levels, insulin infusion increases amino acid disposal, and that the insulin sensitivity and responsiveness of amino acid disposal decrease with development (Wray-Cahen et al., 1997). This response suggests that the developmental change in the insulin sensitivity of whole-body amino acid disposal may underlie the developmental change in the efficiency of utilization of dietary amino acids for protein deposition. Furthermore, raising insulin concentrations in the neonatal pig to levels typical of the fed state increases the rate of skeletal muscle protein synthesis to within the range normally present in the fed state, even when amino acids and glucose are maintained at fasting levels (Wray-Cahen et al., 1998). This response to insulin, like the response to feeding, is attenuated with development and is greater in muscles that are composed primarily of FG fibres, and is not specific to myofibrillar proteins (Davis et al., 2001). The insulin signalling cascade (fig. 6) leading to the stimulation of protein synthesis is initiated by insulin binding to its receptor. This leads to autophosphorylation of the receptor, the activation of insulin receptor tyrosine kinase, and the subsequent phosphorylation of several cytosolic substrates including insulin receptor substrate (IRS)-1 and -2 (Sun et al., 1991; White and Kahn, 1994). IRS-1 and -2 serve as “docking proteins”, transmitting insulin signals to several proteins that contain Src-homology 2 (SH2) domains (Backer et al., 1992; Sun et al., 1993) including phosphatidylinositol (PI) 3-kinase, which catalyses the phosphorylation of PI. The activation of PI 3-kinase triggers the activation of components of the insulin signalling pathway leading to translation initiation, i.e. protein kinase B (Akt) and mTOR.

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Fig. 6.

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Insulin signalling pathway leading to translation initiation.

Studies focusing on the developmental changes in the insulin signalling pathway that leads to translation initiation have shown that in the pig the abundance of insulin receptor protein in muscle during the early suckling period is 2-fold higher than at weaning (Suryawan et al., 2001). Although the abundance of IRS-1 and IRS-2 does not change with development, the abundance of the downstream signalling proteins, protein kinase B and mTOR, decreases with development (Kimball et al., 2002). This developmental decline in the abundance of insulin receptor, protein kinase B, and mTOR in skeletal muscle likely contributes to the overall decline in the responsiveness of muscle protein synthesis to feeding that occurs over the course of development. Because insulin mediates the postprandial elevation in skeletal muscle protein synthesis and this response decreases with development (Wray-Cahen et al., 1998; Davis et al., 2001), it is not surprising that the feeding-induced activation of the insulin signalling pathway that regulates protein synthesis decreases with development. Thus, the feeding-induced activation of the insulin receptor, IRS-1, IRS-2, PI 3-kinase, and protein kinase B in skeletal muscle decreases with development (Suryawan et al., 2001; Kimball et al., 2002), in parallel with the developmental decline in the feeding-induced activation of translation initiation factors and protein synthesis (Davis et al., 1996). This suggests that the developmental decline in the postprandial stimulation of protein synthesis in skeletal muscle results from a reduction in the capacity of the intracellular insulin signalling pathway to transduce to the translational apparatus the stimulus provided by the feeding-induced rise in insulin and/or amino acid concentrations. A number of studies performed in cell culture, in the perfused hindlimb, and in intact growing rats have demonstrated that the stimulation of protein synthesis by insulin involves increased phosphorylation of the translational repressor protein, 4E-BP1, reduced interaction with eIF4E, and increased assembly of the mRNA cap-binding complex, eIF4G:eIF4E (Kimball et al., 1994, 1997). Furthermore, insulin increases phosphorylation of S6K1, thereby increasing the translation of specific proteins involved in the regulation of translation. Recent in vivo studies performed in neonatal pigs support these findings and further show that the insulin-induced

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changes in factors regulating translation initiation as well as the upstream components of the insulin signalling pathway occur in a dose-response manner within the physiological range (Suryawan et al., 2001; O’Connor et al., 2003). Recently, however, studies in adult rats suggest that while insulin increases the phosphorylation of S6K1, insulin does not alter 4E-BP1 phosphorylation (Long et al., 2000). This lack of effect of insulin on 4E-BP1 phosphorylation and, by inference, eIF4F formation, is not surprising as physiological hyperinsulinaemia has no effect on muscle protein synthesis in adults. Thus, insulin plays an important role in the regulation of protein synthesis in muscle of growing animals, but its importance during adulthood is less apparent. 4.3. Amino acids Although amino acids are the precursors for the synthesis of proteins, they also play a key role as nutritional signals in the regulation of muscle protein synthesis. Amino acids have the capability to stimulate muscle protein synthesis throughout a substantial part of the life cycle, in contrast to the developmental decline and loss of the capability of insulin to stimulate muscle protein synthesis with age. In weaned but still growing rats (Preedy and Garlick, 1986), adult humans and rats (Bennet et al., 1990; McNulty et al., 1993; Vary et al., 1999), and elderly people (Volpi et al., 1998), acute amino acid infusion, either alone or concurrent with insulin infusion, stimulates protein synthesis in skeletal muscle. Recent studies suggest, however, that the magnitude of the stimulation of muscle protein synthesis by amino acids may decrease in the early postnatal period (Davis et al., 2002a). When a balanced amino acid mixture is infused into fasted, growing pigs, muscle protein synthesis increases and this response to amino acid infusion decreases with development, in parallel with the developmental decline in the feeding-induced stimulation of skeletal muscle protein synthesis (Davis et al., 2002a). In young pigs, the stimulation of skeletal muscle protein synthesis by amino acids is greater in muscles that contain predominately FG muscle fibres than in those that contain primarily SO fibres, and is similar for myofibrillar and sarcoplasmic proteins. The response to amino acid infusion occurs when insulin levels either remain at the fasting level or are raised to the fed level by infusion (O’Connor et al., 2003). Indeed, the magnitude of the increase in muscle protein synthesis with amino acid stimulation is similar to that which occurs with insulin stimulation alone, implying that insulin and amino acids may be interacting with the same signalling pathway within skeletal muscle. Studies performed in cell culture have shown that amino acid availability modulates protein synthesis by regulating both the met-tRNA and mRNA binding steps of translation initiation (Fox et al., 1998; Hara et al., 1998; Kimball et al., 1998; Patti et al., 1998; Jefferson and Kimball, 2001). In vivo studies in mature, food-deprived rats in which a large oral dose of leucine was administered suggest that in muscle, leucine promotes the binding of eIF4G to eIF4E, increases the phosphorylation of 4E-BP1, and represses the association of eIF4E with 4E-BP1 (Anthony et al., 2000). In neonatal pigs, raising amino acids from the fasting to the fed levels in the presence of insulin produced a similar response (O’Connor et al., 2003). In the absence of insulin, amino acids do not affect either the phosphorylation of S6K1 and 4E-BP1, or the association of eIF4E with 4E-BP1 and eIF4G, even though they stimulate muscle protein synthesis. This suggests that amino acids stimulate muscle protein synthesis in growing animals by modulating the availability of eIF4E for 48S ribosomal complex formation, and by processes that do not require enhanced assembly of the mRNA cap-binding complex.

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4.4. Insulin-like growth factors Many (Douglas et al., 1991; Fryburg et al., 1995; Bark et al., 1998; Vary et al., 2000; Davis et al., 2002b), although not all (Oddy and Owens, 1996; Boyle et al., 1998), studies have demonstrated an anabolic effect of IGF-I on protein synthesis in skeletal muscle. However, in some studies reductions in circulating concentrations of amino acids, insulin, and/or glucose during the administration of IGF-I may have limited the ability of IGF-I to stimulate protein synthesis. Thus, when amino acids, glucose, and insulin are maintained at fasting levels, infusion of IGF-I to the level seen in the fed state stimulates muscle protein synthesis in growing swine (Davis et al., 2002b). IGF-I, however, is unlikely to play a role in the feeding-induced stimulation of muscle protein synthesis. First, in contrast to insulin, the rise in circulating IGF-I after feeding is not immediate (Buonomo and Baile, 1991; Goldstein et al., 1991; Davis et al., 1993b, 1996; Svanberg et al., 1996). Second, the postprandial changes in muscle protein synthesis in young animals are positively correlated with changes in circulating insulin, but not IGF-I, concentrations (Davis et al., 1997, 1998). Third, with development, circulating IGF-I levels increase, whereas skeletal muscle protein synthesis rates decrease (Davis et al., 1996). Although circulating IGF-I is unlikely to be a physiologically significant regulator of the feeding-induced stimulation of skeletal muscle protein synthesis, this does not negate the potential role of IGF-I as a long-term regulator of growth, as has been suggested by others (Buonomo and Baile, 1991; Donovan et al., 1991; VandeHaar et al., 1991), or the potential usefulness of IGF-I as an anabolic agent to enhance protein deposition as discussed previously. IGF-I likely stimulates protein synthesis in skeletal muscle by acting on the same signalling pathway as insulin that leads to translation initiation (Dardevet et al., 1996; Vary et al., 2000). The receptors for both IGF-I and insulin share considerable homology of structure and function (Ullrich et al., 1986; Cheatham and Kahn, 1995; LeRoith et al., 1995) and both hormones act on some of the same intracellular signalling pathways (Dardevet et al., 1996; Suryawan et al., 2001). Furthermore, both insulin and IGF-I stimulate protein synthesis by increasing the formation of the active eIF4E . eIF4G complex that regulates the binding of mRNA to the ribosome (Kimball et al., 1997; Vary et al., 2000).

4.5. Growth hormone Growth hormone treatment increases protein deposition, improves nitrogen retention, and enhances the efficiency with which dietary protein is utilized for growth (Campbell et al., 1990; Caperna et al., 1991; Vann et al., 2000a). Furthermore, GH treatment profoundly decreases the synthesis and excretion of urea, and the oxidation of amino acids. Whole-body protein balance is improved in response to GH treatment due to the minimization of protein loss during fasting, and maximization of protein gain during meal absorption (Vann et al., 2000b). GH treatment in GH-deficient (Bier, 1991; Russell-Jones et al., 1998) and normal, mature animals and adult humans (Eisemann et al., 1989; Pell et al., 1990; Fryburg et al., 1991; Bell et al., 1998) increases protein deposition by stimulating whole-body and skeletal muscle protein synthesis. Chronic GH treatment in cattle and swine increases amino acid uptake by the hindquarter (Boisclair et al., 1994; Bush et al., 2003a) and protein synthesis in muscle (Eisemann et al., 1989; Seve et al., 1993) with no change in protein degradation across the hindlimb (Bush et al., 2003a).

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In young, growing swine, GH treatment increases skeletal muscle protein synthesis in the postprandial state, but not in the fasting condition. This increase is due to modulation of translational efficiency by GH and not by ribosome number (Bush et al., 2003b). The GH-induced increase in translation initiation is attributable to modulation of the factors associated with the binding of both mRNA and met-tRNA to the ribosomal complex, that is, the phosphorylation of 4E-BP1, association of eIF4E with eIF4G, and eIF2B activity. Because GH increases circulating IGF-I and insulin concentrations and this increase is greater in the fed than in the fasting state, the GH-induced increase in protein synthesis may involve mediation by IGF-I and/or insulin, or may be due to a direct effect of GH. In fact, GH indirectly activates some of the same signalling components as insulin and IGF-I, i.e. IRS-1 and -2, PI 3-kinase, protein kinase B, and S6K1 (Anderson, 1993; Yenush and White, 1997). In addition, the increased substrate availability, i.e. amino acids, provided in the fed condition may be permissive for the GH-induced increase in muscle protein synthesis. 4.6. Colostrum Colostrum provides a rich source of nutrients for the newborn mammal that supports the rapid growth and accretion of body protein during the first few days of postnatal life (Burrin et al., 1997b). In addition to nutrients, colostrum also contains maternal immunoglobulins that for many species are essential for passive immunity, and a variety of bioactive components that include insulin, IGF-I, IGF-II, and epidermal growth factor. Although the benefits of the consumption of nutrients and immune factors are readily apparent, the functional significance of the numerous hormones and growth factors present in colostrum is unclear. Studies that have compared the growth of newborns have demonstrated an enhanced anabolic response in association with the feeding of colostrum, especially of the visceral organs (Widdowson and Crabb, 1976; Widdowson et al., 1976). Given their mitogenic and anabolic properties, this response was often attributed to the presence of trophic factors in the colostrum. However, it must also be considered that the consumption of colostrum entails the ingestion of a larger quantity of nutrients than that typically provided by mature milk or, indeed, many formulas. Studies designed to distinguish between the trophic effects of macronutrient intake and those due to factors in colostrum (Burrin et al., 1995; Fiorotto et al., 2000b) showed that in newborn pigs, feeding stimulates protein synthesis in all tissues, but the stimulation of protein synthesis in skeletal muscle is greater when colostrum, as opposed to a nutrient-matched formula or mature sow’s milk, is fed. This suggests that the enhanced stimulation of skeletal muscle protein synthesis in newborn pigs fed colostrum, as opposed to other feeds, is not due solely to the provision of macronutrients. Furthermore, the stimulation of protein synthesis by colostrum feeding was restricted specifically to the myofibrillar proteins, unlike the general stimulation of protein synthesis by feeding which incurred a proportional stimulation of the synthesis of both sarcoplasmic and myofibrillar proteins (Fiorotto et al., 2000b). Feeding also resulted in a general increase in muscle mRNA concentration, but in the colostrum-fed piglets the enhanced synthesis rate of myofibrillar proteins was associated with a disproportionate increase in the abundance of myofibrillar mRNA, as exemplified by total MHC mRNAs. Additionally, colostrum augmented the effect of feeding on protein synthesis by promoting a greater accretion of ribosomes. Thus, feeding colostrum has both quantitative consequences for the anabolic process in the skeletal musculature of the newborn animal and qualitative consequences, with potential implications for the development of muscle function. Improvement of skeletal muscle

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function is advantageous insofar as it is critical for the development of the newborn’s ability to survive independently from its mother. The effects observed are likely attributable to nonnutritive factors present in colostrum, although these have not yet been identified. However, a number of potential factors, including insulin, IGF-I, thyroid hormone, and growth hormone, have been excluded. Identification of the mechanisms underlying this phenomenon will be critical for advancing our understanding of the biological role of early mammary secretions in the regulation of neonatal growth and in establishing how diet contributes to the regulation of skeletal muscle growth in early postnatal life.

5. FUTURE PERSPECTIVES There are numerous issues concerning the regulation of skeletal muscle growth and metabolism that need to be explored further. From the point of view of agriculture, the relative significance of these is determined by the economic benefits to be gained. The ultimate aim is to enhance feed efficiency. This, however, needs to be accomplished without compromising meat quality, especially tenderness and fat content. However, it is becoming increasingly evident that consumers are becoming more resistant to the use by the livestock industry of anabolic agents, growth promoters, and antibiotics, and frequently are prepared to pay a premium for products in which they have not been used. Although one may question the validity of these concerns, it must be acknowledged that they are widespread and, therefore, should not be ignored. In this regard, the application of genomics and proteomics to select for breeding stock with desirable traits, and improved husbandry practices to reduce mortality and morbidity in the birth to weaning period, are likely to be the most productive approaches. Given the large increase in fish consumption, research on the growth and composition of muscle of different fish varieties deserves substantially more attention. Enhancement of muscle growth can be accomplished either by increasing the number of myofibres, or by promoting myofibre hypertrophy. As should be evident, the former is a prenatal event and is dictated by maternal and genetic factors. Thus, continued research on the regulation of cell cycle progression and withdrawal of individual myoblast lineages, as well as the factors that control terminal differentiation, are likely to yield relevant information, especially when this can be merged with genomic trait analysis. Because mechanistic studies are difficult to conduct in vivo, in normal animals, much of the basic research on these mechanisms must be performed in cell and tissue culture. However, the widespread use of genetically engineered mice has been most productive and helpful in this regard because, although far removed from livestock animals, transgenic mice provide an important and apposite tool with which to assess the relevance of specific cellular events in the context of the whole animal. “The Myostatin Knockout Story” presents an excellent example of the usefulness of this approach. For some mammalian species, the ability to increase muscle fibre number has limitations, however, because the enhanced fetal growth may increase maternal morbidity and/or compromise maternal lactational capacity. In principle, therefore, enhanced postnatal muscle hypertrophy would be preferable. As we have presented, postnatal hypertrophy is dictated by two key factors, satellite cell number and protein accretion. Satellite cell number is the balance between the continued division of these cells acquired during the third phase of myoblast determination, and their loss, either by terminal differentiation and fusion into the myofibre, or by apoptosis. Although substantial progress has been made in understanding the factors that regulate satellite cell division, there are still many unanswered questions. Satellite cells have their origins in the latter part of fetal life and, therefore, are likely to be influenced

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by maternal variables; however, relatively little is known about the influence of maternal physiology and metabolism on the satellite cells of the progeny. The factors, especially environmental factors, that dictate terminal differentiation and apoptosis of satellite cells, and the extent to which these processes can be manipulated through husbandry practices and diet, are much less clearly understood, and warrant a closer examination as they are likely to have long-term consequences. More recently it is has been demonstrated that under certain in vitro conditions, satellite cells can change lineage and form adipocytes. Clearly this has significant consequences not only for overall muscle growth potential, but also for the composition of meat. The extent to which this occurs in vivo, and the conditions that would favour such a change, merit further attention. The rate of protein accretion is the balance between protein synthesis and degradation. We have demonstrated that in young animals the rate of protein synthesis is the principal regulatory factor. This unique feature is attributable to the ability of the immature muscle to markedly increase translation when food is available. The latter is critical because it enables amino acids to be diverted towards protein synthesis rather than to be oxidized. Thus, dietary protein can be used with greater efficiency, provided the composition of amino acids and energy intake are optimal. Thus, from the nutritional standpoint, the ability to meter the amino acid composition of dietary protein to meet the needs for growth versus maintenance during development can enable maximal exploitation of this high synthetic capacity of the immature muscle. The characteristics of the immature muscle that enable this synthetic response are primarily its high ribosomal content and an enhanced sensitivity and responsiveness of muscle protein synthesis to insulin. Clearly, therefore, further understanding of those factors that are responsible for these unique features of the immature muscle, and their down-regulation with maturation, would be warranted. Moreover, it is equally important that the impact of environmental variables such as infection, temperature, activity (duration, type, and intensity), and dietary nutrients other than protein and energy (e.g. micronutrients, modified lipids, and various non-nutritive factors present in foodstuffs) on protein synthesis during this anabolic phase of growth be investigated. Our emphasis on protein synthesis rather than degradation does not negate the importance of the latter in the regulation of protein accretion. Indeed, the regulation of protein degradation potentially represents a much more energetically efficient approach for improving the efficiency of muscle protein deposition (Goll et al., 1989) beyond the early postnatal period. However, much less is understood about the in vivo regulation of protein degradation, especially the factors that regulate myofibrillar breakdown, and the variability in these mechanisms between muscles and among different species. In addition to the consequences for protein deposition, protein degradation has consequences for meat quality because those enzymes that are responsible for the degradation of muscle myofibrillar proteins are also important determinants of post-mortem meat tenderisation. The evidence would suggest that in domestic animals muscle hypertrophy resulting from suppression of protein degradation in vivo can compromise meat tenderness. Examples of this negative consequence of suppressing protein degradation is the callipyge lamb in which the degree of hypertrophy of certain muscles is positively correlated to calpastatin expression, and increased toughness. The effects of some β-adrenergic agonists in certain species is similar to the effect of the callipyge gene. Thus, a clear understanding of the interplay between the structural characteristics of a muscle, the relative contribution of protein synthesis versus degradation to its overall growth, the variation among species, and how these aspects of muscle structure and metabolism are influenced by environmental factors and husbandry practices, are subjects that merit further study.

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Recently there has been much effort refocused on skeletal muscle metabolism in humans with the recognition that there is an inevitable depletion of skeletal muscle with ageing (sarcopenia). The consequence of this loss is quite severe as it results in a loss of strength, flexibility, and overall mobility, which thereby compromises the individual’s quality of life. The resulting decrease in activity not only exacerbates the muscle loss, but also decreases basal and activity-related energy expenditure, which therefore enhances the propensity for excessive fat deposition and glucose intolerance. The causes of sarcopenia appear to be extensive and include the loss in the replicative capacity of satellite cells, age-related increases in factors that are antagonistic to muscle growth, such as myostatin and Id factors, and a loss in the body’s capacity to produce anabolic agents such as growth hormone and testosterone. The relative importance of these, however, is far from clear. Additionally, or possibly in consequence to these changes, skeletal muscle loses its regenerative capacity with ageing. As the average life expectancy of humans increases, understanding the causes of sarcopenia and the development of therapies and modalities to mitigate its occurrence has enormous economic implications. Importantly, from the metabolic perspective, a better appreciation of the nutrient needs and dietary regimens that are required to sustain optimal muscle metabolism are warranted.

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Whole animal and tissue proteolysis in growing animals V. E. Baracos Department of Oncology, University of Alberta, 11560 University Avenue, Edmonton, Alberta, Canada T6G 1Z2

While it is convenient to conceptualize protein synthesis as being associated with growth and protein degradation as being associated only with atrophy or senescence, both processes proceed continuously in all tissues at all stages of life. Since the highest rates of protein degradation occur during the most rapid growth, protein deposition is inefficient. Our current understanding of proteolytic processes comes in large part from studies of skeletal muscle, including methods and approaches for its determination, contributing proteases and regulators, physiological controls, and post-mortem proteolytic events contributing to meat quality. Because of the contribution of protein catabolism to deposition of marketable muscle tissue, to its energetic cost, and to product quality, there is interest in different strategies increasing or modulating the rate of animal growth, especially the relative rates of protein synthesis and catabolism in skeletal muscle.

1. PROTEIN DEGRADATION: A KEY DETERMINANT OF GROWTH, METABOLIC RATE, AND GROWTH EFFICIENCY This review covers a variety of topics pertaining to protein degradation and regulation of growth in animals. Numerous excellent reviews are cited, and I have not attempted to cover in detail domains for which recent synthesis articles are available. Not all of the work relevant to the topic of growth and protein degradation has been conducted in domestic animal species. One of the reasons for this is that there are many difficulties associated with measures of protein degradation and these approaches are generally more difficult to implement in domestic animals because of their cost, invasiveness, and need for the use of stable or radioactive isotopes and related analytical equipment not routinely available in Animal Science Departments, such as isotope ratio mass spectrometry (Patterson et al., 1997). The reader is thus strongly encouraged to be open-minded to the broader scope of the protein degradation literature, in all species. A fundamental concept in metabolism is that all proteins are in a continual state of turnover. While protein synthesis is associated with growth and protein degradation is a dominant feature of atrophy or senescence, both processes proceed continuously. In fact the highest rates

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of protein degradation occur during the most rapid growth and protein deposition. This means that a large fraction of proteins synthesized in any period of growth are broken back down. The amount of protein formed and broken down again over any period of time is dependent on developmental stage, species, and organ. For example, protein breakdown rates in individual tissues of animals and poultry undergoing high rates of growth vary from <10%/day to >80%/day. This means that in a matter of 10 days, an animal at this stage will have broken down and resynthesized a slowly turning over tissue once and a rapidly turning over tissue eight times! The vast majority of animal protein formed, thus never goes to market. The results of Lapierre et al. (1999) further illustrate the impact of protein degradation across different growth rates. These authors evaluated whole-body protein metabolism in relation to intake (0.6, 1.0, and 1.6 × maintenance requirements) in growing beef steers. Protein retention in the whole body increased with intake, as a result of a greater increase in protein synthesis compared with protein degradation. Protein breakdown had a major impact, as 65% of the protein synthesized was degraded when intake varied from 1.0 to 1.6 times maintenance. Protein degradation is sometimes determined on a tissue- or organ-specific basis (i.e. Biolo et al., 1994; Samuels and Baracos, 1996; Zhang et al., 1996a,b; Lapierre et al., 1999), although relatively few tissue-specific determinations have been done in domestic species. Where this has been looked at, there is considerable emphasis on the skeletal muscles, since this organ is the main product of meat animal agriculture. Splanchnic tissues drained by the portal and hepatic veins are organ sites amenable to determination of protein degradation by tracer techniques (i.e. Lapierre et al., 1999). These organ systems are also a considerable focus, since because of their high turnover rates they constitute a major fraction of whole-body catabolism. In spite of its relatively small size, the liver in a growing monogastric can comprise 25% of whole-body protein catabolism. In growing cattle, the total splanchnic tissues inclusive of the liver accounted for 44% of whole-body turnover (25% from the portal-drained viscera and 19% from the liver) (Lapierre et al., 1999). There is increased protein synthesis in gut epithelium of cattle in response to feeding (Kelly et al., 1995), and this has implications for energy expenditure. When the degradation of protein is considered, the production of animal protein seems startlingly inefficient. This apparently wasteful metabolism, however, performs essential functions. Protein turnover is metabolically costly but is thought to convey flexibility in protein and amino acid metabolism. Protein breakdown is a particular feature of remodelling, such as during involution or metamorphosis when entire structures or organs are removed or replaced. Protein breakdown provides a means to be able to make a rapid change in the metabolic mass of any protein or group of proteins. Proteins not needed, non-functional, or damaged may be rapidly removed by activating their catabolism. Proteins may be rapidly induced, by activation of their synthesis and simultaneous suppression of their catabolism. Amino acids can be mobilized by degradation of existing proteins, and used for the synthesis of other proteins or other purposes such as gluconeogenesis. This is an essential function, which is capable of providing a continuous source of essential and non-essential amino acids, in a fashion independent of dietary intake. Continuous turnover of proteins incurs a considerable cost in ATP (Mitch and Goldberg, 1996). This cost in ATP may be largely associated with the energetic costs of protein synthesis; however, it is now recognized that at least some elements of the process of protein degradation also require ATP (Mitch and Goldberg, 1996). Energy economy could be improved by reducing flow through cyclical metabolic pathways that use ATP, such as protein turnover (Gill et al., 1989). Because of the contribution of protein catabolism to deposition of marketable muscle tissue and to its energetic cost, there is interest in different strategies increasing or modulating the rate of animal growth, especially the relative rates of protein synthesis and catabolism in skeletal muscle.

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2. MECHANISMS OF DEGRADATION: PROTEOLYTIC SYSTEMS Intracellular proteolytic systems are extensively characterized in a wide variety of cells, tissues, and species, including domestic livestock. The intracellular proteolytic systems of muscle that degrade the myofibrillar proteins are now well characterized, and are the main focus of this section. Three intracellular proteolytic systems are known to have the potential to contribute to myofibrillar protein degradation: the lysosomal system, the calcium-dependent proteolytic system (Tan et al., 1988), and the ATP-ubiquitin–proteasome-dependent proteolytic system (Attaix et al., 1998). There is a low level of lysosomal proteolytic activity in skeletal muscle and its overall contribution to catabolism in this tissue is small, except in the case of tissue injury (Farges et al., 2002). Cytosolic Ca2+-activated proteases (calpains) and their inhibitors have been extensively studied in muscle of domestic animals, because of their putative relationship with postmortem proteolysis and hence of meat quality (see below). The calpains constitute a large family comprising ubiquitous, tissue-specific, and atypical calpains (reviewed by Sorimachi et al., 1997; Kinbara et al., 1998). The calpains are cysteine proteases with a Ca2+ requirement for activation, in either the millimolar or micromolar concentration range. There are also atypical calpains, such as p94 (also called calpain 3), a mammalian calpain homologue predominantly expressed in skeletal muscle, which has been shown to be responsible for a form of limb-girdle muscular dystrophy. Calpastatin is a specific inhibitor of the calpains and the isolation of this protein from animal species such as cattle, cloning of its complementary DNA, and nucleotide sequencing have been completed (Killefer and Koomaraie, 1994). The contribution of this system to overall muscle protein breakdown in vivo is difficult to estimate; however, in incubated muscles inhibition of this system decreases protein degradation by less than 10% in most studies. Although some authors suggest that calpains are rate-limiting for release of filaments from the myofibrillar superstructure, if this were rate-limiting for myofibrillar proteins to be degraded, inhibition of this system would be expected to block all myofibrillar proteolysis and this is clearly not so (Attaix et al., 1998, 2001). Muscle protein catabolism appears primarily mediated by the ATP-dependent ubiquitin– proteasome system, which is responsible for degrading the bulk of intracellular proteins including myofibrillar proteins. This conclusion is based on the use of specific proteasome inhibitors, which are able to block upwards of 60% of total myofibrillar protein catabolism (Attaix et al., 1998, 2001, 2002). This has been well established in a wide range of animal models (reviewed by Mitch and Goldberg, 1996; Attaix et al., 2002). The ubiquitination/ deubiquitination system is a complex machine responsible for the specific tagging and proofreading of substrates degraded by the proteasome. Polyubiquitination of substrates targets them for degradation by the proteasome, a multiprotein complex conserved from archaebacteria to humans. Ubiquitin is an evolutionarily highly conserved 76-amino-acid polypeptide that is abundant in all eukaryotic cells. The initial step in the ubiquitin pathway is ATP-dependent and involves the linkage of ubiquitin to a ubiquitin-activating enzyme, or E1, in a high-energy thioester bond. Ubiquitin is then transferred in a second thioester linkage to a ubiquitinconjugating enzyme, which in turn catalyses the transfer of ubiquitin to the substrate protein in a covalent bond. In some cases, substrate polyubiquitination requires another enzyme, the ubiquitin ligase (Bodine et al., 2001; Gomes et al., 2001). The ubiquitin ligase can participate in the hierarchic transfer of ubiquitin into the substrate, or can function as an adaptor to facilitate positioning and transfer of ubiquitin from the ubiquitin-conjugating enzyme directly onto the substrate. The substrates tagged by ubiquitin are then recognized by the proteasome and degraded into peptides. How this proteolytic pathway degrades muscle proteins, and

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more particularly contractile proteins, remains largely unknown. Information on the ubiquitinconjugating enzymes and ubiquitin ligases that operate in muscle is still scarce. Similarly, neither the signals that target myofibrillar proteins for breakdown nor the precise substrates of the pathway have been identified. Finally, the possible relationships between the ubiquitin proteasome pathway and the lysosomal cathepsins and calpains are not well understood. The matrix metalloproteinases (MMP) represent a family of enzymes responsible for connective tissue catabolism. Extensive studies in a variety of tissues suggest that the regulation of MMP activities is complex. MMP are secreted in a latent form as zymogens and activated sequentially in a cascade initiated by other proteases including plasmin or membrane-type MMP (MT-MMP). A third level of regulation involves local production of polypeptide tissue inhibitors of metalloproteinases. The matrix metalloproteinase system involved in intramuscular connective tissue degradation has effectively just been described (Balcerzak et al., 2001). The genetic and physiological modulation of this system is barely characterized.

3. DETERMINATION OF PROTEIN CATABOLISM: WHOLE BODY, TISSUES, INDIVIDUAL PROTEINS Measurement of protein catabolism is technically and conceptually difficult. It is not the intent of this chapter to cover all of the methodological considerations; however, it is important to understand thoroughly the inherent limitations of the method used, in the interpretation of any given set of results. The clearest picture is based on multiple independent approaches giving the same overall conclusion. A physiologically relevant alteration in rates of protein catabolism may be small, and a major problem is the size of these changes relative to the error term of the measurement. 3.1. Degradation by difference: protein synthesis + net protein accretion (loss) It should be noted at the outset that if the experimental system can be shown to be in a steady state (i.e. no net protein accretion or loss), rates of protein synthesis and degradation are by definition identical. In this case protein synthesis is a useful surrogate for measures of protein degradation. Under non-steady-state conditions, protein degradation may be estimated as the difference between protein synthesis and net protein gain or loss (i.e. Samuels and Baracos, 1995; Wheeler et al., 2000). Protein gain or loss over time is determined by serial slaughter of groups of animals on the experimental treatments and protein synthesis is determined in each group immediately before animals are killed, often using the “flooding dose” approach. Using this method, the degradative rates of many organs and tissues can be estimated. This method is applicable to small, inexpensive animals such as chicks or lambs and has been extensively used in laboratory rodents. Calculated degradation rates include the summed errors inherent in the directly measured variables, and may be considerable. 3.1.1. Urinary excretion of 3-methylhistidine Post-translationally modified amino acids released on protein catabolism are not re-incorporated into proteins and provide an index of catabolism of the proteins of which they are characteristic. Measurement of urinary 3-methylhistidine (3-MH) excretion is used to estimate myofibrillar protein breakdown. Similarly, urinary OH− proline reflects the appearance of this amino acid from the catabolism of connective tissue proteins, mainly collagen (Funaba et al., 1996). This approach requires quantitative collection of urine and is based on the

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assumption that no metabolism of 3-MH occurs once it is released from actin and myosin. This is true in most species, but in sheep and swine a proportion is retained in muscle as a dipeptide, balenine. In neither of these species does urinary 3-MH yield any data on protein breakdown. Rathmacher and Nissen (1998) proposed a compartmental model of 3-MH that is applicable in domestic animals and does not involve the collection of urine. In this approach, 3-MH metabolism in cattle, swine, and sheep was defined from a single bolus infusion of a stable isotope, 3-[methyl-2H3]-methylhistidine. Following the bolus dose of the stable isotope tracer, serial blood and urine samples are collected. At least three exponentials were required to describe the plasma decay curve adequately. A simple three-compartment model described the plasma kinetics of 3-[methyl-2H3]-MH/3-MH for cattle with one urinary exit from the plasma compartment. The de novo production of 3-MH as calculated by the compartmental model in cattle was not different when compared to total urinary 3-MH production. A plasmaurinary kinetic three-compartment model with two exits was used for sheep with a urinary exit out of the plasma compartment and a balenine exit out of a tissue compartment. A plasma three-compartment model was used in swine with an exit out of a tissue compartment. The kinetic parameters reflect the differences in known physiology of 3-MH metabolism of the respective species. Steady-state model calculations define masses and fluxes of 3-MH between three compartments and, importantly, the de novo production of 3-MH. 3.2. Isotopic tracer approaches for whole-body and tissue catabolism in vivo A primed, constant infusion of an isotopically labelled amino acid such as leucine or phenylalanine may be used to estimate whole protein degradation (i.e. Lapierre et al., 1999; Vann et al., 2000). Splanchnic tissues drained by the portal and hepatic veins (i.e. Lapierre et al., 1999) and the hindlimb drained by the femoral vein (i.e. Savary et al., 2001) are organ sites amenable to determination of protein degradation by techniques based on arterio-venous differences combined with radioactive or stable isotope tracers. Wolfe and co-workers have produced a steady stream of methodological advances in this area (Biolo et al., 1994; Ferrando et al., 1995; Zhang et al., 1996a,b; Patterson et al., 1997). Zhang et al. (1996a) developed an attractive method to measure the fractional breakdown rate of muscle protein. This method involves infusing labelled amino acid to reach an isotopic equilibrium and then observing its decay in the arterial blood and muscle intracellular pool. The calculation of fractional breakdown rate is based on the rate at which tracer released from breakdown dilutes the intracellular enrichment using a modified precursor-product equation. The measured fractional breakdown rates were in agreement with the results from the arteriovenous balance method. This provides a feasible approach for measurement of muscle protein catabolism. This method can be combined with the tracer incorporation method to measure both breakdown and synthesis in the same infusion study. One limitation of tracer/arterio-venous balance approaches is that the degradative rates of the individual organs and tissues within the organ system(s) within the studied vascular bed cannot be descriminated. While limb protein metabolism is often interpreted as equivalent to skeletal muscle protein metabolism, skin protein synthesis and degradation accounted for approximately 10−15% of the total leg protein kinetics in different species (Baracos et al., 1991; Biolo et al., 1994). 3.3. Protease gene expression Protein degradation is clearly regulated, at least to some extent, at the level of gene expression, and in a wide variety of physiological and pathological states expression of various

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elements of proteolytic systems varies with measured overall degradative rates. Regulation of protease gene expression in muscle has been the subject of several elegant studies and comprehensive review articles by Attaix and co-workers (1997, 1998, 2001, 2002; Larbaud et al., 2001). While assessment of gene expression is not a primary measure of degradation, this approach has applications where it is presently not possible to make direct determinations. A small amount of data on local protease gene expression is emerging in tissues of the gastrointestinal tract, which are suggestive of regulation of proteolysis at this level. For example, Samuels et al. (1996) measured mRNA levels for components of the lysosomal (cathepsins B and D), Ca2+-activated (m-calpain), and ubiquitin-dependent (ubiquitin, 14 kDa ubiquitinconjugating enzyme E2, and C8 and C9 proteasome subunits) proteolytic pathways, in the small intestine of rats during food deprivation. mRNA levels for most of these components increased during fasting, suggesting that a co-ordinated activation of multiple proteolytic systems contributed to intestinal protein wasting. Adegoke et al. (1999) tested the effects of a luminal infusion of an amino acid mixture on protease mRNA in jejeunal mucosa of piglets after overnight food deprivation. Amino acids acutely suppressed mucosal levels of mRNA encoding ubiquitin, 14 kDa ubiquitin-conjugating enzyme, and the C9 subunit of the proteasome by 20–30%, demonstrating the sensitivity of components of the ATP-ubiquitin proteolytic pathway to acute regulation by nutrients. 3.3.1.

In vitro techniques

Aside from poultry (i.e. Baracos et al., 1989), the in vitro incubation techniques developed by A.L. Goldberg and used widely in muscles of laboratory rodents are not applicable in domestic animal species. A major attribute of this system has been the ability to study the differential regulation of the lysosomal, Ca2+-dependent and ubiquitin/proteasome-dependent proteolytic pathways using inhibitors. (i.e. Larbaud et al., 2001).

4. PROTEIN DEGRADATION IN RELATION TO GENETIC MAKE-UP Protein turnover rates are subject to genetic variation, and differences in protein turnover may explain part of the inherent differences in efficiency and growth of different animal breeds (Reeds et al., 1998). For example, Wheeler et al. (2000) evaluated the effect of the callipyge phenotype in lambs on protein kinetics. These authors studied callipyge and normal lambs at 5, 8, and 11 weeks of age. The synthesis rates of proteins in various tissues were measured using a primed, continuous infusion of [2H5]phenylalanine. Rates of protein degradation were estimated by difference between protein synthesis and net protein accretion. Enhanced muscle growth seems to be maintained in callipyge lambs by reduced protein degradation. Consistent with this observation, Koohmaraie et al. (1995) reported that the activity of calpastatin is about 80% higher in the callipyge phenotype. Unpublished work from our group suggests that different breeds of cattle may exhibit characteristic differences in protein degradation. When animals were fed identical amounts of metabolizable energy and protein/kg BW·75, Brahman × Angus cross cattle showed a lower level of urinary 3-MH excretion (1.82 mg/d/kg BW·75) than Charolais (3.06 mg/d/kg BW·75) (SE = 0.25; P < 0.009). Lower protein degradation, together with a tendency towards lower metabolic rate, could be responsible for increased protein deposition and consequently a higher growth rate observed in the Brahman × Angus cattle.

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5. REGULATION OF DEGRADATION: ENDOCRINE AND AUTOCRINE CONTROLS Rates of protein degradation are precisely regulated and there are multiple sites of hormonal and metabolic controls. In general, circulating hormones have an anabolic or catabolic effect on a tissue, affecting rates of protein synthesis, degradation, or both. Tissues may be in a steady state, enter into a catabolic state (wasting) or grow, in response to a concert of hormones and factors in a given physiological or pathological state. Note that these factors are diverse and may be hormones, growth factors, substrates, and metabolites. Tissue-specific factors, such as contractile activity and stretch, also greatly influence muscle protein turnover. The list of factors affecting the process of protein catabolism is most fully understood for skeletal muscle (table 1). This symphony of signals act collectively to inform muscle protein catabolism in three information subsets: 1. Contractile activity. Two components of muscular activity, active contraction and passive stretch, are perceived as anabolic signals. Lack of activity and shortening of muscle result in activation of the degradation of contractile proteins. This regulation allows for the maintenance of a muscle mass appropriate to the level of work. 2. Nutritional status/glycemia. Since muscle protein comprises the principal gluconeogenic precursor, the need for muscle protein mobilization is conveyed through the factors

Table 1 Regulatory factors in muscle protein synthesis and degradation Synthesis Factors related to level of contractile work Contractile activity Stretch Disuse (inactivity) Gonadal steroids Factors related to nutritional status/glycemia Insulin Insulin-like growth factor I Growth hormone Glucose Ketone bodies Glutamine Branched-chain amino acids Glucagon Glucocorticoids β-Adrenergic agonists Thyroid hormones (normal) Thyroid (excess) Factors related to the presence of injury/inflammation Prostaglandin E2 Prostaglandin F2α Interleukin-1β Interleukin-6 Tumor necrosis factor α Interferon γ

Degradation

Overall promotes

↑ ↑ ↓ ↑

↓ → ↑ ↓

Protein deposition Protein deposition Atrophy Protein deposition

↑ ↑ ↑ → → ↑ ↑ ↓ → ↑ ↑ ↑

↓ ↓ → ↓ ↓ ↓ ↓ → ↑ ↓ ↑ ↑↑

Protein deposition Protein deposition Protein deposition Protein deposition Protein deposition Protein deposition Protein deposition Atrophy Atrophy Protein deposition Protein deposition Atrophy

→ ↑ ↓ ↓ ↓ ↓

↑ → ↑ ↑ ↑ ↑

Atrophy Protein deposition Atrophy Atrophy Atrophy Atrophy

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regulating glycemia and gluconeogenesis. Since muscle protein comprises a reserve of amino acids and protein in case of food deprivation, muscle catabolism is sensitive to indices of food intake, which indicate a state of plenty where growth may occur, or a state of fasting or nutritional deprivation when net catabolism is required. 3. Stress, infection, and injury. Disease or injury impose additional metabolic demands when food intake may also be low or zero. Factors arising in the context of the stress response and immune and inflammatory responses, such as cytokines and prostaglandins, communicate to muscle the need for extra protein catabolism (Castaneda, 2002). All of the above factors are simultaneously at play. When muscle is catabolized, several metabolic changes (reduced food intake, impaired mobility, and perturbations in the production or responsiveness of catabolic and anabolic hormones, cytokines, and/or proteolysis-inducing factors) act in concert. In the context of this complex regulatory system, it is often difficult to identify the primary and secondary factors, and a complete understanding of their interactions remains to be developed.

6. PROTEIN DEGRADATION, NUTRITIONAL STATUS, AND GROWTH 6.1. Feeding and diet The rapid loss of skeletal muscle protein during acute starvation occurs primarily through increased rates of protein breakdown and activation of the ubiquitin–proteasome-dependent proteolytic process (Wing et al., 1995). The levels of ubiquitin-conjugated proteins increased 50−250% after food deprivation in various muscles. Like rates of proteolysis, the amount of ubiquitin–protein conjugates and the fraction of ubiquitin conjugated to proteins increased progressively during food deprivation and returned to normal within 1 day of refeeding. Larbaud et al. (1996) showed that euglycemic hyperinsulinemia and hyperaminoacidemia decrease skeletal muscle ubiquitin mRNA in goats, suggesting insulin and amino acids as possible mediators of this effect. Restricted feeding during growth constitutes a stress to energy metabolism, and energy utilization is curtailed in this circumstance by reduction in growth. In sheep and cattle, protein turnover is positively related to plane of nutrition (Lobley et al., 1992; Reecy et al., 1996). The turnover of actomyosin, the major myofibril constituent, is modulated in animals tested on various planes of nutrition. Lobley et al. (2000) demonstrated that both fractional degradation rate and fractional synthesis rate were lower in muscles of steers with a low growth rate (1 kg/d) on restricted feeding in comparison with high growth rate (1.4 kg/d). This and other studies (i.e. Boisclair et al., 1993) emphasized myofibrillar turnover and muscle; however, the connective tissue protein catabolism is similarly affected. Lambs with a low growth rate present less active matrix metalloproteinase-2, suggesting a decrease in collagen catabolism (Sylvestre et al., 2002). Other studies suggested lower collagen turnover and reduced deposition of neo-synthesized, immature, and non-cross-linked collagen related with a low growth rate (Aberle et al., 1981; Crouse et al., 1985; Miller et al., 1987; McCormick, 1994). The quality of dietary protein has an impact on protein degradation. Branched-chain amino acids appear to have a specific regulatory effect on protein degradation and decrease the rate of this process (Ferrando et al., 1995). Diets with protein of inferior quality may increase protein breakdown in skeletal muscle (Lohrke et al., 2001). These authors studied the activation of skeletal muscle protein breakdown in pigs fed isoenergetic and isonitrogenous diets based on soy protein isolate compared with casein.

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6.2. Role of the somatotrophic axis The somatotrophic axis plays a key role in the co-ordination of protein metabolism during postnatal growth (reviewed by Breier, 1999). Acute growth hormone treatment improves the partitioning of nutrients by increasing protein synthesis and decreasing protein degradation. Short-term infusion of IGF-1 also reduces whole-body protein breakdown and increases protein synthesis, and Boyle et al. (1998) have also shown this to be true in fetal sheep during late gestation. More recently, Vann et al. (2000) suggest that growth hormone increases protein balance by lowering body protein degradation in fed, growing pigs. Pair-fed, weightmatched growing swine were treated with porcine growth hormone (150 μg/kg/d) or vehicle for a week. Growth hormone treatment increased the efficiency with which the diet was used for growth, but did not alter protein synthesis in skeletal muscles, liver, or jejunum. In the absence of any changes in protein synthesis at these sites, the results suggest that in the fed state, growth hormone treatment of growing swine increases protein deposition primarily through a suppression of protein degradation.

6.3. Stress Domestic animals are exposed to behavioural, environmental, and infectious stressors. One of the metabolic hallmarks of these stresses is the catabolic response in skeletal muscle, mainly reflecting increased protein breakdown, in particular myofibrillar protein breakdown (reviewed by Hasselgren, 2002). Among different intracellular proteolytic pathways, the energy-ubiquitin-dependent pathway is particularly important for the regulation of muscle protein breakdown during acute infection. The gene expression of ubiquitin-conjugating enzyme E214k, ubiquitin ligase E3α, and several components of the proteasome is up-regulated and the activity of the proteasome is increased in muscle during infection. An increased understanding of the molecular regulation of muscle wasting in stress may help in the future to mitigate or prevent catabolic losses in animal production.

7. PROMOTION OF THE EFFICIENCY AND RATE OF GROWTH BY MANIPULATION OF PROTEIN DEGRADATION It is clearly important that proteins must be broken down; however, the physiological range of protein catabolism rates is quite broad. It is of particular interest that some breeds of animals and physiological situations are associated with low breakdown rates. The breeds of animals with lower rates of protein degradation appear to be those breeds adapted to harsh environments and a low availability or quality of forage. Animal selection and breeding based upon efficiency of protein deposition may potentially be used to develop this trait. It has been known for about a decade that β-adrenergic agonists act in part through suppression of protein degradation (Bardsley et al., 1992; Parr et al., 1992; Mills, 2002). β-Adrenergic agonists are structurally similar to the catecholamines epinephrine and norepinephrine and bind with high affinity to β-adrenergic receptors in adipose and muscle tissue. This class of compound includes agents such as clenbuterol, cimaterol, and ractopamine. Ractopamine was the first β-adrenergic receptor ligand to be cleared for use in pigs in the USA, about 4 years ago. Ractopamine consistently increases muscle protein accretion in pigs and while the mechanism responsible for increased protein accretion is not clear, cumulative evidence points to a direct effect, possibly on both protein synthesis and degradation. One reason why the role of protein catabolism is not entirely clear is that while β-agonists cause

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large differences in protein accretion, these are manifest over long periods of time, and the marginal difference in degradation rates required to result in these changes would be quite small. β-Agonist treatment does cause large increases in protease inhibitor activity and gene expression, and these changes are suggestive. For example, cimaterol treatment of Friesian steers (Parr et al., 1992) caused significant increases in muscle mass (+37%) and calpastatin specific activity (+76%). Total RNA was unchanged, but there was a 96% overall increase in calpastatin mRNA in muscle from treated animals. If protein breakdown could be minimized, this would be an attractive way of promoting protein deposition as well as a means of lowering the metabolic cost of maintaining any given protein mass. Thus protein degradation would be an attractive target for growth promotants, which should properly be called “anticatabolic” rather than “anabolic” factors. As we obtain more details regarding the proteolytic processes and their regulation, the possibility of identifying molecular targets for anticatabolic agents seems tangible. A likely site of such targets would be the ubiquitin–proteasome system and in particular the recently identified class of muscle-specific ubiquitin ligases (Bodine et al., 2001). Two unique ubiquitin ligases, MuRF1, a RING finger protein, and MAFbx (Bodine et al., 2001), also called Atrogin-1 (Gomes et al., 2001), of the SCF family, have been reported to play a role in muscle atrophy. Unlike other known ubiquitin ligases found in many tissues, these enzymes appear to be expressed mainly in muscle cells, especially skeletal muscle. Multiple ubiquitin ligases may operate in skeletal muscle, possibly to connect protein catabolism to different classes of external stimuli. This is suggested by the reported findings (Bodine et al., 2001) that null mutation of either MAFbx or MuRF1 in mice led to resistance to denervation-induced muscle atrophy. The ubiquitin–proteasome system appears to be central in muscle protein degradation, regardless of the humoral signal for the system’s activation. The intracellular signal transduction from multiple factors converges upon a common proteolytic pathway, of which ubiquitin ligases are likely to be a critical element. Further studies are required to better understand the importance of the ubiquitin ligase family, including identifying the physiological substrates for these enzymes in skeletal muscle, elucidating signalling events that regulate their activity, and analysing the effects of specific inhibition through gene ablation and/or the design of selective small molecule inhibitors. Ubiquitin ligases may be attractive molecular targets for manipulation of proteolysis since there are isoforms specific to muscle. These features may potentially allow for local suppression of muscle catabolism without affecting the basal proteolytic processes in non-muscle tissues or associated with essential functions.

8. PROTEIN DEGRADATION AND POST-MORTEM PROTEOLYSIS Catabolic processes are modified, but not interrupted, at death. While the activity of ATPdependent processes (and proteases) would cease, the vast majority of proteolytic enzymes can continue to express activity. This autolysis is prefaced by the pre-mortem level of proteolytic activities, but thereafter evolves in a manner dissimilar to in vivo events, because of changes in tissue temperature, pH, and the loss of structural integrity. The identity of all of the active enzymes and the substrates to which they have access is only partly understood (Ho et al., 1994). It has long been believed that proteases play a key role in post-mortem tenderization of meat (reviewed by Koohmaraie, 1992), and this concept is supported by the observation that low levels of pre-mortem proteolysis are associated with reduced degradation during meat maturation. Tissue growth made to be more efficient by reducing proteolytic activity

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(i.e. β-adrenergic agonist-induced muscle hypertrophy) manifested lowered rates of postmortem proteolysis. Koohmaraie et al. (1991) showed that the pattern of post-mortem proteolysis was altered by β-adrenergic agonists. In β-agonist-treated lambs, post-mortem storage was not associated with increased myofibril fragmentation index or degradation of desmin and troponin-T. These results indicate that the ability of the muscle to undergo postmortem proteolysis has been dramatically reduced with β-adrenergic agonist feeding. Similarly, enhanced muscle growth seems to be maintained in callipyge lambs by reduced protein degradation, and Koohmaraie et al. (1995) suggested a causal relationship between this effect and increased shear force in meat of calipyge lambs. The relationship between pre-mortem proteolysis, post-mortem proteolysis, and meat quality is far from being fully explored. This would be a worthy target for future experimentation, since an ability to modulate the structural integrity of tissue elements that confer toughness to meat would have considerable value.

9. FUTURE PERSPECTIVES A means of reducing protein degradation to its physiological minimum during growth holds the potential to increase the efficiency of animal production by a large factor. A means of activating protein degradation in the peri-mortem period holds the potential to increase the quality of meat. Both of these outcomes could have a large economic impact. Given the potential impact for animal growth and production, it is perhaps surprising that the research momentum on proteolysis is not emanating from the agricultural research community. A pharmaceutical industry strongly motivated to produce therapies for inappropriate degradation, muscle atrophy, and wasting syndromes (i.e. Bodine et al., 2001) is presently making large financial commitments in this area. Whatever the source, new developments in our understanding of proteolysis are showing the way towards targets for intervention in these processes, and animal agriculture may benefit greatly if it is poised to capture the relevant information.

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Lapierre, H., Bernier, J.F., Dubreuil, P., Reynolds, C.K., Farmer, C., Ouellet, D.R., Lobley, G.E., 1999. The effect of intake on protein metabolism across splanchnic tissues in growing beef steers. Brit. J. Nutr. 81, 457−466. Larbaud, D., Balage, M., Taillandier, D., Combaret, L., Grizard, J., Attaix, D., 2001. Differential regulation of the lysosomal, Ca2+-dependent and ubiquitin/proteasome-dependent proteolytic pathways in fast-twitch and slow-twitch rat muscle following hyperinsulinaemia. Clin. Sci. 101, 551−558. Larbaud, D., Debras, E., Taillandier, D., Samuels, S.E., Temparis, S., Champredon, C., Grizard, J., Attaix, D., 1996. Euglycemic hyperinsulinemia and hyperaminoacidemia decrease skeletal muscle ubiquitin mRNA in goats. Amer. J. Physiol. 271, E505−E512. Lobley, G.E., Harris, P.M., Skene, P.A., Brown, D., Milne, E., Calder, A.G., Anderson, S.E., Garlick, P.J., Nevison, I., Connell, A., 1992. Response in tissue protein-synthesis to submaintenance and supramaintenance intake in young growing sheep: comparison of large-dose and continuous-infusion techniques. Brit. J. Nutr. 68, 373−388. Lobley, G.E., Sinclair, K.D., Grant, C.M., Miller, L., Mantle, D., Calder, A.G., Warkup, C.C., Maltin, C.A., 2000. The effects of breed and level of nutrition on whole-body and muscle protein metabolism in pure-bred Aberdeen Angus and Charolais beef steers. Brit. J. Nutr. 84, 275−284. Lohrke, B., Saggau, E., Schadereit, R., Beyer, M., Bellmann, O., Kuhla, S., Hagemeister, H., 2001. Activation of skeletal muscle protein breakdown following consumption of soyabean protein in pigs. Brit. J. Nutr. 85, 447−457. McCormick, R.J., 1994. The flexibility of the collagen compartment of muscle. Meat Sci. 36, 79−91. Miller, M.F., Cross, H.R., Crouse, J.D., Jenkins, T.G., 1987. Effect of feed energy intake on collagen characteristics and muscle quality of mature cows. Meat Sci. 21, 287−294. Mills, S.E., 2002. Biological basis of the ractopamine response. J. Anim. Sci. 80, Suppl. 2, E28−E32. Mitch, W.E., Goldberg, A.L., 1996. Mechanisms of muscle wasting: the role of the ubiquitin-proteasome pathway. N. Engl. J. Med. 335, 1897−1905. Parr, T., Bardsley, R.G., Gilmour, R.S., Buttery, P.J., 1992. Changes in calpain and calpastatin mRNA induced by beta-adrenergic stimulation of bovine skeletal muscle. Eur. J. Biochem. 208, 333−339. Patterson, B.W., Zhang, X.J., Chen, Y., Klein, S., Wolfe, R.R., 1997. Measurement of very low stable isotope enrichments by gas chromatography/mass spectrometry: application to measurement of muscle protein synthesis. Metab. Clin. Exp. 46, 943−948. Rathmacher, J.A., Nissen, S.L., 1998. Development and application of a compartmental model of 3-methylhistidine metabolism in humans and domestic animals. Adv. Exp. Med. Biol. 445, 303−324. Reecy, J.M., Williams, J.E., Kerley, M.S., MacDonald, R.S., Thornton, W.H., Davis, J.L., 1996. The effect of postruminal amino acid flow on muscle cell proliferation and protein turnover. J. Anim. Sci. 74, 2158−2169. Reeds, P.J., Burrin, D.G., Davis, T.A., Stoll, B., 1998. Amino acid metabolism and the energetics of growth. Arch. Anim. Nutr. 51, 187−197. Samuels, S.E., Baracos, V.E., 1995. Tissue protein-turnover is altered during catch-up growth following Escherichia coli infection in weaning rats. J. Nutr. 125, 520−530. Samuels, S.E., Taillandier, D., Aurousseau, E., Cherel, Y., Le Maho, Y., Arnal, M., Attaix, D., 1996. Gastrointestinal tract protein synthesis and mRNA levels for proteolytic systems in adult fasted rats. Amer. J. Physiol. 271, E232−E238. Savary, I.C., Hoskin, S.O., Dennison, N., Lobley, G.E., 2001. Lysine metabolism across the hindquarters of sheep: effect of intake on transfers from plasma and red blood cells. Brit. J. Nutr. 85, 565−573. Sorimachi, H., Ishiura, S., Suzuki, K., 1997. Structure and physiological function of calpains (Review). Biochem. J. 328, 721−732. Sylvestre, M.N., Balcerzak, D., Feidt, C., Baracos, V.E., Brun-Bellut, J., 2002. Elevated rate of collagen solubilization and post-mortem degradation in muscles of lambs with high growth rates: possible relationship with activity of matrix metalloproteinases. J. Anim. Sci. 80, 1871–1878. Tan, F.C., Goll, D.E., Otsuka, Y., 1988. Some properties of the millimolar Ca2+-dependent proteinase from bovine cardiac muscle. J. Mol. Cell. Cardiol. 20, 983–997. Vann, R.C., Nguyen, H.V., Reeds, P.J., Burrin, D.G., Fiorotto, M.L., Steele, N.C., Deaver, D.R., Davis, T.A., 2000. Somatotropin increases protein balance by lowering body protein degradation in fed, growing pigs. Amer. J. Physiol. 278, E477−E783.

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Wheeler, T.L., Savell, J.W., Fiorotto, M.L., 2000. Protein kinetics in callipyge lambs. J. Anim. Sci. 78, 78−87. Wing, S.S., Haas, A.L., Goldberg, A.L., 1995. Increase in ubiquitin-protein conjugates concomitant with the increase in proteolysis in rat skeletal muscle during starvation and atrophy denervation. Biochem. J. 307, 639−645. Zhang, X.J., Chinkes, D.L., Sakurai, Y., Wolfe, R.R., 1996a. An isotopic method for measurement of muscle protein fractional breakdown rate in vivo. Amer. J. Physiol. 270, E759−E767. Zhang, X.J., Sakurai, Y., Wolfe, R.R., 1996b. An animal model for measurement of protein metabolism in the skin. Surgery 119, 326−332.

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Cytokine regulation of protein accretion in growing animals R. W. Johnson and J. Escobar Department of Animal Sciences, University of Illinois, Urbana, IL 61801, USA

Inflammatory cytokines secreted by activated leukocytes are the critical molecules that enable the immune system to influence disparate physiological systems that are important for determining protein accretion in growing animals. The inflammatory cytokines, interleukin-1, interleukin-6, and tumor necrosis factor α, reduce feed intake, interfere with the somatotropic axis, reduce skeletal muscle protein synthesis, and enhance skeletal muscle protein degradation. The purpose of this chapter is to discuss how the immune system regulates feed intake and arbitrates the balance between skeletal muscle protein synthesis and degradation, so as to provide a biological explanation for why sick animals do not grow well.

1. INTRODUCTION Skeletal muscle protein accretion is the net result of both protein synthesis and degradation. Both events occur constantly in normal skeletal muscle, but the mechanisms regulating protein synthesis and degradation are distinct and therefore can be influenced independently. It is now evident that the mechanisms that control skeletal muscle protein synthesis and degradation are subject to regulation by the immune system (Johnson, 1997). Infectious pathogens stimulate the immune system, and the immune system in turn actively suppresses feed intake and skeletal muscle protein accretion. The general notion is that nutrients that were allocated to support skeletal muscle protein accretion are reassigned to metabolic processes that support the immune system, which at the time is a higher biological priority (Klasing, 1988). This places the immune system at the interface of environmental pathogens and animal growth (Broussard et al., 2001). Inflammatory cytokines secreted by activated leukocytes are the critical molecules that enable the immune system to regulate feed intake and nutrient allocation. Because inflammatory cytokines reduce voluntary feed intake, and thus the nutrients available to support protein accretion, this issue will be briefly discussed. Feed intake alone, however, cannot account for the decreased protein accretion witnessed in sick animals because inflammatory cytokines also affect protein metabolism by several tissues, including skeletal muscle.

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Therefore, the effects of inflammatory cytokines on skeletal muscle protein synthesis will be discussed. Some attention will be given to the somatotropic axis because inflammatory cytokines regulate animals’ capacity to accrete skeletal muscle protein in part by reducing the amount of growth hormone (GH) and insulin-like growth factor-I (IGF-I) available to skeletal muscle and by reducing the sensitivity of receptors for GH-releasing hormone (GHRH), GH, and IGF-I. Because the collective actions of cytokines lead to inhibition of mRNA translation initiation – an obvious prerequisite for skeletal muscle protein accretion – this issue will be briefly covered. Finally, the inflammatory cytokines that inhibit protein synthesis concomitantly enhance skeletal muscle protein degradation. Therefore, the effects of infection and inflammatory cytokines on the ATP-ubiquitin-dependent, calcium-dependent (calpains), and lysosomal (cathepsins) proteolytic pathways will be discussed as well. The purpose of this chapter is to discuss how the immune system regulates feed intake and arbitrates the balance between skeletal muscle protein synthesis and degradation, so as to provide a biological explanation for why sick animals do not grow well.

2. CYTOKINES ORCHESTRATE ANIMALS’ RESPONSES TO INFECTIONS Agricultural animals live surrounded by pathogens and routinely become infected, but because of a well-developed defense system only occasionally do they show clinical signs of illness. Still, they are constantly challenged by pathogens and must contend with subclinical infections on a daily basis. This never-ending mêlée between the animal’s immune system and pathogens is costly because there is a negative relationship between animal productivity and the pathogenic environment: as pathogens in the environment increase, animal productivity decreases (fig. 1). The animal’s immune system “senses” (Blalock, 1984) the pathogenic environment and biological functions, including feed intake and growth, are adjusted accordingly. To appreciate how the pathogenic environment impinges upon skeletal muscle protein accretion, a superficial understanding of the animal’s primary and secondary defenses is obligatory. The body surfaces are made up of epithelial cells that provide a physical barrier between the internal milieu and the external pathogen-containing environment. Epithelial cells form the outer layer of skin and line gastrointestinal, respiratory, and genitourinary tracts. For infection to occur, primary pathogens must penetrate one of these barriers. Surface epithelia provide mechanical, chemical, and microbiological protection against infectious pathogens (table 1). The immune system provides a secondary defense that deals with organisms once they have entered the body proper. The innate immune response is the first secondary defense to be mounted. For example, complement and certain acute-phase proteins bind and help destroy some pathogens, and macrophages trap, engulf, and destroy others. Activated macrophages also secrete cytokines that cause inflammation, which among other things attracts other phagocytic cells (e.g. neutrophils and monocytes). These cytokines are collectively called inflammatory cytokines. They include interleukin-1α/β (IL-1), interleukin-6 (IL-6), and tumor

Fig. 1. As pathogens in the environment increase, animal productivity decreases.

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Table 1 Epithelial barriers to infection Mechanical Chemical

Microbiological

Epithelial cells joined by tight junctions Ciliated epithelial cells and mucin that trap and remove pathogens Bactericidal enzymes in saliva, sweat, tears, and gut Low pH in stomach Antibacterial peptides Normal flora in gastrointestinal tract produce antibacterial substances and compete against pathogenic microorganisms

necrosis factor α (TNFα). Virus-infected cells produce interferon (IFN) α and γ cytokines that interfere with viral replication. The interferons increase expression of major histocompability (MHC) class I molecules on the surface of virally infected cells, thereby flagging these infected cells for killing by cytotoxic T cells. They also activate natural killer (NK) cells that recognize and kill virally infected cells. Often this set of responses suffices to eliminate or at least contain the infection. If the infection cannot be contained it will spread to the lymphatic system where macrophages and other specialized antigen-presenting cells (e.g. dendritic cells) present the antigen to lymphocytes so they can initiate the second secondary defense – adaptive immunity. The adaptive immune system is more efficient at eliminating pathogens from the host body as compared to the innate immune system. When macrophages ingest and degrade pathogenic microorganisms they process antigen from the pathogen and present it to lymphocytes. This is a key step for proliferation of the B cells that in turn differentiate into plasma cells that will produce antigen-specific antibody as well as forming memory B cells that provide long-lasting immunity. This component of adaptive immunity is called humoral or antibody-mediated immunity. An antigen also stimulates T cells to form cytotoxic T cells and memory T cells. This component of adaptive immunity – cell-mediated immunity – is most effective against intracellular pathogens such as viruses. Antigen binds a specific receptor on a T cell, stimulating that cell to differentiate and proliferate. The result is formation of cytotoxic T cells and memory T cells with receptors appropriate for the subject antigen. Inflammatory cytokines have critical roles in orchestrating both innate and adaptive immune responses (table 2). For example, macrophages secrete IL-1β and TNFα to cause inflammation in order to facilitate movement of other effector cells to the infection site. IL-6 produced by macrophages stimulates hepatocytes to synthesize and secrete acute-phase proteins, which bind and help remove certain bacteria. IL-6 also stimulates B cells to differentiate into antibody-producing plasma cells. IFNα and γ inhibit virus replication and activate NK cells which hunt down and kill virally infected cells. And IL-1β stimulates T lymphocytes to express IL-2 and its receptor – a critical step for T-cell proliferation. A surprising finding in the late 1970s and early 1980s was that cytokines produced by activated leukocytes affect disparate physiological systems and orchestrate a systemic response that also helps protect the host animal. The systemic response initiated by inflammatory cytokines has profound effects on animal metabolism (table 2). How these cytokines might influence skeletal muscle protein accretion in young animals is discussed herein.

3. CYTOKINES INHIBIT ANIMAL GROWTH Animals with infections have reduced appetites, reduced growth rates, and convert feed to product in an inefficient manner. Indeed, feed intake and growth are usually inversely related

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Table 2 Immunological and metabolic effects of cytokines produced by macrophages Cytokine

Major immunological effects

Major metabolic effects

Interleukin-1

Inflammation Activates lymphocytes T-cell proliferation

Interleukin-6

Activates lymphocytes B-cell differentiation Antibody production Acute-phase protein synthesis Inflammation

Muscle protein degradation Reduced muscle protein synthesis Fever Anorexia Hypoferremia Hypozincemia Hypercupremia Muscle protein degradation Reduced muscle protein synthesis Fever Acute-phase protein synthesis Muscle protein degradation Reduced muscle protein synthesis Fever Anorexia Lipolysis Not generally considered to have significant metabolic effects

Tumor necrosis factor α

Interferon α/γ

Activates natural killer cells Inhibits virus replication

to the level of interaction between the host immune system and pathogens (fig. 1). This is why animals kept in poorly sanitized environments that afford a high degree of host–pathogen interaction eat less and grow more slowly than their counterparts kept in cleaner environments. This indicates that the immune system “senses” the pathogenic environment and interacts with the brain and other disparate physiological systems to regulate feed intake and growth. Initial studies showed that activation of the hypothalamic–pituitary–adrenal (HPA) axis, fever, and behavioral signs of illness (e.g. hypersomnia) could be induced by injecting animals with cell-free supernatants collected from activated leukocytes (reviewed by Hart, 1988). The biologically active molecule in the conditioned supernatants was subsequently determined to be endogenous pyrogen – a protein eventually renamed IL-1. Thus, the immune system conveys its message to other physiological systems via inflammatory cytokines. It is now dogma that inflammatory cytokines inhibit animal growth. Administration of inflammatory stimuli that increase circulating levels of TNFα, IL-1, and IL-6 (e.g. lipopolysaccharide, LPS), or injection of recombinant TNFα, IL-1, and IL-6, decrease skeletal muscle protein accretion. These cytokines can reduce skeletal muscle protein accretion in several ways. 3.1. Inflammatory cytokines decrease appetite A prolonged reduction in feed intake depletes protein and fat reserves. In AIDS and certain neoplastic diseases, loss of lean body mass is correlated with increased morbidity and mortality (Dewys et al., 1980; Delmore, 1997; Roubenoff, 2000). One thought is that decreased nutrient intake reduces growth rate, or in adult animals perpetuates loss of skeletal muscle mass, by limiting the supply of amino acids for protein synthesis. When decreased voluntary feed intake induced by an inflammatory challenge is accounted for by covariate analysis or by including a pair-fed control treatment, approximately 20–30% of the decreased growth can be attributed to a reduction in nutrient intake (Ballinger et al., 2000). The inflammatory

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cytokines produced by activated mononuclear phagocytic cells, IL-1, IL-6, and TNFα, reduce appetite and feed intake. The fact that IL-1, IL-6, and TNFα all reduce appetite illustrates an important characteristic of this group of cytokines – redundancy. Indeed, blocking any one or two of the three cytokines did not prevent LPS-induced anorexia (Swiergiel and Dunn, 1999). Only when all three were antagonized simultaneously was LPS-induced anorexia prevented (Swiergiel and Dunn, 1999). Still, based on dose-response studies, IL-1 appears to be most potent at reducing appetite. Recombinant IL-1 injected peripherally (i.v., i.p., or s.c.) reduces animals’ feed intake, similar to what occurs during an acute infection (Plata-Salaman et al., 1988). The decrease in feed intake is due to a decrease in meal frequency and size (Langhans et al., 1993). Cytokines can directly change the activity of hypothalamic neurons that mediate feed intake, or affect neurochemicals and neuropeptides that are implicated in the control of feed intake. For example, peripheral injection of IL-1 caused anorexia and increased the steady-state level of corticotropin-releasing hormone (CRH) mRNA in the hypothalamus (Suda et al., 1990). A CRH antagonist administered intracerebroventricularly (ICV) partially blocked IL-1β-induced anorexia (Uehara et al., 1989). IL-1 also decreases hypothalamic neuropeptide Y – a potent appetite-stimulating factor (Gayle et al., 1997). And LPS stimulates the release of α-melanocyte-stimulating hormone, which has been shown to enhance LPS-induced anorexia (Huang et al., 1999). 3.1.1. Reduced feed intake is an adaptive response to infection Many explanations for why sick animals reduce feed intake are based on teleology. For example, wild animals may expend considerable energy foraging or hunting for feed. Thus, feeding behavior would enhance heat loss and thwart the beneficial fever response, place weakened, vulnerable animals in harm’s way of predators, and perhaps facilitate disease transmission within a group. However, there is tangible evidence that the loss of appetite benefits sick animals, too. In one study, researchers experimentally infected mice with Listeria monocytogenes (LD50) and let some consume feed ad libitum, while others were intubated and force-fed to the level of free-feeding, noninfected controls (Murray and Murray, 1979). Mice allowed to consume feed ad libitum ate 58% of the controls and were much more likely to survive than those force-fed: nearly 100% of infected, force-fed mice died, whereas only about 50% of infected, ad libitum-fed mice died. Furthermore, there was a positive relationship between weight loss and survival for the infected mice with ad libitum access to feed. In some cases, survival appears to be positively related to anorexia and weight loss, provided it does not persist too long. In general, the behavioral and metabolic responses to acute infection are beneficial because they inhibit the pathogen and enhance animals’ immunological defenses (fig. 2). Animals seem to employ their nutritional wisdom and simply eat what they can use. In other words, feed intake in a growing animal might be determined by its capacity to accrete protein. When rats were injected with LPS or IL-1 and allowed to self-select between macronutrients during a 4 h meal period, they decreased total caloric intake by about 50% but ingested relatively less protein and more carbohydrate; relative fat intake was unchanged (Aubert et al., 1995). The fact that animals disproportionately reduced protein intake compared to other macronutrients during an inflammatory challenge suggests a shift in metabolic priorities and nutrient needs. Furthermore, increasing the diet concentration of limiting amino acids to account for decreased appetite of chicks and pigs under immunological stress is not effective for increasing whole-body protein accretion (Williams et al., 1997a,b,c; Webel et al., 1998). Cytokines apparently reduce the animal’s capacity to accrete protein and feed intake is adjusted accordingly.

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Fig. 2. Mononuclear phagocytic cells produce inflammatory cytokines when activated by pathogens. The cytokine molecules act in the brain to reorganize the animal’s behavioral priorities. The sickness behavior syndrome that results is an adaptive response that enhances the animal’s immunological defenses and inhibits proliferation of the pathogen. Thus, sickness behavior enhances disease resistance and promotes recovery (Johnson, 2002).

3.1.2. How do cytokines affect feed intake regulatory centers? Cytokines produced in the periphery can interact directly with central feed intake regulatory centers by entering the circulatory system and moving from the blood into the brain (fig. 3). Recombinant inflammatory cytokines administered directly into the brain via an indwelling ICV cannula, for example, induce anorexia, suggesting that cytokines act centrally to reduce feed intake (Plata-Salaman, 1988). Moreover infusing the IL-1 receptor antagonist ICV in order to block IL-1 receptors in the brain inhibited anorexia caused by inflammation in the periphery (Kent et al., 1992; McHugh et al., 1994). Because inflammatory cytokine proteins are 17–26 kD in size, they are ordinarily too large to diffuse passively from the blood, across the blood–brain barrier, into the brain. However, pathogens or cytokines might promote passive movement of cytokine from the blood into the brain by increasing the permeability of the blood–brain barrier (de Vries et al., 1996). There is also evidence that cytokines are actively transported from the blood into the brain (fig. 3). For example, Banks and colleagues injected radiolabeled cytokine (e.g. IL-1, IL-6, and TNFα) intravenously and were able to recover from the brain a portion of what was injected (Banks et al., 1991, 1994a,b; Gutierrez et al., 1993). The transport mechanism for each cytokine was saturable and the transport of radiolabeled cytokine could be competitively blocked by intravenous injection of unlabeled cytokine. Peripheral cytokines may also access the brain through circumventricular organs, which are devoid of blood–brain barrier (fig. 3). Here, peripherally produced cytokines diffuse into the brain or stimulate glial cells, causing them to produce inflammatory molecules (e.g. prostaglandins and cytokines), which diffuse into the brain. Based on extensive temporal and spatial mapping of Fos expression (a marker for neural activity) and IL-1, Konsman et al. (1999) proposed that cytokines produced in the periphery affect the brain according to the principles of volume transmission. In this model, IL-1 or other inflammatory mediators in the periphery induce IL-1 production in the choroid plexus and circumventricular organs. The cytokine then slowly diffuses into the brain by volume transmission, along the way activating neurons and neural pathways that result in anorexia (Konsman and Dantzer, 2001). Consistent with this hypothesis, inflammatory stimuli in the periphery (e.g. LPS and inflammatory

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Fig. 3. Cytokines produced in the periphery can convey a message to the brain in several ways. Peripheral cytokines may cross the blood–brain barrier by diffusion or active transport. In addition, peripheral cytokines may activate the vagus nerve, which in turn induces cells in the brain (e.g. microglia) to produce cytokines. Finally, peripheral cytokines may stimulate the release of hormones that are able to cross the blood–brain barrier. Adapted from Johnson (2002).

cytokines) induce de novo synthesis of IL-1, IL-6, and TNFα in the brain of the mouse and rat (Ban et al., 1992; Laye et al., 1994). For example, inflammatory stimuli in the periphery induce perivascular microglial cells to express cytokines (van Dam et al., 1992). Moreover, anorectic rats bearing prostate adenocarcinoma tumor cells had increased IL-1 mRNA in the cerebellum, cortex, and hypothalamus. Cytokines in the periphery can also convey a message to the brain via the vagus nerve (fig. 3). After i.p. LPS challenge, dendritic cells and macrophages that are closely associated with the abdominal vagus express IL-1 protein (Goehler et al., 1999). IL-1 binding sites are evident in several regions of the vagus as well (Goehler et al., 1997). When activated by peripheral cytokines the vagus can activate specific neural pathways that are involved in sickness behavior. Activation of the vagus also appears to stimulate microglia in the brain to produce cytokines. If the vagus nerve is severed just below the diaphragm in rats, the expression of cytokines in the brain and the sickness behavior that normally occurs after intraperitoneal injection of LPS is inhibited (Laye et al., 1995). Plasma levels of cytokines are elevated in LPS-injected vagotomized rats, indicating that the neural signal is needed for the induction of sickness. The neural signal may be necessary for the induction of cytokines in the brain, or may sensitize the brain to cytokines produced in the periphery. The neural pathways activated in the brain by the vagus nerve for rapid immune-to-brain signaling have been recently described in some detail (Dantzer, 2001a,b). These pathways appear to be responsible for activating the HPA axis and depressing behavior in response to infection. Cytokines originating in the periphery act on other peripheral targets as well, which in turn reduce appetite (fig. 3). For instance, leptin is a 16 kD protein secreted by adipocytes. By acting in the hypothalamus to reduce appetite and increase energy expenditure, leptin plays an important role in long-term energy balance. Mice have increased circulating levels of

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leptin when the immune system is stimulated with LPS (see Johnson and Finck, 2001). This effect of LPS is cytokine-dependent because mice with a mutated toll-like receptor 4 gene – a defect that prevents them from secreting cytokines in response to LPS – do not have increased leptin when challenged with LPS. However, when mice with the mutation are injected with recombinant TNFα, circulating leptin increases (Finck et al., 1998). TNFα acts directly on adipocytes via the p55 TNF receptor to induce expression of leptin (Finck and Johnson, 2000). It is reasonable to postulate that a cytokine-induced elevation in circulating leptin is involved in the cytokine-induced anorexia. Accordingly, the anorectic response to LPS is attenuated in mice lacking the leptin receptor (Faggioni et al., 1997). However, mice with a mutated leptin gene reduce feed intake similar to mice with a fully functional leptin gene after LPS injection (Faggioni et al., 1997), so this issue is not fully resolved. 3.2. Inflammatory cytokines decrease protein accretion in growing animals Inflammatory cytokines are pleiotropic molecules that can either increase or decrease protein synthesis, depending on the target tissue. In general, protein synthesis is decreased in skeletal muscle and is increased in liver, lung, and heart. In the liver, for example, inflammatory cytokines induce a marked increase in acute-phase protein synthesis. The weight of liver and total liver protein is increased in animals chronically infused with IL-1, TNFα, or the two cytokines together. However, animals infused with cytokines lose body weight because muscle protein accretion is reduced due to a decrease in protein synthesis and an increase in protein degradation. The liver represents roughly 3% of animals’ total body mass whereas skeletal muscle represents 40–45%. Thus, the net effect of increased circulating cytokines in a growing animal is a decrease in whole-body protein accretion. Administration of IL-1 and TNFα – alone or in combination – results in increased urinary nitrogen excretion and skeletal muscle catabolism accompanied by weight loss in rats (Flores et al., 1989; Ling et al., 1997). The effects of cytokines on protein kinetics are vastly different from those induced by fasting or feed restriction, where peripheral proteins are spared and visceral proteins are degraded. Chronic treatment with either TNFα or IL-1 results in a redistribution of body protein. Rats that were injected twice daily for 7 days with LPS, TNFα, or IL-1 lost a comparable amount of weight to respective pair-fed animals. However, the LPS and cytokine-treated animals had accelerated skeletal muscle protein degradation but preserved liver protein content, which was not the case for pair-fed animals (Fong et al., 1989). The decrease in skeletal muscle protein under inflammatory conditions is associated with decreases in steady-state levels of muscle mRNA for myofibrillar proteins myosin heavy chain, myosin light chain, actin, and in 18S and 28S subunits of ribosomal RNA. Similarly, the body weight of transgenic mice that overexpress IL-6 is comparable to that of wild-type controls at 16 weeks of age. However, the transgenic mice have reduced gastrocnemius muscle weights and suffer from severe muscle atrophy, which is prevented by treatment with an antagonistic anti-mouse IL-6 receptor antibody (Tsujinaka et al., 1996). Furthermore, implantation of a TNFα-secreting tumor in the hind leg muscles of nude mice led within 50 days to profound fat and protein loss (Tracey et al., 1990). In sepsis, protein synthesis and translational efficiency are reduced in gastrocnemius muscle, and prior treatment with TNF-binding protein (TNFBP) prevented these effects (Cooney et al., 1999). Prior treatment with IL-1 receptor antagonist also prevented the inhibitory effects of sepsis on protein synthesis (Vary et al., 1996). The release of one cytokine often initiates a cascade of cytokine synthesis and release. For example, animals challenged with E. coli or endotoxin and treated with TNFBP had reduced plasma levels of IL-1 and IL-6 (Roth et al., 1998; Solorzano et al., 1998).

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Consistent with these results, when pigs were challenged with LPS there was a marked increase in circulating TNFα and IL-6 that preceded a 3-fold increase in plasma urea nitrogen (PUN). Because pigs were fasted, the increase in PUN was interpreted to suggest an increase in skeletal muscle protein degradation (Webel et al., 1997). The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) preferentially infects and replicates within mononuclear phagocytic cells (i.e. macrophages). Mononuclear phagocytic cells infected by PRRSV produce copious amounts of inflammatory cytokines (van Reeth et al., 1999; van Reeth and Nauwynck, 2000). Whole-body protein accretion is markedly reduced in nursery pigs infected with the PRRSV. There is a high negative correlation between protein accretion and circulating IL-1 and IL-6 (Escobar et al., 2002). The influence of cytokines on protein synthesis and degradation seems to be dependent on skeletal muscle fiber type. Slow-twitch, or Type I muscle fibers are designed to work repetitively and generally use oxygen to fuel metabolic processes. Fast-twitch, or Type II muscle fibers contract at a high rate of speed and work well in the absence of oxygen. Vary and Kimball (1992) demonstrated that muscles consisting of fast-twitch fibers (gastrocnemius and psoas) were subject to breakdown during sepsis whereas protein kinetics was unaffected by sepsis in muscles consisting of slow-twitch fibers (soleus and heart). The specific effects of inflammatory cytokines on fast-twitch and slow-twitch fibers may be important in domestic food-producing animals. For example, the longissimus muscle in domestic pigs contains fewer slow-twitch fibers and more fast-twitch fibers than wild boars of the same age (EssenGustavsson and Lindholm, 1984). Pigs and chickens intended for meat production have been selected for maximal lean growth rate and increased breast-meat yield, respectively. Because pigs selected for maximal lean growth rate have a greater proportion of muscles containing fast-twitch vs slow-twitch muscles (Rahelic and Puac, 1981), and chickens selected for maximal breast-meat yield likewise have higher levels of fast-twitch fibers, the effects of cytokines on muscle tissue growth are potentially more deleterious in leaner, more modern genotypes. It appears that a portion of the amino acids released by skeletal muscle as a result of protein degradation are taken up by leukocytes to support cell proliferation and by the liver to support acute-phase protein synthesis (fig. 4). For example, the inflammatory cytokines, IL-1, IL-6, and TNFα, increase the rate of hepatic amino acid uptake (Argiles et al., 1989; Argiles and Lopez-Soriano, 1990) and protein synthesis (Klasing and Austic, 1984; Geiger et al., 1988; Ballmer et al., 1991). Reeds et al. (1994) proposed that a significant portion of nitrogen excreted during an inflammatory response was the result of excessive demands for the aromatic amino acids, phenylalanine, tyrosine, and tryptophan. Their conclusions were based on a comparison of the amino acid profiles of the major acute-phase proteins produced by humans and the amino acid profile of mixed muscle protein. Analysis of the acute-phase proteins indicated that four of the six proteins contained high levels of phenylalanine, five of the proteins were rich in tryptophan, and three contained high levels of tyrosine. By calculating the quantity of amino acids incorporated into a typical acute-phase protein mixture (850 mg/kg BW), they calculated that 1980 mg of muscle protein per kg body weight would need to be liberated to supply an adequate quantity of phenylalanine for the increased hepatic protein synthesis. The amino acids that are released in excess of the need for acute-phase protein production (1980–850 mg) are catabolized because they cannot be used for protein resynthesis due to the phenylalanine limitation, with the end result being an excessive excretion of nitrogen. Assuming that animals have a similar pattern and quantity of acute-phase proteins, it is apparent that an infectious insult could result in a substantial amount of skeletal muscle protein degradation and nitrogen excretion. For example, for a 100 kg pig there would

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Fig. 4. Inflammatory cytokines inhibit skeletal muscle protein synthesis and enhance its degradation. A portion of the freed amino acids (AA; e.g. glutamine) is taken up by leukocytes to support cell proliferation and by the liver to support acute-phase protein (APP) synthesis and other metabolic processes.

be roughly 200 g of protein broken down to supply amino acids for acute-phase protein synthesis, and approximately 13 g nitrogen would be excreted. Glutamine is another example of how amino acids are repartitioned by cytokines (fig. 4). Skeletal muscle is the major repository of glutamine. During infection, there is a 2-fold increase in glutamine release from skeletal muscle. Despite a significant increase in endogenous glutamine biosynthesis in skeletal muscle, intracellular glutamine becomes depleted. At the same time there is an 8- to 10-fold increase in hepatic glutamine uptake where it can be used for (1) biosynthesis of nonessential amino acids; (2) gluconeogensesis; (3) energy; and (4) biosynthesis of urea, which is ultimately excreted.

4. CYTOKINES AND MUSCLE PROTEIN SYNTHESIS AND DEGRADATION When protein degradation remains constant, decreased protein accretion can occur when substrates necessary for protein synthesis are limiting or when signals that promote protein synthesis are thwarted. When protein synthesis remains constant, decreased protein accretion can occur when signals that promote protein degradation are enhanced. Protein synthesis and degradation can be influenced independently, so protein accretion is most profoundly affected when there is a decrease in muscle protein synthesis and a concomitant increase in muscle protein degradation. If the amount of degradation exceeds that of synthesis, muscle wasting occurs. Inflammatory cytokines are uniquely qualified to manipulate protein accretion because they have the ability to simultaneously influence both protein synthesis and degradation. Thus, the immune system, by production of inflammatory cytokines, is able to adjust animal growth according to the level of immunological challenge. 4.1. Inflammatory cytokines inhibit skeletal muscle protein synthesis 4.1.1. Inflammatory cytokines inhibit the GH–IGF-I axis One way cytokines inhibit skeletal muscle protein synthesis is by adversely affecting the somatotropic axis (fig. 5). Growth hormone induces IGF-I secretion, a potent growth factor that is responsible for a wide range of anabolic processes. Insulin-like growth factor-I increases skeletal muscle mass by binding the type I IGF-I receptor and initiating a cascade of intracellular signaling events that ultimately initiate protein synthesis. For a detailed

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Fig. 5. Inflammatory cytokines can inhibit skeletal muscle protein synthesis by interfering with the secretion of growth hormone (GH) and insulin-like growth factor (IGF)-I. Cytokines also lead to GH and IGF-I receptor resistance.

description of IGF-I receptor signaling, the reader is referred to recent reviews (LeRoith, 2000; Broussard et al., 2001; Nakae et al., 2001). In short, the IGF-I receptor belongs to the family of tyrosine kinase receptors. It is composed of two ligand-binding extracellular α subunits and two transmembrane-spanning β subunits that have tyrosine kinase activity. Following binding of the IGF-I to the extracellular receptor α subunit, the receptor dimerizes, forms a heterotetramer (βααβ), and the tyrosine residues in the kinase domain of the β chains are autophosphorylated. The tyrosine phosphorylated IGF-I receptor causes tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 – docking molecules that can recruit and bind the p85 regulatory subunit of phosphatidylinositol 3′-kinase (PI 3-kinase). The IRS docking molecules can lead to a sustained activation of PI 3-kinase, which is key in connecting several intracellular pathways that promote cell survival, differentiation, and protein synthesis. In general, inflammatory stimuli reduce IGF-I levels and reduce sensitivity of receptors for GHRH, GH, and IGF-I. Receptors for IL-1 are present in the anterior pituitary and are localized exclusively to somatotrophs – cells that produce GH (French et al., 1996). Stimulation of the immune system with LPS causes species-specific changes in circulating GH levels. In humans and sheep, GH is increased, but in other animals including cattle, chickens, and rats it is decreased. The effects of LPS and cytokines on GH are discussed elsewhere and the general conclusion is that the effects are variable and not consistent among species (Broussard et al., 2001). Of seemingly greater importance is IGF-I, which is consistently depressed in immunologically challenged animals. Pigs injected with LPS or infected with Salmonella typhimurium showed a marked decrease in serum IGF-I levels, but little or no change in circulating GH (Balaji et al., 2000; Wright et al., 2000). An uncoupling of GH and IGF-I secretion has been reported in a number of species and is due to the impaired ability of GH to induce hepatic IGF-I synthesis. TNFα and IL-1 decrease hepatic GH receptors and may inhibit post-receptor signaling events necessary for IGF-I synthesis and release. Interleukin-6 also profoundly affects IGF-I. Transgenic mice that overexpressed IL-6 and wild-type mice injected with recombinant IL-6 had decreased IGF-I levels and stunted growth (De Benedetti et al., 1997).

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Mice injected with IL-6 had decreased IGF-I levels even when feed intake was not depressed, indicating that the decrease in IGF-I in sick animals is not necessarily due to reduced feed intake. Inflammatory cytokines can also act directly on skeletal muscle and induce IGF-I receptor resistance. Bona fide receptors for IL-1, IL-6, TNFα, and IFNγ are present in skeletal muscle (Zhang et al., 2000; Alvarez et al., 2002a) and a model for how cytokines might interfere with IGF-I receptor signaling has been proposed (Broussard et al., 2001). TNFα completely inhibits the IGF-I-induced increase in protein synthesis of human myoblasts (Frost et al., 1997). Apparently, TNFα impairs the ability of IGF-I receptors to exert their biological effect when IGF-I ligand binds. TNFα reduces IGF-I-induced tyrosine phosphorylation of the IGF-I receptor and IRS (Venters et al., 1999). This decreases PI 3-kinase activity and thus inhibits the ability of IGF-I to promote protein synthesis. The IGF-I receptor resistance partially explains why administration of recombinant IGF-I to rats with colitis failed to restore linear growth to that of control rats (Ballinger et al., 2000). However, most circulating IGF-I is bound to IGF-binding protein 3 (IGFBP3). A recent study showed that administration of IGF-I/IGFBP3 binary complex enhanced protein synthesis in septic rats, suggesting that the decreased responsiveness of muscle to exogenous IGF-I may be due to both an induction of IGF-I receptor resistance and decreased circulating levels of important IGF-I-binding proteins (Svanberg et al., 2000). 4.1.2. Inflammatory cytokines inhibit the initiation of mRNA translation The synthesis of new protein – an obvious prerequisite to protein accretion – begins with the initiation of mRNA translation (i.e. translation initiation; fig. 6). In eukaryotic cells, translation initiation involves more than a dozen proteins referred to as eukaryotic initiation factors (eIF). For a complete up-to-date description of translation initiation and how the process is

Fig. 6. A truncated schematic diagram of the translation initiation process. See text for details on how cytokines inhibit the initiation of protein synthesis (eukaryotic initiation factor, eIF; binding protein, BP).

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influenced by nutrients and hormones, the reader is referred to excellent reviews by Kimball (2002) and Shah et al. (2000). In short, translation initiation consists of four major steps (Cooney et al., 1997): (1) dissociation of the 80S ribosomal complex into the 40S and 60S ribosomal subunits; (2) binding of met-tRNAi (methyonil-tRNA initiator) to the 40S ribosomal subunit to form the 43S pre-initiation complex; (3) binding of mRNA to the 43S pre-initiation complex: and (4) association of the 60S ribosomal subunit to form the active ribosome (fig. 6). Several recent reports indicate that steps 2 and 3 are affected most during infection, so these are discussed below. 4.1.2.1. Formation of the 43S pre-initiation complex The primary function of eIF2 is to bind met-tRNAi to the 40S ribosomal subunit. Thus, eIF2 can regulate translation initiation in two ways. First, a decrease in the amount of intracellular eIF2 or its activity will reduce translation initiation. However, to our best knowledge a reduction in eIF2 during infection when skeletal muscle protein synthesis is reduced has not been reported. Second, eIF2 activity is controlled by eIF2B, whose function is to exchange GDP for GTP in eIF2 (i.e., eIF2 . GDP + eIF2B + GTP → eIF2 . GTP + eIF2B + GDP). Only the eIF2 . GTP complex is able to bind mRNA. Therefore, a reduction in eIF2 . GTP complex will decrease the translation initiation process. Indeed, a decrease in eIF2B was accompanied by a decrease in protein synthesis and translational efficiency in the gastrocnemius muscle of septic rats (Vary et al., 1994; Voisin et al., 1996b). In the same study, treatment of septic rats with IL-1 receptor antagonist returned eIF2B and protein synthesis and translational efficiency in the gastrocnemius muscle to control levels. In a separate but similar study, injection of LPS did not affect eIF2B levels in the gastrocnemius muscle, but eIF2B activity was decreased as was the rate of protein synthesis and translational efficiency (Lang et al., 2000). Thus, a change in either the amount or activity of eIF2B is sufficient to influence skeletal muscle protein synthesis. Similar results were reported when rats were infused with TNFα (Lang et al., 2002). Administration of the TNFα antagonist, TNFBP, restored the translational efficiency and protein synthesis in septic rats to control levels (Cooney et al., 1999). Furthermore, TNFBP prevented the decrease in eIF2B in gastrocnemius muscle of septic rats. 4.1.2.2. Binding of mRNA to the 43S pre-initiation complex The primary function of eIF4E is to bind mRNA to form the eIF4E . mRNA complex, which binds to eIF4G and eIF4A to form the active eIF4F (Gingras et al., 1999). The 43S pre-initiation complex directly binds to eIF4F. The eIF4E is bound to its repressors 4E-binding protein (4E-BP1), 4E-BP2, and 4E-BP3, where 4E-BP1 is the predominant form in skeletal muscle (Vary and Kimball, 2000). Kinases phosphorylate 4E-BP1 and free eIF4E. Thus, phosphorylation of 4E-BP1 and the level and activity of eIF4E are important factors that can influence protein synthesis. Interference of this signaling cascade by cytokines might partially explain the decreased protein synthesis in sick animals. However, it is not clear if the phosphorylation state of 4E-BP1 and the level and activity of eIF4E are affected during infection (Vary and Kimball, 2000; Vary et al., 2001). For example, on the one hand, IGF-I stimulates protein synthesis in skeletal muscle by inducing an intracellular signaling cascade (reviewed by Broussard et al., 2001) that ultimately results in the phosphorylation of 4E-BP1 and release of eIF4E. In septic rats, phosphorylation of 4E-BP1 is markedly reduced in the gastrocnemius muscle so there is more 4E-BP1 associated with eIF4E. Accordingly, both protein synthesis and translational efficiency are reduced (Svanberg et al., 2000). Administration of IGF-I/IGFBP3 binary complex to septic rats restored translational efficiency without affecting the phosphorylation state of 4E-BP1 or the association of eIF4E with 4E-BP1 (Svanberg et al., 2000). Thus, how IGF-I/IGFBP3 increases protein synthesis during sepsis is not yet known. On the

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other hand, insulin induces hyperphosphorylation of 4E-BP1 during sepsis, which causes the predicted dissociation of eIF4E . 4E-BP1 complex (Vary et al., 2001). However, formation of eIF4E . eIF4G complex, which is necessary for protein synthesis, was drastically reduced in septic rats despite the availability of eIF4E (Vary et al., 2001). The reduced binding of eIF4E to eIF4G may be important for inhibition of protein synthesis during infection. In summary, anti-cytokine treatment during sepsis appears to prevent the decrease in eIF2B and enhance protein synthesis and translational efficiency. In contrast, administration of IGF-I/IGFBP3 binary complex enhances protein synthesis and translational efficiency during infection even though the amount of eIF2B is reduced. Thus, regulation of eIF2B during sepsis might be independent of IGF-I but under direct cytokine control. 4.2. Inflammatory cytokines enhance skeletal muscle protein degradation 4.2.1. ATP-ubiquitin-dependent proteolytic pathway The majority of proteolysis in skeletal muscle, including short-lived and long-lived myofibrillar proteins, is ATP-dependent and involves the cofactor ubiquitin (Ub) and the 26S proteasome complex (Solomon and Goldberg, 1996; Hasselgren and Fischer, 2001; fig. 7). The functionality, structural features, regulation, and modes of action of the 26S proteasome have been extensively reviewed (Gorbea et al., 1999; Tanahashi et al., 1999; Voges et al., 1999). In short, an enzyme (E1) activates Ub in the presence of ATP to produce Ub-adenylate, which subsequently is transferred to one of several Ub-carrier enzymes (E2). Ubiquitin is attached to the ε-amino group in a lysine residue of a target protein directly by Ub-carrier enzymes or through the action of Ub-ligases (E3). Attachment of more than one Ub (poly-Ub) is generally observed in E3-dependent reactions. Poly-Ub is the preferred signal for protein degradation within the 26S proteasome. Protein degradation by the 26S proteasome results in many small

Fig. 7. A schematic diagram of the ATP–ubiquitin-dependent proteolytic pathway. See text for details on how cytokines influence this proteolytic pathway (ubiquitin, Ub; enzyme, E).

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peptide chains that can be further degraded to free amino acids. During or after proteasome degradation, Ub is released from the remnant of the target protein and is recycled for later use. Not all Ub-tagged proteins are degraded because Ub can be freed by the action of specific isopeptidases. The specificity of the ubiquitination process is thought to be primarily due to the many different enzyme isoforms. Because infection is associated with proteolysis, the role of the ATP-Ub-dependent pathway in skeletal muscle has been investigated. The increase in skeletal muscle protein degradation in septic or tumor-bearing rats is paralleled by an increase in ATP-Ub-dependent proteolysis (Temparis et al., 1994; Tiao et al., 1994). For example, protein degradation was increased in extensor digitorum longus muscle isolated from septic rats compare to controls (Tawa et al., 1997; Hobler et al., 1998). Protein degradation was inhibited when the muscle was incubated in the presence of specific proteasome inhibitors, MG101 (N-acetyl-Leu-Leu-norleucinal; Hobler et al., 1998), lactacystin (Hobler et al., 1998), or MG132 (Tawa et al., 1997). Proteolysis increases progressively in denervated muscle. Proteolysis in denervated soleus muscle was inhibited 68% by the proteasome inhibitor, MG132 (Tawa et al., 1997). These results strongly indicate the crucial role of the ATP-Ub-dependent pathway in skeletal muscle proteolysis. As indicated earlier, skeletal muscle expresses receptors for IL-1, IL-6, TNFα, and IFNγ (Zhang et al., 2000; Alvarez et al., 2002a). Inflammatory cytokines appear to play a key role in activating the ATP-Ub-dependent proteolytic pathway (Llovera et al., 1998a); however, the specific role of each cytokine is not clear. Tumor necrosis factor α but not IL-1 increased Ub mRNA in gastrocnemius muscle of rats 3 h after bolus injection (Garcia-Martinez et al., 1995). However, when examined 6 h after bolus injection, Ub mRNA in gastrocnemius muscle was increased by TNFα, IL-1, and IFNγ, but not IL-6 (Llovera et al., 1998a). Tumor necrosis factor α has been studied for its role in protein degradation more than any other cytokine, having been initially referred to as cachectin. For example, acute treatment of rats with TNFα increased protein ubiquitination (Garcia-Martinez et al., 1993) and enhanced the degradation of both total and myofibrillar proteins (Zamir et al., 1992; Fischer et al., 2001). And incubation of C2C12 murine myotubes with TNFα reduced total cellular protein content (Li et al., 1998; Li and Reid, 2000; Alvarez et al., 2002b). The protein degradation induced by TNFα is partially due to activation of the ATP-Ub-dependent proteolytic pathway. During tumor growth, muscle wasting is associated with the activation of the ATP-Ub-dependent proteolytic pathway, which is mediated via cytokines. Immunoneutralization of TNFα with a polyclonal anti-TNF antibody blocked the increase in steady-state levels of Ub mRNA in gastrocnemius muscle of tumor-bearing rats (Llovera et al., 1996). Xanthine derivatives such as pentoxifylline have been used to inhibit TNFα production. A new xanthine derivative, torbafylline, decreased plasma concentration of TNFα in rats injected with LPS and in Yoshida sarcoma-bearing rats (Combaret et al., 2002). The decrease in circulating TNFα was paralleled by decreases in muscle Ub mRNA, Ub-conjugated myofibrillar proteins, proteasome mRNA (C2 subunit of 20S), and proteasome-dependent proteolysis (Combaret et al., 2002). The soluble TNF receptor can serve as a decoy for biologically active TNFα. Binding of TNFα to the soluble receptor prevents the cytokine from binding cell membrane-bound receptors, thus effectively eliminating the cytokine’s bioactivity. Implantation of Lewis lung carcinoma cells caused a decrease in gastrocnemius muscle protein accumulation in both wild-type mice and transgenic mice that overexpressed soluble TNF receptor (Llovera et al., 1998b). However, the reduction in protein accumulation was substantially greater in wild-type mice compared to transgenic mice. In both cases, when protein accumulation was decreased, Ub mRNA in gastrocnemius muscle was increased. Tumor necrosis factor

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receptor type I knockout mice implanted with Lewis lung carcinoma cells had reduced gastrocnemius muscle wasting compared to wild-type controls (Llovera et al., 1998c). Further evidence that IL-6 does not activate the ATP-Ub-dependent proteolytic pathway comes from studies with IL-6 knockout mice. Sepsis induced by cecal ligation and puncture induced a similar increase in total and myofibrillar protein degradation in both wild-type and IL-6 knockout mice, suggesting that IL-6 was not necessary for protein degradation (Williams et al., 1998). Interestingly, in wild-type mice sepsis was accompanied by a marked increase in muscle Ub mRNA, whereas Ub mRNA was only moderately increased in muscle of IL-6 knockout mice. In contrast, administration of IL-6 to rats reportedly increased total and myofibrillar protein degradation (Goodman, 1994). The expression of Ub mRNA, however, is not always indicative of the proteolytic activity of the ATP-Ub-dependent pathway (Hasselgren, 2000). In contrast, transgenic mice that overexpressed IL-6 had severe muscle atrophy and increased levels of Ub mRNA in gastrocnemius muscle. The increase in Ub mRNA levels was reduced when mice were treated with an anti-IL-6 antibody, which also restored muscle mass (Tsujinaka et al., 1996). These contradictory results exemplify the potential difficulties of interpreting results from genetically altered animals. For example, TNFα, which induces proteolysis, was reportedly 3-fold higher in septic IL-6 knockout mice compared to wild-type controls (Fattori et al., 1994). Manipulating a single cytokine often disrupts the normal cytokine cascade. In addition, the ability of the ATP-Ub-dependent pathway to degrade protein may depend on the activity of other proteolytic systems (e.g. Ca2+-dependent). Thus, it might be important to examine the effects of multiple cytokines and multiple proteolytic systems simultaneously. 4.2.2. Calcium (Ca2+)-dependent proteolytic pathway The Ca2+-dependent proteolytic pathway involves the proteases μ-calpain, m-calpain, and calpain 3 (muscle-specific calpain p94). μ-Calpain and m-calpain are so named because they are activated by micro- and millimolar concentrations of Ca2+, respectively. A concentration of 1–20 μM of Ca2+ is required to activate μ-calpain, which exceeds the normal physiological intracellular concentration. Therefore, the calpains are generally inactive, so their role in normal cellular function is largely unknown (Stracher, 1999). Mitochondria and the sarcoplasmic reticulum release large amounts of Ca2+ postmortem and activate the calpains. The calpains are most noted for the proteolytic changes in postmortem muscle, which are important for improving tenderness (Koohmaraie, 1992). The overall contribution of calpains to skeletal muscle protein degradation during infection when protein accretion is decreased is relatively small compared to ATP-Ub-dependent proteolysis. The calpains, however, are important for degradation of certain muscle proteins including the sarcomeric proteins (Huang and Forsberg, 1998). Even though the total muscle protein degraded by calpains is relatively small, it is important because degradation of sarcomeric proteins may facilitate degradation of myofilaments. In other words, the activity of the Ca2+-dependent pathway may facilitate the activity of the ATP-Ub-dependent pathway. It has been proposed that the Ca2+-dependent release of myofilaments from the sarcomere of myofibrils is the rate-limiting event for ATP-Ub-dependent proteasome degradation of the myofilaments (Hasselgren et al., 2002). Indeed, it appears that the activity of the Ca2+dependent proteolytic pathway is increased during infection and that blockade of this pathway markedly reduces total skeletal muscle protein degradation. Skeletal muscle m-calpain was increased in septic rats experiencing accelerated muscle protein degradation (Voisin et al., 1996a). In a recent study, steady-state mRNA levels

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of μ-calpain, m-calpain, and calpain 3 were increased and the Z-disks disrupted in extensor digitorum longus muscle of septic rats (Williams et al., 1999). The addition of dantrolene, an inhibitor of the release of Ca2+ from intracellular stores to the cytoplasm, significantly reduced myofilament release from the sarcomere in septic rats, suggesting involvement of a Ca2+-dependent proteolytic pathway – probably calpain (Williams et al., 1999). In support of this, dantrolene prevented the sepsis-induced increase in muscle Ca2+ levels, mRNA levels for m-calpain, μ-calpain, and calpain 3, the increase in release of myofilaments, and total protein degradation in extensor digitorum longus muscle (Fischer et al., 2001). Interestingly, dantrolene reduced serum TNFα as well, suggesting an important but yet to be defined relationship between cytokines, Ca2+-dependent proteolysis, and muscle wasting. 4.2.3. Lysosomal proteolytic pathway Skeletal muscle contains relatively few lysosomes, but the involvement of the lysosomal proteolytic pathway in skeletal muscle protein degradation during disease has been investigated nonetheless. The main lysosomal proteases are cathepsins B, H, L, and D, which play a major role in the degradation of long-lived, soluble, and integral membrane proteins. Cathepsins, however, are unable to degrade myofibrillar proteins (Furuno et al., 1990), so the lysosomal proteolytic pathway’s contribution to disease-induced muscle wasting is considered to be relatively small. Inconsistent findings have been reported for the activity of the lysosomal proteolytic pathway in skeletal muscle of wasting animals (Llovera et al., 1994, 1995; Temparis et al., 1994; Baracos et al., 1995; Voisin et al., 1996a). For example, muscle levels of cathepsin B were increased in septic rats compared to pair-fed controls (Voisin et al., 1996a), whereas cathepsin B and B + L activities and cathepsin B mRNA were unchanged in wasting tumor-bearing rats (Temparis et al., 1994). Transgenic mice that overexpressed IL-6 suffered from muscle atrophy and had increased steady-state mRNA levels of cathepsins B and L as well as ubiquitin. Injecting IL-6 transgenic mice with an IL-6 receptor-blocking antibody decreased cathepsin (B and L) and Ub mRNA. In injured muscle, lysosomal proteolysis appears to be mediated by enzymes produced primarily by infiltrating macrophages (Farges et al., 2002). Enzymes from macrophages may not be a prerequisite, however. Incubation of C2C12 myotubes with IL-6 reduced the half-life of long-lived proteins and increased cathepsin B + L activity (Ebisui et al., 1995). It is possible that the lysosomal pathway participates in muscle wasting indirectly by controlling degradation of intracellular regulatory proteins.

5. FUTURE PERSPECTIVES Most of what is known about the in vivo effects of cytokines on skeletal muscle protein synthesis and degradation has been derived from adult animal models of sepsis or cancer – diseases that are often characterized by severe muscle wasting. These are important models for human medicine and have served to amplify the host’s responses to acute immune system activation, which is useful for elucidating the underlying mechanisms involved. It must be noted, however, that these diseases are only marginally significant to growing domestic foodproducing animals. In general, the most significant diseases in growing agricultural animals do not cause muscle wasting, but instead cause a significant and chronic decrease in skeletal muscle protein accretion. Thus, despite the presence of infectious disease, farm animals often continue to grow. In any case, it is reasonable to postulate that the same machinery that results in muscle wasting in sepsis, for example, is involved in decreasing protein accretion in infected slow-growing animals. Therefore, results of studies on muscle wasting might be

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germane to increasing protein synthesis and reducing protein degradation in slow-growing animals. However, it is not clear if the “markers” of protein synthesis and degradation evident in animals undergoing severe muscle wasting are detectable in farm animals that are merely accreting skeletal muscle protein at a less than maximal rate due to infection. To defend the host animal from infectious pathogens, the immune system and liver use some of the amino acids made available by the effects of cytokines on skeletal muscle. Thus, in growing animals we believe that the repartitioning of nutrients is a constructive adaptation that enables the animal to contend against the pathogen and continue to accrete protein, albeit at a decreased rate. It should be possible to develop novel nutrition programs that better match the animal’s metabolic state(s) during an infection, so that protein accretion is maintained while the needs of the defense systems are met. However, the nutrient requirements for agriculture animals are, for the most part, based on experiments conducted in laboratory situations where exposure to infectious pathogens and other stresses is minimized. Therefore, the estimated nutrient requirements for animals have been established to maximize production of healthy animals. If the nutritional requirements of slow-growing infected animals can be precisely defined, it will be possible to formulate cost-effective diets that maximize protein accretion under the given circumstance. Of course the goal should be to minimize infectious disease in animal production systems. However, because many infectious pathogens are endemic, it will be necessary to understand how and why the immune system regulates protein accretion in growing animals. This is particularly important because even animals with subclinical infections have reduced growth. How cytokines simultaneously influence systems involved in protein synthesis and degradation in growing animals is needed because certainly the whole is greater than the sum of their effects.

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Amino acid metabolism in the small intestine: biochemical bases and nutritional significance1 G. Wua, D. A. Knabea, and N. E. Flynnb aDepartment of Animal Science and Faculty of Nutrition, Texas A & M University, College Station, Texas, TX 77843-2471, USA bDepartment of Chemistry and Biochemistry, Angelo State University, San Angelo, TX 76909, USA

The small intestine is a highly differentiated and complex organ, which is not only responsible for the terminal digestion and absorption of nutrients, but also plays an important role in amino acid metabolism. Most of glutamine and almost all of glutamate and aspartate in the diet are catabolized by the small intestinal mucosa in the first pass. The small intestinal mucosa also degrades enteral arginine, ornithine, proline, branched-chain amino acids and lysine, and perhaps enteral methionine, phenylalanine, threonine, glycine, and serine, such that 30−50% of these dietary amino acids do not enter the portal circulation. In the postabsorptive state, the small intestine actively takes up arterial glutamine and releases ammonia, alanine, citrulline, and proline as the major nitrogenous products. The intestine-derived citrulline is effectively utilized for arginine synthesis by extrahepatic cells and organs (e.g. the kidneys). This is of nutritional significance, particularly for suckling neonates because the milk of most species, including the pig, cattle, sheep, rat, and human, is remarkably deficient in arginine. In addition to hepatic gluconeogenesis, the alanine released by the small intestine plays a key role in the extensive recycling of nitrogen between the liver and the gut. Because dietary amino acids are major fuels for the small intestinal mucosa, and are essential precursors for intestinal synthesis of proteins, glutathione, polyamines, nitric oxide, purine, and pyrimidine nucleotides, intestinal amino acid metabolism is obligatory for maintaining intestinal mucosal mass, function, and integrity. However, the extensive catabolism of enteral amino acids by the 1We

thank our students, technicians, and collaborators who have contributed to the work cited here. Our research on intestinal amino acid metabolism was supported, in part, by USDA National Research Initiative competitive grants No. 92-37206-8004, No. 94-37206-1100, No. 97-35206-5096, No. 2001-35203-11247 and No. 2003-35206-13694 (GW), by Hatch projects No. 8200 (GW) and No. 6601 (DAK) from the Texas Agricultural Experiment Station, by grants from the Houston Livestock Show and Rodeo (GW and DAK), and by a Texas A & M University Faculty Fellowship (GW). This paper is dedicated to the memory of Dr. Peter J. Reeds, our dear friend and mentor.

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small intestine substantially reduces their availability to extraintestinal tissues and selectively alters the patterns of amino acids that enter the systemic circulation. This has important practical implications for the utilization efficiency and recommended requirements of dietary protein and amino acids by animals, including humans.

1. INTRODUCTION The small intestine is a highly differentiated and complex organ, which is responsible for the terminal digestion and absorption of dietary nutrients and therefore is essential to health, growth, development, reproduction, and sustaining life of the organism (Madara, 1991). Enterocytes (epithelial absorptive cells of the small intestine) constitute >80% of the mucosal epithelial cell population (Cheng and Leblond, 1974; Klein and McKenzie, 1983) and have high rates of intracellular protein turnover and cell proliferation (Smith and Jarvis, 1978; Burrin and Reeds, 1997). Interestingly, the apical and basolateral membranes of each enterocyte are chemically, biochemically, and physically distinct (Madara, 1991). Such a polar organization of the enterocyte allows it to selectively receive nutrients from two sources: the arterial blood across its basolateral membrane and the intestinal lumen across its brush border membrane. This has important practical implications for choosing the route of feeding (e.g. enteral vs parenteral) for nutrient delivery to animals. The gut is also the barrier separating the internal milieu of the organism from the external environment, therefore excluding food-borne pathogens and preventing the translocation of luminal microorganisms into the circulation. As the largest lymphoid organ in the body, the small intestine participates in immune surveillance of the intestinal epithelial layer and regulation of the mucosal response to foreign antigens (Mowat, 1987). The pioneering studies of Windmueller and coworkers in the 1970s have demonstrated extensive intestinal catabolism of glutamine, glutamate, and aspartate (see Windmueller, 1982, for review). In recent years, there has been growing recognition that the small intestinal mucosa also degrades enteral arginine, ornithine, proline, branched-chain amino acids (BCAA), and lysine, and perhaps enteral methionine, phenylalanine, threonine, glycine, and serine, such that 30−50% of these dietary amino acids do not enter the portal circulation (Wu, 1998a; Reeds and Burrin, 2001). The major objective of this chapter is to review recent work on intestinal amino acid metabolism, with an emphasis on its biochemical bases and nutritional significance.

2. AMINO ACID METABOLISM IN THE SMALL INTESTINE Amino acids can be classified into four major groups on the basis of their metabolic fates in the small intestinal mucosa of rats, pigs, and ruminants (sheep and cattle): (1) amino acids that are neither synthesized nor degraded; (2) amino acids that are synthesized but not degraded; (3) amino acids that are degraded but not synthesized; and (4) amino acids that are both degraded and synthesized (table 1). Note that there are species differences in intestinal amino acid metabolism (Wu, 1998b). 2.1. Amino acids that are neither synthesized nor degraded by the intestinal mucosa 2.1.1. Asparagine There is no synthesis of asparagine from glutamine and aspartate in enterocytes of rats, pigs (Wu et al., 1995b; Wu, 1998b), and ruminants (sheep and cattle) (Wu, unpublished data).

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Table 1 Metabolic fates of amino acid in the small intestine Metabolic fates

Amino acids

Neither synthesized nor degraded Synthesized but not degraded Degraded but not synthesized

Asparagine, cysteine, histidine, tryptophan Tyrosine Branched-chain amino acids (isoleucine, leucine, valine), lysine, methionine, phenylalanine, threonine Alanine, arginine, aspartate, citrulline, glutamate, glutamine, glycine, ornithine, proline, serine

Both degraded and synthesized

Asparagine is not degraded by the rat small intestine, because of the absence of asparaginase; thus in rats all of the asparagine absorbed by enterocytes from the intestinal lumen appears in the intestinal venous blood intact (Windmueller, 1982). There is no release of asparagine by the postabsorptive small intestine of pigs (Wu et al., 1994a) and rats (Brosnan et al., 1983), indicating the absence of asparagine synthesis by the gut. Interestingly, asparaginase activity is detectable in the canine small intestine, and asparagine is hydrolyzed to aspartate plus ammonia in the guinea pig small intestine (Windmueller, 1982), indicating species differences in intestinal asparagine metabolism. However, in both dogs and guinea pigs, intestinal mucosal asparagine catabolism is quantitatively low. 2.1.2. Cysteine, tryptophan, and histidine Cysteine, tryptophan, and histidine are neither synthesized nor degraded by enterocytes of rats, pigs, sheep, and cattle (Wu, unpublished data). However, cysteine is used for glutathione synthesis in enterocytes (Reeds et al., 1997). Because there are mixed cell populations in the small intestine, the infiltrating mast cells can decarboxylate histidine to produce histamine in response to immunological activation (Wu, 1998b), thereby contributing to intestinal histidine utilization by intestinal mucosal cells and the portal-drained viscera (PDV). 2.2. Amino acids that are synthesized but not degraded by the intestinal mucosa 2.2.1. Tyrosine Tyrosine is synthesized from phenylalanine by phenylalanine hydroxylase, a tetrahydrobiopterin-dependent enzyme. This enzyme is restricted primarily to the liver but is also expressed in the kidney and pancreas (Tourian et al., 1969). Phenylalanine hydroxylase was previously found to be absent from the small intestine (Tourian et al., 1969). However, in these earlier studies, protease inhibitors were not used for preparing intestinal extracts or the enzyme assay, and thus intestinal activity of phenylalanine hydroxylase should be reexamined. We have recently found that phenylalanine was converted into tyrosine in enterocytes of pigs, rats, sheep, and cattle (Wu, unpublished data), indicating the presence of intestinal phenylalanine hydroxylation. In support of this view, there is significant output of tyrosine (167% of dietary intake) by the PDV of the milk protein-fed pig (Stoll et al., 1998) and of tyrosine (28%) by the small intestine of sheep fed a 20% crude protein diet (Tagari and Bergman, 1978).

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2.3. Amino acids that are degraded but not synthesized by the intestinal mucosa 2.3.1.

BCAA

Studies from a number of species have documented BCAA catabolism by the small intestine of nonruminant animals. For example, 30% of the total ingested dietary leucine is extracted by the dog small intestine in the first pass, 55% of which enters the transamination reaction (Yu et al., 1990). In adult humans, 20−30% of enterally delivered leucine is taken up in the first pass within the splanchnic region (mainly the small intestine) during the postabsorptive state or feeding (Hoerr et al., 1993). Similarly, in growing pigs fed a milk protein-based diet, 40% of leucine, 30% of isoleucine, and 40% of valine in the diet are sequestered by the PDV in the first pass, and <20% of the sequestered BCAA are utilized for mucosal protein synthesis (Stoll et al., 1998). These results suggest substantial catabolism of dietary BCAA by the small intestine of humans, dogs, and pigs. In more recent studies, we found that in pig enterocytes, most of the transaminated BCAA were released as branched-chain α-ketoacid (BCKA) (Wu, unpublished data), indicating a low rate of oxidative decarboxylation of BCKA by the small intestinal mucosa. In contrast to nonruminant animals, BCAA catabolism is negligible in the small intestinal mucosa of fed or fasted sheep (Pell et al., 1986; Cappelli et al., 1997). Both BCAA transaminase, which initiates BCAA degradation, and BCKA dehydrogenase, which oxidatively decarboxylates BCKA, are present in the nonruminant small intestinal mucosa (Khatra et al., 1977; Harper et al., 1984). The specific activities of BCAA transaminase and BCKA dehydrogenase are relatively low in the small intestine, compared with skeletal muscle and liver, respectively. However, this should not negate a quantitatively important role of the small intestine in BCAA catabolism in the whole animal, partly due to a relatively large mass of the gut. On the basis of the intestinal activities of BCAA transaminase and BCKA dehydrogenase, most of the BCKA produced by enterocytes likely enters the portal circulation and are then utilized by the liver for complete oxidation and/or gluconeogenesis. 2.3.2. Lysine, methionine, phenylalanine, and threonine The degradation of these four amino acids has recently been demonstrated in the small intestine, such that ~50% of dietary lysine and methionine, 45% of dietary phenylalanine, and 60% of dietary threonine are extracted in the first pass by the PDV of milk protein-fed pigs (Stoll et al., 1998). Only <20% of the extracted amino acids are utilized for mucosal protein synthesis, whereas one-third of the extracted amino acids are catabolized by the small intestinal mucosa such that intestinal metabolism dominates the splanchnic extraction of lysine, methionine, phenylalanine, and threonine in pigs (Stoll et al., 1997, 1998). Using stable isotopes, van Goudoever et al. (2000) showed that intestinal oxidation of enteral lysine contributed one-third of total body lysine oxidation in growing pigs fed a high-protein diet, but was virtually absent in pigs fed a low-protein diet. These results indicate adaptive regulation of intestinal lysine metabolism. In support of intestinal lysine oxidation, Pink et al. (2002) recently reported the production of CO2 from [14C]lysine in mitochondria of pig enterocytes. In adult humans, 30% and 58% of enterally delivered lysine and phenylalanine are extracted in the first pass, respectively, within the splanchnic bed (Biolo et al., 1992; Hoerr et al., 1993). Remarkably, in sheep fed 16−20% crude protein diets, 30−40% of lysine, 37−43% of phenylalanine, 55−73% of threonine, and 69−71% of methionine that disappeared from the small intestinal lumen did not enter the portal circulation (Tagari and Bergman, 1978), suggesting extensive catabolism of these amino acids by the ovine small intestine.

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There is evidence for the presence of key enzymes responsible for the degradation of lysine, methionine, phenylalanine, and threonine in the intestinal mucosa. For example, Pink et al. (2002) recently detected lysine α-ketoglutarate reductase activity in the pig small intestinal mucosa when the enzyme assay was conducted in the presence of protease inhibitors. Also, both methionine transamination (Mitchell and Benevenga, 1978) and the transsulfuration pathway (Luk et al., 1980) are present in the rat small intestinal mucosa. In addition, glutamine transaminase K, whose major substrates include glutamine, phenylalanine, and methionine, is widespread in mammalian tissues, including the small intestine of rats (Cooper and Meister, 1977) and pigs (Wu, unpublished data). Furthermore, there is an inherent phenylalanine transaminase activity in porcine aspartate transaminase isoenzymes (Shrawder and Martinez-Carrion, 1972). More recently, we were able to detect threonine dehydrogenase activity in mitochondria of pig enterocytes when protease inhibitors were used for preparing cell extracts and the enzyme assay (Wu, unpublished data).

2.4. Amino acids that are both degraded and synthesized by the intestinal mucosa 2.4.1. Glutamine, glutamate, aspartate, and alanine The pioneering studies of Windmueller and coworkers have clearly demonstrated that the small intestine extensively catabolizes enteral glutamine, glutamate, and aspartate as well as arterial glutamine, and releases large amounts of alanine, citrulline, and proline (Windmueller, 1982). For example, the small intestine of postabsorptive rats extracts 30% of arterial glutamine in a single pass, which accounts for 30% of whole-body glutamine utilization (Windmueller and Spaeth, 1975). Interestingly, intestinal utilization of arterial glutamine appears to be lower in ruminants, compared with nonruminants (Gate et al., 1999). Approximately 55%, 66%, and almost 100% of enterally delivered glutamine are sequestered in the first pass by the small intestine of adult humans (Matthews et al., 1993), adult rats (Windmueller and Spaeth, 1975), and growing pigs (Stoll et al., 1998), respectively. In contrast, there is no significant uptake of arterial glutamate and aspartate by the small intestine. Remarkably, 98% and >99% of luminal glutamate and aspartate (6 mM) are catabolized in a single pass by the rat jejunum, respectively (Windmueller, 1982). Similarly, 96% and 95% of enterally delivered glutamate is extracted in the first pass by the human splanchnic bed (Battezzati et al., 1995) and by the porcine PDV (Reeds et al., 1996), respectively. Likewise, there is negligible appearance of intra-abomasum infused glutamate in the portal circulation of sheep (Tagari and Bergman, 1978). Furthermore, the PDV of growing beef steers extracts virtually all diet- and rumen-derived glutamate, glutamine, and aspartate (Lapierre et al., 2000). Thus, most of glutamine and almost all glutamate and aspartate in the diet do not enter portal circulation in both ruminant and nonruminant animals. The metabolic fate of glutamine, glutamate, and aspartate has been quantified in the small intestine. Ammonia, citrulline, alanine, and proline released by the rat jejunum account for 38%, 28%, 24%, and 7% of the metabolized glutamine nitrogen, respectively. In postabsorptive pigs, the small intestine takes up arterial glutamine and releases not only ammonia, citrulline, alanine, and proline but also arginine, glutamate, and aspartate (Wu et al., 1994a). In vitro studies have also shown that pig enterocytes extensively utilize glutamine and produce ammonia, glutamate, alanine, aspartate, CO2, ornithine, citrulline, arginine, and proline, and rates of glutamine catabolism are greater in cells from newborn pigs compared with suckling and postweanling pigs (Wu et al., 1995b). Ammonia, glutamate, alanine, aspartate, CO2, ornithine, citrulline, arginine, and proline are also produced from glutamine in enterocytes of sheep and cattle (Wu, unpublished data). Interestingly, there is no production of ammonia

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from glutamate and aspartate in the small intestine (Windmueller, 1982), suggesting a dominant role of transamination in their catabolism. In the rat small intestine, CO2, lactate, alanine, and glucose account for 56−64%, 16−20%, 4−8%, and 2−10% of the total catabolized carbons of luminal glutamine, glutamate, and aspartate, respectively. Under conditions similar to a meal, oxidation of arterial glutamine, luminal glutamine plus glutamate plus aspartate, and luminal glucose contribute 38%, 39%, and 6% of the CO2 produced by the rat small intestine, respectively (Windmueller, 1982). Similarly, the oxidation of enteral glutamate accounts for 36% of the total CO2 production by the PDV of fully fed growing pigs, and is the most important single contributor to mucosal oxidative ATP production (Stoll et al., 1999b). Approximately 80% and 65% of enterally delivered glutamate is oxidized to CO2 in the first pass by the human splanchnic bed (Battezzati et al., 1995) and by the porcine PDV (Reeds et al., 1996), respectively, indicating that oxidation dominates intestinal glutamate metabolism. These results demonstrate that amino acids, rather than glucose, are major fuels for the small intestinal mucosa. Syntheses of purine and pyrimidine nucleotides and of glutathione represent physiologically important pathways for intestinal utilization of glutamine and aspartate and of glutamate, respectively (Burrin and Reeds, 1997; Wu, 1998b). Reeds et al. (1997) have shown that luminal glutamate, rather than the glutamate derived from intracellular glutamine degradation, is the preferential source of glutamate for glutathione synthesis in the porcine intestinal mucosa, suggesting that intestinal glutamate utilization is highly compartmentalized. Enzymes for intestinal glutamine degradation have been identified, which include phosphatedependent glutaminase (PDG), carbamoylphosphate synthase II (glutamine) (CPS-II), glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), pyrroline5-carboxylate (P5C) synthase, ornithine aminotransferase (OAT), P5C reductase, ornithine carbamoyltransferase (OCT), carbamoylphosphate synthase I (ammonia) (CPS-I), argininosuccinate synthase (ASS), argininosuccinate lyase (ASL), ornithine decarboxylase (ODC), and Krebs cycle enzymes (Wu and Morris, 1998; Bush et al., 2002; Morris, 2002). In animals, P5C synthase is expressed primarily in enterocytes, indicating a unique role of the small intestine in citrulline production and thus endogenous synthesis of arginine. The intestinal mucosa also contains N-acetylglutamate (NAG) synthase, which synthesizes NAG (an allosteric activator of CPS-I) from glutamate and acetyl-CoA (Wakabayashi et al., 1991). All of the glutamine-metabolic enzymes, except for CPS II, P5C reductase, ASS, and ASL (cytosolic enzymes), are located in mitochondria, whereas GPT and GOT are expressed in both the cytosol and mitochondria of the intestinal mucosa. Glutamine can be synthesized from glutamate and ammonia by glutamine synthetase in avian and mammalian small intestines. For example, chick enterocytes are capable of synthesizing glutamine in the presence of glutamate and ammonia (Porteous, 1980), which may explain the net release of glutamine by the chick small intestine in vivo (Windmueller, 1982). The intestinal synthesis of glutamine, coupled with a low rate of intestinal glutamine degradation, helps explain a relatively high plasma concentration of free glutamine (1.1 mM) in chicks (Wu et al., 1995a). In addition, a small amount of [14C]glutamine (1.2% of infused [14C]glutamate) appeared in the sheep portal circulation when abomasum was infused with [14C]glutamate (Tagari and Bergman, 1978). Both immunological and in situ hybridization studies have shown that glutamine synthetase protein and mRNA are located primarily in the intestinal crypt (Roig et al., 1995). Using the IEC-6 cell line (a well-characterized rat small intestinal epithelial cell line), DeMarco et al. (1999) have shown that an inhibition of glutamine synthetase reduces cell proliferation. This result suggests that when extracellular glutamine is absent, the cytosolic synthesis of glutamine from glutamate and NH4+ may play a role in endogenous provision of glutamine for supporting DNA and protein synthesis. However, the

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nutritional significance of such an observation is not entirely clear because the small intestine constantly receives a supply of arterial glutamine. Moreover, the activity of glutamine synthetase in the small intestine is generally very low, compared with PDG (Wu et al., 1994b, 1995b), and some of the glutamine synthesized in the cytosol likely enters mitochondria for extensive catabolism. Thus, although there may be an intracellular (between the cytosol and mitochondrion) or perhaps an intercellular glutamine–glutamate cycle (between crypt and villus cells) in the small intestine, it is unlikely that net synthesis of glutamine in a nutritionally significant quantity occurs in the mammalian gut in vivo. 2.4.2. Arginine, citrulline, ornithine, and proline These four closely related amino acids are interconverted in the small intestinal mucosa. Studies over the past 25 years have established that the production of citrulline by enterocytes of the small intestine plays a crucial role in the endogenous synthesis of arginine (Wu and Morris, 1998). Glutamine/glutamate and proline (abundant amino acids in milk) are the major precursors for citrulline and arginine synthesis in incubated enterocytes from pigs (Wu et al., 1994a; Wu, 1997), sheep, and cattle (Wu, unpublished data). In vivo studies have also demonstrated citrulline and arginine synthesis from enteral proline and glutamate in pigs (Murphy et al., 1996; Brunton et al., 1999) and the release of citrulline by the ruminant small intestine (Bergman and Heitmann, 1978). The citrulline released by the small intestine is not taken up by the liver, and is utilized for arginine synthesis primarily in kidneys (Dhanakoti et al., 1990). Also, uptake of physiological concentrations of arginine by the liver is low due to a low activity of the amino acid transport system y+ in hepatocytes (Wu and Morris, 1998). Importantly, almost all extrahepatic cells are capable of synthesizing arginine from citrulline (Wu and Morris, 1998). Thus, intestine-derived citrulline and arginine are equally effective as sources of arginine for the whole body. The release of citrulline into the portal circulation by the small intestine and the uptake of arterial citrulline by the kidneys for arginine production is referred to as the intestinal–renal axis for endogenous synthesis of arginine. Recent studies with pigs have demonstrated marked developmental changes in intestinal arginine metabolism. First, in 1- to 7-day-old pigs, most of the citrulline synthesized from glutamine and proline in enterocytes is converted locally into arginine because of high activities of both ASS and ASL (Wu and Knabe, 1995). However, in older piglets (14- to 21-day-old), enterocytes release most of the synthesized citrulline due to a low ASS activity (Wu et al., 1994a). Thus, the small intestine shifts from the major site of net arginine synthesis in 1-week-old pigs to the major site of net citrulline synthesis in 2- to 3-week-old pigs. Second, intestinal synthesis of citrulline and arginine decreases by 60−75% in 7-day-old suckling pigs compared with newborn pigs, and declines further in 14- to 21-day-old suckling pigs (Wu, 1997). The metabolic basis for the marked decrease in citrulline and arginine synthesis by enterocytes of 7- to 21-day-old pigs is not known. Because the ratios of small intestinal weight (or mucosal protein weight) to body weight do not change substantially in newborn and suckling piglets (28−32 g small intestine per kg body wt from 1 to 21 days of age), intestinal synthesis of citrulline and arginine per kg body weight remains strikingly low in 7- to 21-day-old piglets compared with 1- to 3-day-old piglets. Consequently, plasma concentrations of arginine and its immediate precursors (ornithine and citrulline) decrease progressively by 20−41%, as the age of sow-reared piglets increases from 3 to 14 days (Flynn et al., 2000). It should be borne in mind that plasma arginine concentration is regulated by a number of factors, including arginine synthesis and degradation, dietary arginine intake, degradation of plasma proteins and peptides, and intracellular protein turnover (protein synthesis and degradation) (Young and El-Khoury, 1995).

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Thus, in suckling piglets, plasma arginine concentration would not be expected to decrease to the same extent as the intestinal synthesis of citrulline and arginine. In contrast to pigs, there are no postnatal decreases in plasma citrulline and arginine concentrations or in rates of intestinal citrulline and arginine synthesis from glutamine between 2- and 7-day-old suckling calves (Wu, unpublished data). For examples, rates of citrulline production from 2 mM glutamine are 149 ±13 and 132 ± 16 nmol/mg DNA per 30 min (means ± SEM, n = 5), respectively, in jejunal enterocytes of 2- and 7-day-old suckling calves. In addition, rates of arginine synthesis from 2 mM glutamine are 185 ± 20 and 169 ± 22 nmol/mg DNA per 30 min (means ± SEM, n = 5), respectively, in jejunal enterocytes of 2- and 7-day-old suckling calves. The marked postnatal decline in intestinal citrulline and arginine synthesis represents an intriguing, and perhaps unique, aspect of amino acid metabolism in neonatal pigs. Third, arginine catabolism in pig enterocytes is limited at birth and during the suckling period due to a negligible arginase activity, but is markedly enhanced at weaning (Wu et al., 1996a) due to the cortisol-mediated induction of arginase (Flynn and Wu, 1997a,b). Consequently, urea is synthesized from extracellular and intramitochondrially generated ammonia and from arginine in enterocytes of weaned pigs (Wu, 1995). This novel finding challenges the textbook view that ureagenesis occurs only in the mammalian liver. The induction of arginase also makes possible the synthesis of proline and polyamines from arginine in enterocytes of postweaning pigs (Wu, 1995; Wu et al., 2000a,b). Because of a relatively high activity of arginase in the small intestinal mucosa of postweaning mammals, 40% of the arginine absorbed by enterocytes from the intestinal lumen is degraded in a single pass in adult rats (Windmueller and Spaeth, 1976). Similarly, in adult humans, 38% of dietary arginine is removed in the first pass within the splanchnic region, and most of the arginine uptake is accounted for by the small intestine (Castillo et al., 1993a). In sheep fed 16−20% crude protein diets, 42−68% of arginine that disappears from the small intestinal lumen does not enter the portal circulation (Tagari and Bergman, 1978). In addition to ornithine production, there is nitric oxide (NO) synthesis from arginine by NO synthase in enterocytes (Wu et al., 1996a) or the small intestine (Alican and Kubes, 1996). This pathway, however, is quantitatively low in enterocytes of newborn, suckling, and weaned pigs (Wu et al., 1996a). Similarly, in healthy adult humans, only 0.34% of the dietary arginine taken up in the first pass within the splanchnic region is utilized for NO production (Castillo et al., 1993b). Despite the presence of arginine decarboxylase in a number of animal tissues (e.g., brain, liver, and kidney) for agmatine synthesis from arginine, this enzyme is absent from pig enterocytes (Wu et al., 1996a). The findings that proline is actively catabolized by porcine enterocytes to produce ornithine, citrulline, arginine, and polyamines (Wu, 1997; Wu et al., 2000a,b) are some of the most exciting developments in intestinal amino acid metabolism in recent years. In pigs, the activities of proline oxidase and OAT are greatest in the small intestinal mucosa (Wu, 1997). Proline oxidase activity is also present in enterocyte mitochondria of rats (Wu, 1997), sheep, and cattle (Wu, unpublished data). Considering the relatively large mass of the small intestine as compared with the liver and kidneys, the intestinal mucosa likely plays a major role in initiating proline degradation in the body. Consistent with this suggestion, Stoll et al. (1998) demonstrated that 38% of dietary proline was extracted in the first pass by the PDV of milk protein-fed piglets. In sheep fed 16−20% crude protein diets, 54−71% of proline that disappeared from the small intestinal lumen did not enter the portal circulation (Tagari and Bergman, 1978). By bridging the urea cycle with the Krebs cycle, arginine and proline metabolism, and polyamine synthesis, OAT plays a central role in intestinal nitrogen and carbon metabolism (fig. 1). We have recently suggested that the intestinal OAT reaction proceeds in

Fig. 1. Intestinal mucosal amino acid metabolism. Enzymes that catalyze the indicated reactions are: (1) phosphate-dependent glutaminase; (2) glutamine synthetase; (3) pyrroline-5carboxylate synthase; (4) ornithine aminotransferase; (5) ornithine carbamoyltransferase; (6) argininosuccinate synthase; (7) argininosuccinate lyase; (8) arginase; (9) ornithine decarboxylase; (10) spermidine synthase; (11) spermine synthase; (12) S-adenosylmethionine synthase; (13) S-adenosylmethionine decarboxylase; (14) nitric oxide synthase; (15) N-acetylglutamate synthase; (16) carbamoylphosphate synthase-I ; (17) proline oxidase; (18) pyrroline-5-carboxylate reductase; (19) branched-chain amino acid transaminase; (20) branched-chain α-ketoacid dehydrogenase; (21) alanine transaminase; (22) aspartate transaminase; (23) purine- and pyrimidine-synthesizing enzymes; (24) aspartate decarboxylase; (25) histidine decarboxylase; (26) α-ketoglutarate dehydrogenase; (27) asparaginase; (28) via aspartate transaminase and Krebs cycle enzymes; (29) possibly via NADP-linked malic enzyme, phosphoenolpyruvate carboxykinase/pyruvate kinase, and oxaloacetate decarboxylase; (30) pyruvate dehydrogenase; (31) via Krebs cycle enzymes; (32) phenylalanine hydroxylase; (33) phenylalanine transaminase; (34) lysine:α-ketoglutarate reductase; (35) serine transaminase; (36) serine dehydratase; (37) via enzymes of gluconeogenesis; (38) serine hydroxymethyltransferase; (39) glutathionesynthesizing enzymes; and (40) glycine cleavage system. The symbol “?” denotes unknown reactions in intestinal mucosa. Abbreviations: Ala, alanine; Asp, aspartate; BCAA, branched-chain amino acids; BCKA, branched-chain α-ketoacid; CoA, coenzyme A; CP, carbamoyl phosphate; DCAM, decarboxylated S-adenosylmethionine; α-KG, α-ketoglutarate; Met, methionine; MTA, methylthioadenosine; MTF, N5,N10-methylenetetrahydrofolate; NAG, N-acetylglutamate; OAA, oxaloacetate; PPYR, phenylpyruvate; Pyr, pyruvate; TF, tetrahydrofolate.

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the direction of net synthesis of either ornithine or P5C, depending on intramitochondrial concentrations of ornithine and P5C (Wu, 1998b; Dekaney et al., 2000). For example, when there is a high concentration of P5C in mitochondria due to the oxidation of large amounts of proline by proline oxidase, the synthesis of ornithine and therefore of citrulline from P5C is favored in enterocytes (Wu, 1997). On the other hand, when there is a high concentration of mitochondrial ornithine due to the hydrolysis of large amounts of arginine by arginase II, the synthesis of P5C and therefore of proline from ornithine is favored in enterocytes (Wu et al., 1996a). Arginine-metabolic enzymes have been identified in the small intestine, and arginase is the major enzyme initiating arginine catabolism in enterocytes (Wu et al., 1996a). Both enzymological and metabolic evidence have established that P5C synthase is a key regulatory enzyme in intestinal synthesis of citrulline from glutamine/glutamate (Wakabayashi et al., 1991; Wu and Morris, 1998). By synthesizing NAG, NAG synthase may be another key regulatory enzyme in intestinal synthesis of citrulline and arginine from glutamine/glutamate and proline. Given the high activity of OCT in the small intestine, it is surprising that the major product of the degradation of extracellular ornithine in enterocytes is proline but not citrulline (Wu et al., 1996a). This may be explained by the following reasons. First, enterocytes have an exceedingly high activity of mitochondrial OAT, but a relatively low activity of mitochondrial CPS I for yielding carbamoyl phosphate (a substrate for OCT) from NH3, HCO−3, and ATP. Thus, intramitochondrial ornithine is preferentially utilized by OAT to form P5C instead of citrulline. Second, enterocytes have a high activity of cytosolic P5C reductase. Therefore, when P5C enters the cytosol from mitochondria, P5C is readily converted into proline by P5C reductase. Thus, dietary or arterial blood ornithine is a poor precursor for intestinal synthesis of citrulline, and does not contribute significantly to maintaining arginine homeostasis in animals (Wu and Morris, 1998). There are marked species differences in intestinal citrulline and proline synthesis, both of which are NADPH-dependent (Wu, 1996). For example, P5C synthase is absent and OAT activity is very low in chick enterocytes (Wu et al., 1995a). This provides the metabolic basis for explaining the absence of citrulline synthesis from glutamate and glutamine in the avian small intestine and thus the nutritional essentiality of arginine for birds. Similarly, the near absence of P5C synthase in the intestinal mucosa of the cat explains the lack of intestinal production of citrulline and thus little endogenous synthesis of arginine in this species (Rogers and Phang, 1985). Strikingly, the rate of conversion of arginine or ornithine into proline in chick enterocytes is only about 4% of that in pig enterocytes owing to low activities of both arginase and OAT (Wu et al., 1995a). These data help explain why ornithine is an ineffective replacement of proline in the chicken diet and why proline is an essential amino acid for the chick (Graber and Baker, 1971, 1973). 2.4.3. Serine and glycine Although the small intestine was not traditionally considered as a major organ for the catabolism of serine and glycine, Stoll et al. (1998) have recently reported that 40% and 50% of dietary serine and glycine are extracted in the first pass by the PDV of the milk protein-fed pig, respectively. Interestingly, <20% of the extracted serine and glycine are utilized for intestinal protein synthesis, and the majority of the extracted amino acids are catabolized (Stoll et al., 1998). In sheep fed a 20% crude protein diet, 28% of serine and 36% of glycine that disappear from the small intestinal lumen do not enter the portal circulation (Tagari and Bergman, 1978). These results suggest that dietary serine and glycine may be substantially catabolized by the small intestine of both monogastric animals and ruminants. There is some published evidence for the presence of key enzymes for intestinal metabolism of serine and glycine. For example, the rat small intestine has been reported to contain

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the activities of serine dehydratase, serine aminotransferase, and serine hydroxymethyltransferase (Kikuchi et al., 1980). The latter interconverts serine into glycine and generates N5, N10-methylenetetrahydrofolate for purine and pyrimidine synthesis. This reaction is important for supporting the high rates of protein synthesis and epithelial cell proliferation in the small intestinal mucosa. Glutathione synthesis also represents a physiologically important pathway for glycine utilization by the small intestinal mucosa (Reeds et al., 1997). Furthermore, the transsulfuration pathway occurs in the small intestinal mucosa (Luk et al., 1980), which can contribute to serine metabolism. Although previous studies could not show the presence of the glycine cleavage system in the rat small intestine (Kikuchi et al., 1980), likely due to the lack of protease inhibitors in the enzyme assay system, we recently detected the activity of the glycine cleavage system for the tetrahydrofolate-dependent production of ammonia and CO2 from glycine in enterocytes of rats, pigs, sheep, and cattle (Wu, unpublished data).

3. SIGNIFICANCE OF INTESTINAL AMINO ACID METABOLISM 3.1. Intestinal integrity and function A characteristic of the small intestine physiology is high rates of intracellular protein turnover and protein secretion by mucosal epithelial cells (Burrin and Reeds, 1997). Although protein in the small intestine accounts for only 5% of whole-body protein, the amount of protein synthesized by the small intestine daily contributes 15−20% of the whole-body protein synthesis (McNurlan and Garlick, 1980; Reeds et al., 1993). This necessitates an adequate supply of both amino acids and energy to the small intestinal mucosa. In support of this notion, the available evidence shows that enteral feeding is the primary source of amino acids for the intestinal mucosa because uptake of amino acids other than glutamine from arterial blood is either low or insignificant (Windmueller, 1982; Wu et al., 1994a). Amino acid metabolism is crucial for intestinal integrity and function through the following mechanisms. First, dietary glutamine, glutamate and aspartate, and arterial blood glutamine are major fuels for the small intestinal mucosa, and are responsible for providing energy required for intestinal ATP-dependent metabolic processes, including active nutrient transport, intracellular protein turnover, as well as epithelial cell proliferation and migration (Burrin and Reeds, 1997; van der Schoor et al., 2001). On the basis of both experimental and clinical evidence, the importance of glutamine for supporting the metabolic function of intestinal mucosa has now generally been accepted (Reeds and Burrin, 2001). Second, ornithine (a product of arginine, glutamine, and proline metabolism) is the immediate precursor for the synthesis of polyamines in the enterocyte (Wu et al., 2000a,b), which are essential to DNA and protein synthesis, as well as to the proliferation, differentiation, and repair of intestinal epithelial cells (Luk et al., 1980; Wu, 1998b). In addition, glutamine, asparagine, and glycine are potent stimulators of intestinal ODC (Kandil et al., 1995), thereby enhancing polyamine synthesis from arginine- and proline-derived ornithine. Third, arginine is the physiological precursor of NO, which plays an important role in regulating intestinal blood flow, integrity, secretion, and epithelial cell migration (Alican and Kubes, 1996), the relaxation of gastrointestinal smooth muscle cells, and feed intake (Morley and Flood, 1991). Fourth, glutamate, glycine, and cysteine are precursors for the synthesis of glutathione, a tripeptide critical for defending the intestinal mucosa against toxic and oxidative damage (Reeds et al., 1997). Because there is compartmentation of glutathione synthesis in enterocytes, and because there is no significant uptake of arterial glutamate, glycine, and cysteine by the small intestine, adequate dietary supply of these amino acids plays a vital role in glutathione synthesis by the intestinal mucosa (Reeds et al., 2000a; Reeds and Burrin, 2001). Fifth, villus enterocytes derive amino acids for protein synthesis and cell proliferation preferentially from the intestinal lumen

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rather than from the arterial blood (Alpers, 1972; Stoll et al., 1999a, 2000; Burrin et al., 2000). This helps explain why parenteral nutrition selectively decreases protein synthesis in the small intestinal mucosa and results in intestinal atrophy (Dudley et al., 1998; Stoll et al., 2000), and further supports the view that enteral feeding of amino acids is obligatory for maintaining intestinal mucosal mass and integrity (Wu, 1998a; Reeds et al., 2000a,b). 3.2. Endogenous synthesis of amino acids 3.2.1. Citrulline and arginine Although arginine is formed via the urea cycle in the mammalian liver, there is no net synthesis of arginine by this organ due to an exceedingly high activity of arginase in hepatocytes (Wu and Morris, 1998). A unique, important aspect of intestinal amino acid metabolism is the synthesis of citrulline and arginine from glutamate, glutamine, and proline in ruminant and nonruminant animals (Bergman and Heitmann, 1978; Wu, 1998a). In both neonates and adults, enterocytes are almost the exclusive source of citrulline for endogenous synthesis of arginine. The near absence of arginase from neonatal enterocytes helps maximize the output of arginine by the small intestine. This is of nutritional significance because arginine is remarkably deficient in the milk of most mammals studied, including primates (human, chimpanzee, gorilla, and rhesus), ruminants (cow, goat, sheep), and other nonprimates (elephant, llama, pig, and rat) (Davis et al., 1994; Wu and Knabe, 1994; Reeds et al., 2000a), owing to extensive catabolism of arginine by lactating mammary tissue (O’Quinn et al., 2002). For example, concentrations of amino acids in sow’s whole milk (containing 18.6% dry matter) on day 7 to day 28 of lactation are as follows (g/L): alanine, 1.89; arginine, 1.43; aspartate plus asparagine, 5.02; cysteine, 0.72; glutamate plus glutamine, 9.42; glycine, 1.16; histidine, 0.92; isoleucine, 2.12; leucine, 4.23; lysine, 3.87; methionine, 0.98; phenylalanine, 1.95; proline, 5.56; serine, 2.30; threonine, 2.08; tryptophan, 0.64; tyrosine, 1.92; and valine, 2.40 (N = 10 sows; Wu and Knabe, unpublished data). Note that the ratio of arginine/lysine in the sow’s milk (0.37) is only 40% of that in the pig body (0.92) (Davis et al., 1993). Arginine requirements by young mammals are particularly high (Rogers et al., 1970; Wu et al., 2000a). The relative contribution of milk vs endogenous synthesis to meeting arginine requirements by the suckling neonate can be estimated on the basis of arginine intake and arginine accretion plus catabolism in the body. For example, for a 7-day-old pig (2.5 kg) which gains 200 g body weight (27.2 g crude protein or 1.88 g arginine) per day (Flynn et al., 2000), catabolizes 0.65 g arginine daily via arginase and NOS pathways (Murch et al., 1996), and utilizes 0.17 g arginine daily for creatine synthesis [calculated on the basis of urinary creatinine excretion (0.38 mmol/kg body wt/day)] (Weiler et al., 1997), the arginine requirement is at least 2.7 g/day (table 2). On the basis of milk consumption by the suckling 7-day-old pig (0.78 L milk/day), arginine content in sow’s milk (1.43 g/L of whole milk) (Wu and Knabe, 1994), and digestibility of arginine in milk protein (90.4%) (Mavromichalis et al., 2001), the intake of sow’s milk provides only 1.01 g arginine/day, or at most 37% of the daily arginine requirement (table 2). Thus, endogenous synthesis of arginine must provide at least 63% of arginine for the suckling piglet. The crucial role of the small intestine in endogenous arginine synthesis is further supported by the following lines of direct evidence. First, an inhibition of intestinal synthesis of citrulline retards the growth of young rats fed an arginine-deficient diet (Hoogenraad et al., 1985). Second, resection of the rat small intestine (removal of 80% of the gut) results in (1) marked deficiencies of citrulline and arginine in plasma and of arginine in skeletal muscle, (2) reduced animal growth, (3) negative nitrogen balance, and (4) hypertension due to a deficiency of arginine for

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Table 2 Relative contributions of milk vs endogenous synthesis of arginine to arginine requirements by the 7-day-old (2.5 kg) sow-reared piglet Arginine requirements and sources Arginine requirements Body weight gain (200 g/day; 27.2 g crude protein)a Arginine catabolism via arginase and NO synthaseb Creatine synthesis (0.38 mmol/kg body wt/day)c Arginine supply from sow’s milk Milk consumption (0.78 L/day; 1.43 g/L whole milk)d Undigestible arginine in sow’s milk (9.6%)e Arginine supply from endogenous synthesis a

Amounts of arginine (g/day) ≥2.7 1.88 0.65 0.17 ≤1.01 1.12 −0.11 ≥1.69

Wu et al. (2000c), b Murch et al. (1996), c Weiler et al. (1997), d Wu and Knabe (1994), e Mavromichalis et al. (2001).

NO synthesis (Wakabayashi et al., 1994a,b). Third, hypoornithinemia, hypocitrullinemia, hypoargininemia, and hyperammonemia occur in patients with short bowel syndrome and chronic renal failure (Yokoyama et al., 1996), due to reduced intestinal production of citrulline and impaired synthesis of arginine from citrulline in kidneys. Fourth, a deficiency of intestinal OAT results in hypoornithinemia, hypocitrullinemia, hypoargininemia, hyperammonemia, and even death in mice and humans during the neonatal period (Wang et al., 1995). Fifth, a recently recognized deficiency of intestinal P5C synthase in humans causes hypoornithinemia, hypocitrullinemia, hypoargininemia, and hyperammonemia, retarded mental development, and death (Kamoun et al., 1998). Sixth, in the sparse-fur mutant mouse, the inborn X-linked deficiency of OCT limits intestinal citrulline synthesis, leads to impaired maturation of intestinal epithelial cells (Malo et al., 1986), and causes retarded postnatal growth and death (DeMars et al., 1976). Finally, an inhibition of intestinal citrulline synthesis for 12 h results in decreased plasma concentrations of ornithine, citrulline, and arginine by 59%, 52%, and 76%, respectively, in 4-day-old neonatal pigs nursed by sows (Flynn and Wu, 1996). On the basis of decreases in plasma concentrations of arginine, ornithine, and citrulline as well as nitrite and nitrate (stable end products of NO oxidation), and a concomitant increase in plasma ammonia concentration, we have suggested that arginine is deficient in 7- to 21-day-old sow-reared piglets (Flynn et al., 2000). Although sow-reared piglets continue to grow during the 21-day suckling period, this does not necessarily mean that arginine supply from milk plus endogenous synthesis meets arginine requirements for maximal growth, as exemplified by submaximal growth and impaired NO synthesis in arginine-deficient young rats (Wu et al., 1999). Indeed, recent artificial rearing data show that the biological potential for neonatal pig growth (from birth to day 21 of age) is at least 74% greater than that for sowreared piglets (Boyd et al., 1995). Both metabolic and growth data indicate that arginine deficiency represents a major obstacle to maximal growth in milk-fed piglets (Kim et al., 2004). In view of reduced arginine supply from endogenous synthesis in suckling compared with newborn piglets and a great potential of arginine to enhance neonatal pig growth, it is of crucial importance to identify an effective means for enhancing intestinal synthesis of citrulline, thereby improving arginine nutrition and growth of sow-reared piglets. Endogenous synthesis of arginine also plays an important role in maintaining arginine homeostasis in postweaning growing animals. In 75-day-old pigs fed a conventional diet containing 0.98% arginine (2.5 times the recommended National Research Council requirement for dietary arginine), an inhibition of intestinal citrulline synthesis decreases plasma concentrations

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of citrulline and arginine by 26% and 22%, respectively (Wu et al., 1997). On the basis of dietary arginine intake, ileal digestibility of dietary arginine, tissue protein accretion, and oxidation of plasma arginine, we have estimated that endogenous synthesis of arginine provides ~50% of the total daily arginine requirement in the postweaning growing pig (Wu et al., 1997). 3.2.2. Proline Proline is synthesized from arginine, ornithine, glutamine, and glutamate in enterocytes of most mammals (Wu, 1998a). The finding that there is no synthesis of proline from intravenously infused glutamate in both pigs and humans (Matthews et al., 1993; Murphy et al., 1996) not only suggests the complex compartmentation of intestinal glutamate metabolism and proline synthesis, but also underscores the essential role for the small intestine in synthesizing proline from enteral glutamate. The dietary essentiality of proline for birds results from (1) a low rate of endogenous synthesis of proline from arginine in the small intestine because of a low activity of intestinal OAT in birds, and (2) the lack of synthesis of proline from glutamate or glutamine in the small intestine because of the absence of P5C synthase (Wu et al., 1995a). The synthesis of proline from arginine and glutamine is low in enterocytes of suckling pigs because of a negligible activity of arginase and P5C synthase, but is markedly increased in cells from postweaning pigs owing to the induction of both enzymes (Wu et al., 1996a). This may also explain, in part, (1) why proline is an essential amino acid for neonatal pigs (2.5 kg of body weight) (Ball et al., 1986), and (2) why proline is a nonessential amino acid for postweaning pigs (5−15 kg of body weight) (Chung and Baker, 1993). 3.2.3. Alanine In nonruminant animals, alanine is an important nitrogenous product of the intestinal catabolism of glutamate, aspartate, and BCAA (Windmueller, 1982; Brosnan et al., 1983), and its carbon skeleton (pyruvate) is derived partially from enteral glucose (Stoll et al., 1999b). Large amounts of alanine are also released by the ruminant small intestine (Bergman and Heitmann, 1978). Thus, alanine transaminase functions primarily for alanine synthesis in the small intestinal mucosa of both monogastric animals and ruminants. Because alanine is a major amino acid for hepatic gluconeogenesis, the intestine-derived alanine plays an important role in maintaining glucose homeostasis. Alanine released by the small intestine also helps transport the nitrogen and perhaps the carbons of dietary amino acids and arterial glutamine from the small intestine to the liver. Because the liver actively takes up alanine and ammonia from portal and arterial blood and releases glutamine, and because the small intestine substantially utilizes enteral and arterial blood glutamine and releases large amounts of alanine and ammonia during both the postabsorptive state and feeding, there appears to be a “glutamine–alanine cycle” involving the small intestine and the liver. This cycle seems to be analogous to the glucose–lactate cycle (the Cori cycle) which spans the skeletal muscle and liver. However, it should be pointed out that the carbons and nitrogen of the alanine released by the small intestine are utilized preferentially for the synthesis of glucose and urea, respectively, rather than for glutamine synthesis, in the liver. Thus, the splanchnic “glutamine–alanine” cycle is indeed not a true metabolic cycle, but illustrates a key role of alanine in the extensive recycling of nitrogen between the liver and the gut (Wu, 1998a). 3.3. Availability of dietary amino acids to extraintestinal tissues A theme that has emerged from this review is that intestinal mucosal amino acid catabolism plays an important role in regulating the availability of dietary amino acids to

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extraintestinal tissues. This novel concept has important implications for protein and amino acid nutrition in animals. First, the extensive catabolism of dietary essential amino acids by the small intestine results in a decrease in their nutritional efficiency. For example, the particularly high requirement of dietary arginine by the postweaning growing animal results, in part, from the extensive hydrolysis of the absorbed dietary arginine by enterocytes (Wu et al., 1997). Also, the extensive degradation of dietary glutamine limits the role for enteral glutamine feeding to increase its plasma concentrations in many monogastric mammals, including the rat and pig (Wu et al., 1996b; Wu, 1998b). In addition, infection with Trichostrongylus colubriformis in 6- to 9-month-old lambs increases the catabolism of luminal leucine by the gastrointestinal tract and reduces the availability of diet- and rumen-derived leucine for other tissues, which provides a metabolic basis for the decreases in nitrogen retention and growth rates under conditions of subclinical nematode infection (Yu et al., 2000). In support of the concept of first-pass intestinal catabolism of essential amino acids, recent in vivo studies have shown that methionine and threonine requirements are 35% and 45% higher, respectively, during oral compared with parenteral feeding in neonatal pigs (Bertolo et al., 1998; Shoveller et al., 2000). Most recently, Elango et al. (2002) reported that the parenteral requirement for total BCAA is only 56% of the enteral requirement in neonatal pigs, indicating that 44% of total BCAA is extracted by the first-pass metabolism in the gut. There is a positive correlation between first-pass intestinal catabolism of dietary amino acids and mucosal mass (Stoll et al., 1998). Thus, factors that affect intestinal mass (e.g. antibiotics, growth hormone, insulin-like growth factor-I, and diabetes) may have an important impact on the requirements of dietary amino acids (Wu, 1998b). For example, in pigs fed antimicrobial agents (antibiotics and chemotherapeutics), a decrease in the small intestinal mucosal mass is associated with an increase in whole-body growth rate (Yen et al., 1985; Cromwell, 2001). This raises a possibility that reduced catabolism of dietary amino acids by the small intestine is a mechanism responsible for the growth-promoting effect of antimicrobial agents. Second, intestinal amino acid metabolism modulates the entry of absorbed dietary amino acids into portal circulation. Therefore, the pattern of amino acids in the diet differs remarkably from that in the intestinal tissue, portal circulation, and the extraintestinal organs (Le Floc’h and Seve, 2000; Daenzer et al., 2001). Also, the pattern of amino acids in tissue proteins or animal products (e.g. egg, milk, wool, and meat) is not necessarily similar to the ideal pattern of dietary amino acid requirements in animals. In addition, there are profound differences in organ or plasma amino acid concentrations between enteral and parenteral feeding in neonates, including infants and piglets (Bertolo et al., 2000; Wu et al., 2001). Third, there are developmental changes, disease-associated alterations, and species differences in intestinal amino acid catabolism. Thus, these factors should be taken into consideration in recommending dietary amino acid requirements and in refining in vivo models of amino acid and protein metabolism. This is graphically demonstrated, for example, by the dynamic changes in dietary requirements of arginine and proline by developmental pigs (Wu, 1998b; Wu et al., 2000c), by increased requirements of arginine and proline for wound healing in burned patients (Young and El-Khoury, 1995), and by the nutritional essentiality of arginine and proline for birds (Wu and Morris, 1998).

4. FUTURE PERSPECTIVES Much has been learned in recent years regarding intestinal amino acid metabolism. However, there are many important and yet challenging questions in this rapidly growing and fruitful

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area of investigation. First, recent intriguing findings of the extensive first-pass extraction of dietary essential amino acids and cysteine by the porcine PDV and human splanchnic bed raise an important question regarding their catabolism in the small intestinal mucosa. This novel concept should be firmly established by biochemical studies with isolated enterocytes. In addition, microorganisms in the intestinal lumen may substantially contribute to the catabolism of enteral essential amino acids and cysteine and such microbial pathways should be quantified. Second, in view of the recently recognized deficiency of arginine (an essential amino acid for neonates) in sow-reared piglets (Flynn et al., 2000), further studies are necessary to elucidate the mechanisms responsible for the marked decline in intestinal synthesis of citrulline and arginine in suckling piglets. This new knowledge will undoubtedly help design new, effective means to enhance arginine supply to the piglets and therefore improve their postnatal growth. Third, much work is required to define hormonal and nutritional regulation of intestinal amino acid metabolism at molecular, cellular, and whole-body levels. This will be facilitated by the recent availability of porcine small intestinal epithelial cells (Lu et al., 2002) and of mammalian cDNAs for key regulatory enzymes [e.g. arginase II (Morris, 2002), P5C synthase (Aral et al., 1996), and NAG synthase (Caldovic et al., 2002)], and by recent biochemical and molecular biology techniques (e.g. proteomics, metabolomics, and microarrays) (Phelps et al., 2002). We predict that exciting new knowledge on the regulation of intestinal amino acid metabolism will be discovered in the coming years, which will help design new means to improve the efficiency of protein and amino acid utilization by animals, including humans.

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Role of intestinal first-pass metabolism on whole-body amino acid requirements R. F. P. Bertoloa, P. B. Pencharzc,d and R. O. Ballb,d a Department

of Biochemistry, Memorial University of Newfoundland, St. John’s, NewfoundLand, Canada A1B 3X9 b Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5 c Department of Paediatrics, University of Toronto, Toronto, Ontario, Canada M5G 1X8 d The Research Institute, The Hospital for Sick Children, Toronto, Department of Nutritional Sciences, University of Toronto, Toronto, Ontario, Canada

The small intestine utilizes a different profile of amino acids compared to whole-body requirements. Quantifying the gut requirements for amino acids is critical to understand the limiting availability of these amino acids during periods of rapid growth in animals. Many methods have been employed to determine amino acid requirements in man and animals including growth assays, nitrogen balance and amino acid oxidation methods. The most versatile approach is the indicator amino acid oxidation technique which can be safely employed in many vulnerable populations. The amino acid requirements of the gut have been estimated using this technique in the parenterally fed piglet, which is a model of a gut-deficient animal, and comparing requirements to enterally fed controls. The gut’s requirement for threonine is proportionately greatest of the amino acids tested due to its role in mucin synthesis. The sulphur and branched-chain amino acids are also significantly utilized by the gut. Tryptophan, lysine, phenylalanine and tyrosine utilization by the gut is not significant. The availability of threonine and sulphur amino acids may be limiting for growth in situations of gut stress or disease due to the higher maintenance requirements during such gut challenges.

1. INTRODUCTION The small intestine has classically been regarded as a digestive organ responsible for the absorption of nutrients from foods. Only recently has the gastrointestinal tract, whose metabolism is dominated by the small intestine, been studied as a significant metabolic tissue with

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great impact on whole-body metabolism. Through co-ordinated inter-organ pathways, the gastrointestinal tract is involved in the synthesis, conversion and catabolism of amino acids to be used by other tissues in the body. In addition to this critical role in whole-body nutriture, the gut also requires vast amounts of particular amino acids for maintenance and growth. The profile of these amino acid requirements does not seem to parallel those for growth and maintenance of the rest of the body. Rather, because of the gut’s specific functions in digestion, absorption and immunity, the gut requires a different profile of amino acids. Quantifying these requirements has become an important goal in understanding the role of the gut in amino acid availability for the rest of the body. In particular, this availability becomes of paramount concern in situations of gut disease or stress where increased maintenance requirements can limit the availability of certain amino acids for whole-body growth and physiological functions.

2. METHODS TO MEASURE AMINO ACID REQUIREMENTS Many methods have been employed in humans and animals to determine amino acid requirements. The advantages and disadvantages of many of these techniques have been extensively reviewed by others (Lewis, 1992; Fuller and Garlick, 1994; Young and el-Khoury, 1995; Zello et al., 1995; Waterlow, 1999). Many of these methods were originally developed in animals and then modified for humans. However, it is important to note that most animal research on amino acid requirements has primarily focused on the growing phase for economic reasons, whereas human research has almost exclusively focused on the adult phase which comprises most of the lifespan. The basic strategy employed by almost all studies involved with the determination of amino acid requirements includes the feeding of graded levels of the test amino acid and the measurement of a specific biological response. The choice of biological response depends on many factors including species, age, health status, sample availability as well as ethical considerations, analytical equipment availability, financial constraints and practicality. In choosing the biological response, the most important aspect of amino acid metabolism to consider is the need for an amino acid for incorporation into protein. If all other essential nutrients, especially energy and other amino acids, are at or above requirement levels, then whole-body protein synthesis will occur at a level determined by the intake of the most limiting amino acid (i.e. the test amino acid). If the intake of this test amino acid is below its requirement, then protein synthesis will be reduced and the intake of all other essential amino acids will be in relative excess; because amino acids cannot be stored, this excess must be catabolized by the body and excreted as bicarbonate and ammonia via carbon dioxide and urea, respectively. Increasing the intake of the test amino acid will result in greater protein synthesis and the concomitant reduction in excess amino acid catabolism indicated by lower carbon dioxide and urea excretion. At intakes above its requirement, the test amino acid will no longer be the first limiting one and additional intakes will not result in greater protein synthesis. At these intakes, the test amino acid itself is in relative excess and must be catabolized to carbon dioxide and urea. This general scheme has been used to develop almost all techniques employed to determine amino acid requirements, including growth assays, serial slaughter, nitrogen balance, plasma amino acids, plasma urea, direct amino acid oxidation, amino acid balance, and indicator amino acid oxidation (fig. 1). It is also important to note that the chosen biological response should be amenable to statistical modelling techniques so that an objective estimate for an amino acid requirement can be determined, preferably with an estimate of population variance. Because the pattern of the biological response is rarely predictable over deficient to excess intakes of the test amino acid, several statistical models have been proposed and employed. The overall response is

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Fig. 1. Graphical representation of various response curves to increasing intakes of the test amino acid. The “breakpoint” requirement is usually determined using two-phase linear regression.

often argued, from a biological standpoint, to be a quadratic model; however, a two-phase linear regression breakpoint model sometimes fits better. In practice, when both models fit well, the breakpoint estimate is usually similar between techniques (Baker et al., 2002). Using either model, the requirement can be determined within individuals and then averaged for a requirement estimate with population variance. Or the model can be applied to a complete data set of many animals over many different intakes and the error of the fit could be used to predict the population variance. In any event, it is obvious that the more data are available for statistical manipulation, the more versatile the modelling can be. This issue of statistical manipulation is not a trivial one. Indeed, it has been recently demonstrated that re-analysis of the classic nitrogen balance studies of Rose and Jones yielded very different conclusions about the amino acid requirements in humans using the exact same data (Rand and Young, 1999; Di Buono et al., 2001a). These studies have clearly demonstrated that the importance of the chosen statistical model is almost as important as the data. 2.1. Growth assays Because the primary role of an amino acid is its incorporation into protein, the measurement of protein synthesis itself, during varying intakes of the limiting amino acid, can be considered to be the most direct of approaches. As such, growth assays, and more specifically the serial slaughter technique, have often been considered as the “gold standard” of techniques in the determination of amino acid requirements in animals. As the test amino acid intake is increased towards its requirement, then more protein is synthesized which leads to increased lean tissue deposition. In young animals with minimal fat deposition, growth is almost directly proportional to lean tissue deposition and the growth assay is appropriate. Obviously, as animals approach maturity, more fat is deposited and this proportional relationship between lean and body growth is not constant. To resolve this discrepancy, the serial slaughter technique employs body composition analysis to accurately determine lean tissue content. Using a reference group of animals analysed at the starting body weight, lean tissue accretion can be determined. However, the main drawback to this approach is the necessity of using

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different animals at different time points. Also, body tissue analysis for amino acids is not very accurate given the heterogeneity of ground body samples and the problems associated with protein-bound amino acid analysis. So although these techniques are simple and direct, they are limited to fast-growing lean animals with low and constant fat deposition or must involve large numbers of slaughtered growing animals of similar genetic background to minimize the inter-animal variation. These techniques are not useful in adult, non-growing animals or in animals with special conditions (i.e. gestation, lactation, egg production, disease, etc.).

2.2. Nitrogen balance When protein synthesis is limited, excess amino acids are catabolized to their metabolic endproducts which for all amino acids include bicarbonate and ammonia. The measurement of these excreted biological products provides an indirect and inverse measurement of changes in protein synthesis and thus can be used to determine amino acid requirements. Ammonia enters the nitrogen pool of the body and is excreted primarily as urea in mammals and as uric acid in birds. Because such excreta are relatively easy to collect and analyse for total nitrogen, the nitrogen balance method was one of the first to be developed for assessment of amino acid requirements in humans and animals. The amino acid requirement can be determined from either balance calculations or over a range of test amino acid intakes. The balance approach regresses nitrogen balance on test amino acid intake and defines the requirement as the intake level at which optimum balance is achieved (i.e. zero or positive balance in adults). A considerable drawback with balance calculations is the need to make an assumption of unmeasurable losses (sweat, skin, nails, hair, etc.). Indeed, some of the original landmark experiments by Rose and Jones to determine human amino acid requirements did not include such an assumption; when these data were later corrected for an estimate of these losses, amino acid requirement estimates more than doubled (Young and Marchini, 1990; Rand and Young, 1999). And because two large numbers are being subtracted (nitrogen intake and nitrogen output), the difference is relatively very small and this assumption becomes extremely important. An alternative approach not involving this correction is to measure the qualitative change in nitrogen balance over a range of test amino acid intakes. Nitrogen output is high when protein synthesis is limited by a single amino acid because the other amino acids are in relative excess and are catabolized. As the test amino acid is added incrementally to the diet, nitrogen output decreases until the requirement is met and further increments will not stimulate protein synthesis and nitrogen balance will remain constant (assuming isonitrogenous diets). The many technical advantages and disadvantages of the nitrogen balance technique have been extensively reviewed over the years (Waterlow, 1999; Tome and Bos, 2000) and will not be discussed here. However, several important points need to be mentioned in the present chapter. An important advantage of the nitrogen balance technique is that unlike growth assays, this method can be successfully applied to adult as well as young species. The major disadvantage of the technique is that because of the very large urea pool in most species, its response to dietary manipulation is rather slow and thus adaptations of a week or greater are generally required. If one is studying 6 or 7 test amino acid levels, this long adaptation results in a lengthy experimental period which is not useful in special physiological situations such as gestation, lactation or disease progression. In addition, this technique is also inappropriate in vulnerable populations where long-term feeding of deficient diets is not ethical. The nitrogen balance technique has been extensively applied for all indispensable amino acids in many species.

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2.3. Direct amino acid oxidation technique As opposed to the nitrogen balance technique, oxidation methods monitor the excretion of carbon dioxide, the other obligatory end-product of amino acid catabolism. However, the basic principle is similar in concept to other methods to determine amino acid requirements. At deficient test amino acid intakes, the test amino acid is efficiently utilized for protein synthesis and its oxidation is low and constant. At intakes above requirement, protein synthesis is maximized and excess test amino acid is preferentially oxidized to its end-products. Instead of measuring changes in nitrogen excretion as in the nitrogen balance technique, amino acid oxidation methods measure changes in carbon dioxide excretion in breath. The use of isotopically labelled amino acids allows for an extremely sensitive means of measuring small changes in amino acid oxidation in response to changes in intake. Although all carbons of amino acid skeletons are eventually oxidized, the most sensitive approach is to monitor the expiration of the cleaved carboxyl group at the 1-carbon position. Thus, oxidation of uniformly labelled amino acids results in distribution of the label among many metabolites which makes interpretation difficult and somewhat less sensitive. Alternatively, the oxidation of carboxyl-labelled amino acids is more direct and easier to interpret provided the decarboxylation step is irreversible. However, because one is measuring carbon dioxide in breath, the carboxyl group must also enter general bicarbonate pools which equilibrate readily with carbon dioxide expiration at the lungs. For example, experiments with labelled threonine and methionine have found that non-linear responses are typical with infusion of these amino acids over varying intakes as a result of their more complex degradative pathways (Chavez and Bayley, 1976; Zhao et al., 1986; Storch et al., 1988; Ballevre et al., 1990). Thus, as summarized by Zello et al. (1995), there are general criteria for choosing appropriate carboxyllabelled amino acids for oxidation studies: (1) the amino acid must be indispensable; (2) it must be primarily partitioned between oxidation to carbon dioxide and protein incorporation; and (3) the labelled carboxyl group must be irreversibly oxidized and sufficiently equilibrated with labelled carbon dioxide in breath. These restrictions adequately apply for some indispensable amino acids such as phenylalanine (provided excess dietary tyrosine is fed), lysine and the branched-chain amino acids; as expected, these amino acids have been used extensively in direct oxidation studies. Although the carboxyl group of methionine is irreversibly oxidized, methionine can equilibrate reversibly with homocysteine prior to the irreversible oxidative pathway, thus complicating interpretation of oxidation data. Brookes et al. (1972) were the first to use the oxidation of isotopically labelled amino acids to determine amino acid requirements in rats. Since then, the direct oxidation technique has been used to determine the requirements of several amino acids in growing rats (Kang-Lee and Harper, 1977, 1978; Harper and Benjamin, 1984), adult rats (Simon et al., 1978), young pigs (Kim et al., 1983a,b; Ball and Bayley, 1984, 1986; House et al., 1997a,b), infants (Roberts et al., 2001a) and adult humans (Meguid et al., 1986a,b; Meredith et al., 1986; Zhao et al., 1986; Zello et al., 1990). For animals, the direct oxidation technique has yielded similar or slightly lower amino acid requirements compared to “classical” techniques such as nitrogen balance and growth assays; this finding validates the approach to a certain extent. In contrast, direct oxidation studies in adult humans have yielded much higher (i.e. 2- to 3-fold) requirements for all tested amino acids compared to those proposed by the FAO/WHO/UNU (1985) based upon nitrogen balance studies. This latter discrepancy has been extensively debated and reviewed (Young and Borgonha, 2000). However, one cannot ignore the problems demonstrated in the interpretation of the small amount of original balance data (Rand and Young, 1999). Indeed, the human lysine requirement estimated from the re-analysed

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original nitrogen balance data (Rand and Young, 1999) actually agrees very well with the estimates derived from various techniques using direct oxidation (Meredith et al., 1986), indicator amino acid oxidation (Zello et al., 1993; Duncan et al., 1996), 24 h oxidation (el-Khoury et al., 2000) and indicator amino acid balance (Kurpad et al., 2001). In addition, animal studies employing nitrogen balance techniques, which are numerous and well controlled, result in more reliable and agreeable results compared to those of more recent kinetic techniques. In any event, the direct oxidation technique provides as biologically valid an approach as the nitrogen balance technique and generally agrees with the growth assays performed to date in animals. A very important advantage of the oxidation method compared to the nitrogen balance technique is the more rapid adaptation of the biological response to test amino acid intake changes. Initial studies in direct amino acid oxidation fed particular test amino acid levels for 7–10 days prior to oxidation measurement, analogous to nitrogen balance studies. However, more recently it has been demonstrated that prior adaptation to amino acid intake (Zello et al., 1990; Motil et al., 1994) does not affect the breakpoint estimate of its requirement using the direct oxidation approach. Therefore, only hours of adaptation to a deficient or excess level of test amino acid seems to be necessary to measure changes in oxidation, and thus to determine requirement. To our knowledge, there have been no studies in animals designed to address this adaptation issue using the direct oxidation technique. However, we have successfully employed the direct oxidation technique to measure phenylalanine requirement in parenterally fed piglets using only 16 h of adaptation prior to oxidation measurement (House et al., 1997a). The adaptation issue has been carefully addressed in indicator amino acid oxidation studies in animals and will be discussed below. It is important to note that this issue of sufficient adaptation is the subject of considerable debate and has been reviewed (Young and Marchini, 1990). With long-term adaptation to a deficient diet, the subject will “accommodate” to this situation and possibly become more efficient in its metabolism. The question is: does this accommodation come at the cost of other unmeasured metabolic functions? If such costs were incurred, then it violates Waterlow’s (1985) reasonable definition of adaptation: the process that permits the organism to respond to a dietary change without adverse consequences. We also need to consider the definition of amino acid requirement which has been proposed by Young and Borgonha (2000) as the minimal intake level needed to maintain a specific nutritional criterion such as growth, body composition, body amino acid balance, organ or system function, etc: the choice of nutritional criterion then becomes the subject of debate. In spite of this ongoing debate, the studies in humans have methodically shown that the direct oxidation method has the distinct advantage over the nitrogen balance technique of very short adaptation periods resulting in more time-efficient and cost-effective studies. Although the direct oxidation technique has provided a more sensitive approach to determination of amino acid requirements, its disadvantages have limited its widespread use in many species. Most importantly, as mentioned above, not all indispensable amino acids can be easily used for direct oxidation measurements, limiting its general application. More specifically, of the indispensable amino acids, only phenylalanine (with excess dietary tyrosine), methionine, lysine and the branched-chain amino acids undergo irreversible oxidation of the carboxyl carbon so that amino acid oxidation can be calculated from expired breath carbon dioxide. However, Young and colleagues have attempted to predict the requirements of the other amino acids using previously determined tracer techniques, composition of body proteins and assumed obligatory oxidative amino acid losses (Young and el-Khoury, 1995); these predictions were subsequently validated (Raguso et al., 1999). An additional criticism

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from a kinetic standpoint is that the feeding of deficient to excess amounts of test amino acid changes that amino acid’s pool size dramatically, thereby diluting the tracer being measured. This variable dilution of the tracer in the pool increases variability and reduces sensitivity of oxidation measurements and hence requirement estimates. 2.4. Indicator amino acid oxidation technique The indicator amino acid oxidation (IAAO) technique is an extrapolation of the earlier work with the direct oxidation technique. Both require the accurate measurement of amino acid oxidation by collecting isotopically labelled carbon dioxide in breath. As with the direct oxidation method, the IAAO method is based on the hypothesis that the partitioning of amino acid metabolism between incorporation into protein and catabolism via oxidation is determined by the most limiting amino acid in the diet. However, the difference between the techniques is that instead of measuring oxidation of the test amino acid, the IAAO method measures the oxidation of one of the other amino acids that is also responding to changes in protein synthesis. When the test amino acid is deficient, protein synthesis is limited and other amino acids are in excess. Indispensable amino acids in excess must be catabolized at a level inversely reflecting the rate of protein synthesis which is dictated by the test amino acid intake. By monitoring the oxidation of one of these “indicator” amino acids over a range of test amino acid intakes, one can estimate the test amino acid requirement for protein synthesis. As intake of the test amino acid increases towards requirement, protein synthesis increases which utilizes more of the indicator amino acid resulting in a smaller excess and lower oxidation. Once the test amino acid intake equals requirement, then greater intakes of this amino acid will not lead to greater protein synthesis and therefore indicator amino acid oxidation remains constant. The choice of indicator amino acid depends on its metabolic characteristics. Phenylalanine (see below), lysine (Ball and Bayley, 1984; Roberts et al., 2001b) and leucine (Kurpad et al., 2001) have been used with success to determine various amino acid requirements. Methionine (Brookes et al., 1972) has been employed unsuccessfully due to its complicated metabolic pathways, as mentioned previously. When the requirement of a test amino acid has been determined using more than one of the indicators, the requirement estimates were very similar (Ball and Bayley, 1984; Zello et al., 1993; Kurpad et al., 2001). Phenylalanine has been used most often as the indicator with successful determinations of the requirements for lysine (Kim et al., 1983a; House et al., 1998a), histidine (Kim et al., 1983b), threonine (Kim et al., 1983a; Bertolo et al., 1998), tryptophan (Cvitkovic et al., 2000), methionine and total sulphur amino acids (Kim and Bayley, 1983; Shoveller et al., 2001), branched-chain amino acids (Elango et al., 2002a), arginine (Ball et al., 1986), proline (Ball et al., 1986) and total protein (Ball and Bayley, 1986) in piglets, tryptophan in trout (Were, 1989), lysine in chickens (Coleman et al., 2002), lysine in growing pigs (Bertolo et al., 2001), and in humans, lysine (Zello et al., 1993; Duncan et al., 1996; Kriengsinyos et al., 2002), threonine (Wilson et al., 2000), tryptophan (Lazaris-Brunner et al., 1998), methionine (Di Buono et al., 2001b), total sulphur amino acids (Di Buono et al., 2001a), branched-chain amino acids (Mager et al., 2001, 2002; Riazi et al., 2001) and tyrosine (Bross et al., 2000; Roberts et al., 2001b). The IAAO technique to determine amino acid requirements was first developed in the neonatal piglet by Bayley and colleagues. Following observations that amino acid catabolism depends on the balance of other amino acids (Brookes et al., 1972; Newport et al., 1976), these researchers successfully demonstrated that the IAAO technique can be used to determine the amino acid requirements in piglets. In particular, Kim et al. (1983b) showed that the

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estimate for histidine requirement in piglets was similar using both the direct oxidation and IAAO techniques with phenylalanine as the indicator. In addition, Ball and Bayley (1984) found that either phenylalanine or lysine could be used as an indicator because the oxidation of both responded to varying tryptophan intakes similarly. To further validate the theoretical concept, Ball and Bayley (1984) also demonstrated that liver protein synthesis was inversely correlated with phenylalanine oxidation. In addition, this research group demonstrated that the amino acid requirements for piglets determined using the IAAO technique, namely histidine (Kim et al., 1983b), sulphur amino acids (Kim and Bayley, 1983), lysine and threonine (Kim et al., 1983a), tryptophan (Ball and Bayley, 1984), proline and arginine (Ball et al., 1986) and total protein (Ball and Bayley, 1986), agreed very closely with those determined by classical techniques (NRC, 1979, 1998). From the initial studies of Bayley and colleagues, the IAAO method has been subsequently refined and expanded from the original approach. All indispensable amino acids have been tested using this method; this aspect is the main advantage of the IAAO method over the direct oxidation technique. In addition, the aforementioned criticism of the direct oxidation technique regarding amino acid pool size does not apply to the IAAO approach. Indeed, because the indicator amino acid is fed at the same level over varying test amino acid intakes, its pool size is not permitted to change and therefore the tracer is not variably diluted. This unchanging pool size is probably the main reason why the IAAO approach tends to give requirement estimates with less variability compared to direct oxidation estimates. As with the direct oxidation technique, the adaptation period required to a particular test amino acid intake has been shown to be minimal. Indeed, in recent human studies, the lysine requirement was similar whether hours (Zello et al., 1993; Duncan et al., 1996; Kriengsinyos et al., 2002), 7 days or 21 days of adaptation (Kurpad et al., 2002b) were employed. This finding is profound in context with the aforementioned ongoing debate about adaptation versus accommodation. Furthermore, this adaptation seems to be relatively insensitive to body size or growth. We have also recently found that phenylalanine oxidation after 1.5 days of adaptation (the shortest adaptation tested) to a high or deficient lysine diet was not different up to 8 days of adaptation in both 25 kg growing pigs and 250 kg sows (Bertolo et al., 2001). This distinct advantage of short adaptation over the classical nitrogen balance or growth assays allows great versatility in the application of oxidation techniques, especially to vulnerable populations (Brunton et al., 1998). Indeed, we have recently determined the branched-chain amino acid requirement of children with the inherited genetic disorder, maple syrup urine disease (unpublished), as well as the tyrosine requirements of parenterally fed infants (Roberts et al., 2001a) and the phenylalanine (unpublished) and tyrosine (Bross et al., 2000) requirements of children with phenylketonuria. Because an estimate of population variance is critical to recommending amino acid requirements, the more data in the breakpoint model, the better. Because of the short adaptation time associated with the IAAO method, it is possible to measure amino acid requirements in individuals. To accurately determine a breakpoint in a two-phase linear regression model, at least six test amino acid intakes should be included (i.e. three oxidation measurements per regression). In the nitrogen balance technique, this would require at least 6 weeks of experimentation assuming 7 days of adaptation per diet. Because we have shown that only 1.5 days of adaptation were necessary in the IAAO method (Bertolo et al., 2001), we recently determined the lysine requirement of individual growing pigs in 2 weeks by changing diets and measuring indicator oxidation every other day (Moehn et al., 2001). Similarly, we have demonstrated that individual amino acid requirements of chickens can be determined in less than 3 weeks (Coleman et al., 2002). With the determination of enough individual amino acid

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requirements, an accurate population variance can be calculated; for animal species, this technique allows such a calculation for the first time. Indeed, more accurate diet formulation and animal performance can be achieved with knowledge of such an error within a genetic population of animals. In addition, long-term genetic improvement can also be achieved if animals with low amino acid requirements, or more efficient utilization of dietary amino acids, could be selected and subsequently bred. The obvious potential for such a technique to be exploited by various animal production groups is enormous. 2.5. 24 hour oxidation/indicator amino acid balance The balance technique is based on the principle that in non-growing adults, protein synthesis is balanced with protein breakdown and thus protein or nitrogen intake is balanced with nitrogen excretion. These two balances are connected through the free amino acid pool which is relatively tightly regulated and represents a minute proportion of total body nitrogen. This relationship between the two balances is often expressed by the steady-state flux equation proposed by Waterlow et al. (1978): flux (Q) = synthesis (S) + oxidation/excretion (O) = breakdown (B) + input (I). This equation is rearranged so that protein balance (S − B) = input/output balance (I − O). This latter equation is the basis of the nitrogen balance calculation technique as well as the amino acid balance technique. A recent adaptation of the oxidation techniques incorporates the balance concept described by Waterlow et al. (1978). Young and colleagues have developed a new method involving a 24 h infusion of amino acid tracer and the measurement of labelled carbon dioxide output in adult humans (el-Khoury et al., 1994a,b, 1995). These data are then used to calculate carbon balance at different levels of test amino acid intake. The requirement is taken as the minimal amino acid intake necessary to maintain balance. This amino acid balance is the difference between the intake of the test amino acid and whole-body oxidation of that amino acid. This approach was first employed to determine the leucine requirement in adult humans which compared very well with their previous direct oxidation experiments (Young et al., 1989). Subsequently, the technique was successfully used to verify direct oxidation determinations of aromatic amino acid (Basile-Filho et al., 1997, 1998; Sanchez et al., 1995, 1996) and lysine (el-Khoury et al., 1998, 2000) requirements. These experiments also demonstrated that when the test amino acids were fed at the FAO/WHO/UNU (1985) requirement levels, subjects were in significantly negative amino acid balance, indicating that the present accepted requirements are too low (Young and Borgonha, 2000). This approach was advanced by the same group by applying the IAAO technique. Using 24 h labelled leucine infusions, lysine (Kurpad et al., 2001, 2002b) and threonine (Kurpad et al., 2002a) requirements were determined by measuring leucine oxidation and balance over a range of test amino acid intakes. These new techniques are particularly suited for non-growing adults and account for amino acid metabolism in both the fasting and fed states. However, fasting-state amino acid kinetics are not relevant to young suckling animals (Bertolo et al., 2000a). Furthermore, this approach has only been used in humans and may not be widely applicable to fast-growing meat-producing animals that are in continuous positive nitrogen balance due to high rates of lean tissue deposition. As with the nitrogen balance calculation technique, the most important criticism of the amino acid balance techniques is the reliance on absolute calculations based on various assumptions. With amino acid kinetic calculations, the most important assumption is that the precursor amino acid enrichment in readily accessible body pools (i.e. plasma) is representative of the enrichment of the true intracellular precursors for protein synthesis (i.e. tRNA)

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and oxidation; this assumption is almost always untrue in constant infusion experiments (Wolfe, 1992). Intracellular exchange of labelled amino acids is incomplete and variable among tissues. Indeed, plasma free amino acid enrichments have been found to be 2 to 3 times greater than those for corresponding amino acyl-tRNA enrichments for leucine, lysine and phenylalanine (Caso et al., 2001). Because α-ketoisocaproate (KIC) is synthesized from intracellular leucine only and is released easily into plasma, KIC enrichment has often been proposed as a suitable representation of intracellular leucine enrichments. However, several studies have shown that KIC enrichment is not in equilibrium with the entire intracellular leucine pool (Cobelli et al., 1991; Chinkes et al., 1993) or leucine-tRNA in pigs (Baumann et al., 1994) or rats (Watt et al., 1991). Alternatively, others have used enrichments of amino acids that have been incorporated into apolipoprotein B-100 (Reeds et al., 1992; Cayol et al., 1996; Stoll et al., 1999), fibrinogen (Bennet and Haymond, 1991; Stoll et al., 1999) or albumin (Cayol et al., 1996). These very rapidly synthesized plasma proteins of hepatic origin can reflect isotopic steady state within hours. These enrichments are much lower than KIC enrichments but similar to tRNA measurements. Another issue is the possibility that precursors for oxidation may not equilibrate with tRNA pools, either intracellularly or between tissues, so that different enrichments may need to be measured to accurately calculate balance. The amino acid balance techniques will need to address this precursor enrichments issue. An advantage of the “relative” techniques comparing biological outcomes across dietary intakes is the avoidance of absolute assumptions. Indeed, in direct oxidation and IAAO analysis of amino acid requirements, the most reliable estimate of requirement with the lowest error is when percent dose oxidized is used as the biological outcome (House et al., 1997a,b, 1998a; Bertolo et al., 1998; Lazaris-Brunner et al., 1998; Bross et al., 2000; Roberts et al., 2001a,b). This outcome is in contrast to equivalent measurements of oxidation rate, which employ flux calculations that also use assumptions about the precursor pool. Ultimately, the “black box” approach of total labelled carbons in and total labelled carbons out provides the most reliable requirement estimates. Despite these methodological comparisons, it is important to reiterate that the amino acid balance studies to date have calculated amino acid requirements that are very close to those determined by the more qualitative direct oxidation and IAAO techniques. Therefore, the error associated with these kinetic assumptions and calculations may not be as significant as some have proposed.

3. RECENT DEVELOPMENTS IN THE INDICATOR AMINO ACID OXIDATION TECHNIQUE Considering that all of the techniques used to determine amino acid requirements generally agree in their final estimate, one must choose the most versatile method available for wider application. Given the short adaptation time, applicability to all indispensable amino acids, ease of biological sampling (i.e. breath) and applicability to most populations, the IAAO technique has been successfully adapted for use in a variety of situations and purposes. An important advance in the method was its adaptation for use in parenterally fed piglets as models for parenterally fed infants (Wykes et al., 1993; Bertolo et al., 1998; House et al., 1998a). The original work by Bayley in piglets employed one or two oral bolus feeds which included the isotope dose; the total labelled carbon dioxide excretion was not kinetically quantified, but rather relatively compared over different test amino acid intakes. With parenterally fed piglets, primed constant intravenous infusions of labelled indicator amino acid were introduced to acquire added information through kinetic calculations. With these studies, it was shown that the indicator (phenylalanine) flux rate was unchanging across test amino

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acid intakes (Bertolo et al., 1998; House et al., 1998a). In addition, it was demonstrated that, statistically, percent dose oxidized was the most reliable estimate in terms of variability (House et al., 1997a,b, 1998a; Bertolo et al., 1998); this finding has been confirmed in human studies (Lazaris-Brunner et al., 1998; Bross et al., 2000; Roberts et al., 2001a,b). Recently, we have also directly compared amino acid requirements using either intravenous or oral/gastric infusion of labelled phenylalanine as the indicator amino acid. The breakpoint estimates of the lysine requirement in adult humans were the same whether the indicator was delivered intravenously or orally (Kriengsinyos et al., 2002); similarly, the tryptophan requirements in gastrically fed piglets were similar with intravenous or intragastric infusion of the indicator (Cvitkovic et al., 2000). These subsequent methodological developments were critical in adapting the IAAO technique for vulnerable populations. In order to use the IAAO technique in infants and children, the dietary interventions must be short and the sample collections must be non-invasive. We have recently validated the use of oral isotope dosing and collection of urinary amino acids to measure enrichment as representative of plasma enrichment (Bross et al., 1998). Study diets were fed hourly for 4 h prior to dosing and subsequent half-hourly meals with isotope led to enrichment plateaux within 2 h. This simple non-invasive protocol can be used to study many different vulnerable populations, provided that dietary ingestion and breath collection are feasible. Similarly, this oral dosing protocol has been shown to be very effective in determining amino acid requirements in large pigs where the implantation and maintenance of catheters can be problematic (unpublished data). In addition, such a simplified protocol allows for a broader application of the technique to other experimental models. Such models include neonatal and adult animals, gestating or lactating animals, as well as compromised populations which include disease or surgical interventions. The IAAO technique can also be applied to research investigating other aspects of protein metabolism where the goal is not simply to determine amino acid requirements. Because indicator amino acid oxidation responds to protein synthesis, intracellular changes in test amino acid availability are reflected in changes in indicator oxidation. Recently we have exploited this principle by adapting the technique to determine true metabolic availability of lysine from feedstuffs in growing pigs (Ball et al., 2001). We designed a low-lysine diet with all other indispensable amino acids above requirements. With incremental additions of synthetic lysine, which is assumed to be 100% available, phenylalanine oxidation declined linearly until the requirement was met. Within this deficient range of lysine intakes, we used the linear response equation to predict true availability of lysine from added feedstuffs (fig. 2). When peas were added to the low (50% of requirement) lysine diet so that the total true available lysine content was 90% of lysine requirement, the phenylalanine oxidation corresponded to the availability predicted by ileal digestibility estimates (i.e. the true available amount according to NRC, 1998). When peas were heated to render some lysine unavailable via Maillard products, the phenylalanine oxidation increased and corresponded to an availability of 50% of unheated peas. When synthetic lysine was added to the heated peas, phenylalanine oxidation decreased below that determined with 90% of requirement, demonstrating that the increase in oxidation due to heating was due solely to loss of available lysine. This technique is a new, rapid approach to determining true metabolic availability of lysine that does not require a series of tenuous assumptions about endogenous losses, which are problematic to the true ileal digestibility method. This novel application of IAAO principles to improve estimates of amino acid availability has demonstrated the technique’s versatility. We have also used the basic qualitative principle of the IAAO technique to determine whether amino acid formulations are inadequate. Recently, by adding amino acids suspected

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Fig. 2. Representation of novel technique to determine metabolic amino acid availability by measuring indicator amino acid oxidation.

of being deficient to TPN solutions and monitoring lysine (the indicator) oxidation, we have systematically demonstrated that the amino acid profile of some commercial TPN solutions are inadequate (unpublished data). This approach can be further adapted to simulate the strategy pioneered by Baker and colleagues to determine amino acid requirements by dietary amino acid supplementation and deletion by employing the ideal ratios to lysine (Mavromichalis et al., 1998). The important advantage of the IAAO approach is that multiple modifications can be made to the diets of individual animals because short oxidation measurements are used, as opposed to the more time-consuming growth assays. Because of the relatively simple and direct biological response in the IAAO technique, many more extrapolations and adaptations of the original technique will probably be developed in the future.

4. INTESTINAL FIRST-PASS METABOLISM In addition to being responsible for the digestion and absorption of nutrients, the gut is also a major metabolic organ in the body, responsible for the synthesis, conversion and degradation of amino acids. The gut has a very high metabolic activity and extracts a significant proportion of the absorbed dietary and endogenous amino acids before transport to the portal circulation and the rest of the body. Indeed, although the portal-drained viscera (PDV) (intestines, pancreas, spleen, stomach) represents only ~5−7% of body mass, these tissues disproportionately account for 20–35% of whole-body energy expenditure and protein synthesis (Lobley et al., 1980; McNurlan and Garlick, 1980; Burrin et al., 1990). This significant extraction of dietary amino acids by splanchnic tissues has been demonstrated by comparing amino acid kinetics when isotopes are delivered intravenously or orally. The “splanchnic disappearance” of labelled amino acids when given orally has led many researchers to speculate on the fate of this irreversible loss of label in kinetics experiments. From these types of studies, several researchers have determined that in humans and animals, the splanchnic tissues metabolize between 20% and 50% of dietary essential amino acids (leucine, lysine, phenylalanine) on first-pass (Yu et al., 1990, 1995; Hoerr et al., 1991, 1993; Biolo et al., 1992; Matthews et al., 1993; van Goudoever et al., 2000). In addition, with low-protein diets, this extraction is as high as 70% of lysine intake (van Goudoever et al., 2000). Albeit the data are very impressive, one problem with this approach is that it is difficult to separate the metabolism of the liver from the PDV, although this can be overcome by measuring amino acid

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enrichments and flow in the portal vein (van Goudoever et al., 2000). An additional problem with the technique is that these types of studies vastly underestimate arterial extraction of recirculating enteral isotope and hence overestimate first-pass extraction. Indeed, the elegant study by van Goudoever et al. (2000) demonstrated that a 46% portal mass balance extraction corresponded to a 22% isotopic extraction by the PDV, which when corrected for arterial recirculation amounted to insignificant utilization of dietary lysine on first-pass. The relative importance of nutrient processing by the small intestine (the predominant PDV organ) versus the liver has only recently been elucidated. Indeed, we have demonstrated that the small intestine is more important than the liver in modifying nitrogen utilization when pigs are chronically fed by central vein, portal vein or stomach (Bertolo et al., 1999). In addition, other researchers have demonstrated that intestinal metabolism dominates splanchnic metabolism of phenylalanine (Stoll et al., 1997) and lysine (van Goudoever et al., 2000) in pigs and leucine in dogs (Yu et al., 1990). In addition to dietary extraction of amino acids, the gut also transports an enormous amount of amino acids from the arterial circulation, especially during the post-absorptive state. Isotopic data describing the incorporation of amino acids from both arterial and dietary sources have demonstrated that both sources of precursor amino acids are critical and that the partitioning between them is dependent on specific amino acid and dietary protein level (MacRae et al., 1997; Stoll et al., 1999; van Goudoever et al., 2000). Recent work by van Goudoever et al. (2000) has determined that with high-protein feeding, almost all of the lysine utilized by the PDV was of arterial origin, whereas with low-protein feeding, approximately half of the lysine utilized was from both arterial and dietary sources. It is important to note that level of protein feeding did not affect total lysine use by the PDV, demonstrating an enormous obligatory utilization of amino acids by the gut for normal function and growth. Because of this high obligatory protein turnover in the gut, it follows that with gut challenges (i.e. tissue damage, increased growth, pathogen exposure, dietary anti-nutritional factors, etc.) the amino acid requirements of the gut increase. Indeed, first-pass intestinal extraction of amino acids is proportional to mucosal mass. Stoll et al. (1997, 1999) demonstrated that phenylalanine splanchnic extraction was 50% higher in pigs raised outdoors, where rooting and pathogen challenges are greater, compared to pigs raised in a relatively clean indoor research facility; this higher extraction correlated with measured mucosal mass. Infestation of pathogens is known to affect growth rate as well as gut function. The stimulation of whole-body and gut immune systems must impart a protein synthetic cost to the infected animal. Indeed, sepsis in rats has been shown to stimulate intestinal protein synthesis (von Allmen et al., 1992; Higashiguchi et al., 1994b); in particular, sepsis stimulates the synthesis of endogenous and secretory proteins, including certain gut peptides, in small intestine mucosa (Higashiguchi et al., 1994a). More recently, Yu et al. (2000) have shown that subclinical nematode infection in sheep increased total gastrointestinal tract leucine sequestration by 24% and gastrointestinal tract oxidative losses of leucine by 22–41%. In another study in pigs, the infusion of endotoxin led to enhanced intestinal catabolism of amino acids (Bruins et al., 2000). These intriguing studies suggest a possible mechanism for the growth-stimulating effect of feed-grade antibiotics; increased protein synthesis during infection by the already metabolically demanding intestinal tissues limits the availability of amino acids for extraintestinal lean tissue growth. With the impending discontinuation of prophylactic feeding of antibiotics in animal production, the prevention of subclinical infections or challenges needs to be a priority in the development of alternative strategies in the near future.

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The primary fate of indispensable amino acids is presumably to protein synthesis; however, recent intriguing data have demonstrated that catabolism dominates the first-pass utilization of these amino acids by the gut (Stoll et al., 1998; Wu, 1998; van Goudoever et al., 2000). This seemingly wasteful oxidation of indispensable amino acids amounts to a small but significant proportion of dietary intake (Yu et al., 1992, 2000; Cappelli et al., 1997; van Goudoever et al., 2000), but a large proportion of whole-body amino acid oxidation (van Goudoever et al., 2000). Indeed, we have recently demonstrated that phenylalanine oxidation is significantly greater when labelled phenylalanine is delivered orally, as opposed to intravenously in the IAAO technique (Cvitkovic et al., 2000; Kriengsinyos et al., 2002). This increased oxidation during feeding of adequate diets demonstrates that first-pass catabolism of phenylalanine by the gut is significant. Whatever the fate of indispensable amino acids extracted by the gut, the evidence clearly suggests that this tissue plays a significant role in modulating the profile of amino acids delivered to the rest of the body (Stoll et al., 1998; Wu, 1998; Bertolo et al., 2000b). Much of this role results in a net loss of amino acids to extraintestinal tissues. Presumably, when gut metabolic activity is increased by growth or stress, so is the loss of dietary amino acids from whole-body functions. This aspect of whole-body amino acid requirements has not been fully explored. In addition to gut metabolic activity, the quality of dietary protein (Deutz et al., 1998; Gaudichon et al., 1999; Mariotti et al., 1999) and type of dietary carbohydrate (van der Meulen et al., 1997) also influence first-pass extraction of amino acids. So the actual availability of dietary amino acids for muscular protein synthesis is highly dependent on the metabolic activity of intestinal tissues. It is important to note that this concept is accommodated by our recent adaptation of the IAAO technique mentioned above for determination of true metabolic availability of dietary amino acids from heat-treated feedstuffs. Given the significant demand of the gut for dietary and arterial amino acids, it is obvious that the maintenance and growth of this organ already constitutes a significant proportion of whole-body amino acid requirements. Furthermore, it follows that in certain situations that increase the metabolic activity of the gut, this proportion will increase at the expense of whole-body growth. Indeed, this hypothesis is supported by the abovementioned studies in subclinical nematode infection and the reduced growth rate commonly observed in gastrointestinal disease. If the availability of an indispensable amino acid is already limiting in an animal’s diet, then an unobservable subclinical challenge to the gut could feasibly limit animal growth further. Although this concept seems intuitive, it is very difficult to demonstrate experimentally.

5. IMPACT OF INTESTINAL METABOLISM ON AMINO ACID REQUIREMENTS Many of the approaches recently employed to demonstrate changes in amino acid utilization by the gut or PDV tissues could be adapted to quantify amino acid requirements of these tissues relative to whole-body requirements. It has been well demonstrated that the proportion of dietary amino acids extracted by the gut changes with dietary composition; but a systematic approach to dietary ingredient requirements for the gut has yet to be published. By employing graded intakes of indispensable amino acids, it is possible to determine the minimum amount required by the gut for normal function and growth. To date, our body of literature regarding amino acid requirements during intravenous or intragastric feeding in piglets provides the only attempt, to the authors’ knowledge, to quantify requirements with and without first-pass splanchnic metabolism.

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5.1. The parenterally fed piglet as a model of splanchnic metabolism The emerging evidence that the gut utilizes a significant proportion of dietary amino acids led us to speculate that in situations of gut bypass or stress, the whole-body amino acid requirements must be different. We proposed that total parenteral nutrition (TPN), which bypasses first-pass metabolism by the small intestine and liver and can cause gut atrophy (Johnson et al., 1975; Goldstein et al., 1985; Alverdy, 1995; Bertolo et al., 1999; Burrin et al., 2000), would lead to changes in amino acid requirements that can be measured. Indeed, we (Duffy and Pencharz, 1986; Bertolo et al., 1999) and others (Sim et al., 1979; Lanza-Jacoby et al., 1982; Jeevanandam et al., 1987) have shown that TPN feeding alters whole-body nitrogen metabolism compared to oral feeding. The different nitrogen utilization as a consequence of parenteral feeding may be due to reduced gastrointestinal metabolism associated with gut atrophy and/or due to lack of hepatic first-pass metabolism. With the development of the TPNfed piglet model (Wykes et al., 1993), we subsequently demonstrated using three infusion routes that intestinal atrophy has a greater impact on nitrogen metabolism than liver bypass (Bertolo et al., 1999). Whatever the fate of indispensable amino acids in the gut, an atrophied gut will utilize fewer amino acids and thereby affect whole-body requirements. Therefore, we have proposed that the TPN-fed piglet is a “gut-deficient” model. So the amino acid requirements of TPN-fed piglets approximate the requirements for extraintestinal tissues. When compared to a piglet gastrically fed identical diets, the amino acid requirements for the intact gut could at least be estimated (table 1). However, TPN feeding is only one of many relevant clinical scenarios that lead to compromised gut metabolic capacity. Gut dysfunction can also be caused by malnutrition, diarrhoea, chemotherapy, gastrointestinal surgery and gastrointestinal diseases. Furthermore, with relevance to the animal industry, gut stress is often associated with weaning, especially

Table 1 Effects of gut atrophy and bypass on whole-body amino acid requirements as determined in parenterally and enterally fed piglets using the indicator amino acid oxidation technique

Amino acid Threonineb Methioninec Total sulphursc Branched-chaind Tryptophane Lysinef Phenylalanineg Tyrosineh a

Oral requirement (g/kg/d)

Parenteral requirement (g/kg/d)

0.42 0.25 0.42 2.64 0.13 and 0.11 0.85 0.50 0.30

0.19 0.18 0.29 1.53 0.14 0.79 0.45 0.35

Gut bypass effecta (%) 55 28 31 42 0 7 10 0

This effect includes atrophy from 7 d on TPN as well as bypass of first-pass gut metabolism. Bertolo et al. (1998). c Shoveller et al. (2001). Methionine requirement was determined with excess dietary cysteine; total sulphurs refer to the methionine requirement determined with no dietary cysteine. d Elango et al. (2002a). Leucine, isoleucine and valine dietary ratio (NRC, 1998) was maintained across intakes. e Cvitkovic et al. (2000). Tryptophan requirement using intravenous (0.13) or oral isotope (0.11). f House et al. (1998a). Oral requirement for lysine was estimated from NRC (1998). g House et al. (1997b). Parenteral phenylalanine requirement was determined using direct oxidation technique; oral phenylalanine requirement was estimated from NRC (1998). h House et al. (1997a). Oral tyrosine requirement was estimated from NRC (1998). b

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with the increasingly popular early weaning practice in swine production. Any clinical situation which diminishes gut capacity would affect the metabolism of many amino acids; for example, increased amino acid requirements discovered during TPN feeding should also be applied to the treatment of any of the above conditions. Thus, our research has relevance to many conditions in addition to the TPN-fed neonate. However, we chose to use the TPN-fed piglet as a model for diminished small intestinal capacity because of demonstrated gut atrophy, reproducibility and ease of development compared to gut disease models. With this clinically relevant model, we could extrapolate our results to any situation involving gut dysfunction. 5.2. Threonine Perhaps the most impressive effect of reduced gut metabolism on whole-body amino acid requirement was demonstrated for threonine. Using the TPN-fed versus gastrically fed piglet, we have demonstrated that the threonine requirement is reduced by 55% when the gut is bypassed and atrophied (Bertolo et al., 1998). These data are supported by Stoll et al. (1998), who also observed that the PDV tissues extract 60% of dietary threonine measured by both net portal balance and labelled threonine extraction. In addition, van Goudoever et al. (2000) showed that when pigs were fed the high-protein diet, 84% of threonine was retained by the gut, and on the low-protein diet, all of the threonine was retained. This enormous demand for threonine by the gut is probably reflected by its role in mucin synthesis for maintenance of the luminal mucus layer (Lamont, 1992). Intestinal mucins are continuously secreted by the intestines and are critical in the defence of the mucosa from mechanical and pathogenic insults. The core protein of mucins contains a disproportionate amount of threonine, proline and cysteine (Specian and Oliver, 1991). With parenteral feeding and gut atrophy, mucin synthesis is reduced and so is the gut’s requirement for threonine. Recently we have demonstrated that feeding threonine-deficient diets to gastrically fed piglets reduces gut growth and goblet cell numbers and alters the mucin profile of intestinal mucus; in addition, parenteral threonine cannot completely restore normal gut function and histology compared to enteral threonine (Ball et al., 1999; table 2). This profound impact of gut metabolism on whole-body threonine requirement must also be considered as a minimum effect. Because the parenteral threonine requirement (0.19 g/kg/d) was so much lower than NRC (1998) recommendations (0.53 g/kg/d on a true ileal digestible basis), we introduced the gastrically infused control group which received identical diets to verify NRC estimates for piglets. The requirement for these control pigs (0.42 g/kg/d) was lower than that recommended by NRC, but still substantially higher than the parenteral requirement. This discrepancy with NRC values is not surprising given that the requirements recommended by NRC (1998) include digestibility estimates for corn–soybean diets and adjust these values for endogenous loss estimates, whereas our diet was completely elemental and available. In subsequent IAAO experiments, we have demonstrated that these NRC estimates are proportionately closer to our gastrically fed estimates for tryptophan (0.15 vs 0.15 g/kg/d for NRC), methionine (0.25 vs 0.23 g/kg/d for NRC) and methionine plus cysteine (0.42 vs 0.48 g/kg/d for NRC). The discrepancy in threonine requirements between that recommended by NRC and our oral estimates could be due to the increased sensitivity of the threonine requirement to endogenous losses. Because the mucin protein core is resistant to digestion and is almost completely recovered in ileal digesta (Mantle and Allen, 1981), a major component of endogenous losses at the ileum is mucins which are rich in threonine, proline, serine and cysteine (Specian and Oliver, 1991). Mucin secretion and hence losses are known

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Table 2 Goblet cell parameters in piglets fed adequate oral threonine (IG-A), deficient oral threonine (IG-D) or deficient oral threonine with adequate parenteral threonine (IV-A)a PAS/AB 2.5b Cellsc Duodenum

Ileum

IG-A IG-D IV-A SDpooled IG-A IG-D IV-A SDpooled

18.8a 15.2b 14.9b 2.7 20.9 22.6 27.5 5.9

Staind 39 41 39 13 79 68 82 18

AB 2.5b

AB 1.0b

Cells

Stain

Cells

Stain

17.6a 5.5c 12.6b 4.4 30.1a 13.9b 19.9a,b 7.8

44a 6b 25a,b 12 63a 14b 31a,b 18

16.3a 7.1b 13.5a,b 4.4 24.6a 13.3b 20.7a,b 6.5

27 12 31 15 30 13 31 11

Ball et al. (1999). Data are means for n = 7 piglets. For data with letter superscripts within a row, those not sharing a letter are different (P < 0.05, LSD comparisons). b PAS/AB 2.5: staining is combination of Alcian blue/periodic acid (5 min)-Schiff base (15 min) (PAS) reaction allowing unsubstituted α-glycol-rich neutral mucins (pink) and acidic mucins (blue) to be differentiated; AB 2.5: 1% Alcian blue (AB, pH 2.5, 1 h) for the localization of carboxylated and/or sulphated acidic mucins; AB 1.0: 1% Alcian blue (AB, pH 1.0, 1 h) for the selective identification of sulphomucins. c Goblet cells in the mucosa stained with PAS/AB 2.5, AB 2.5 or AB 1.0 were counted in 10 well-oriented crypt-villus units ~25 μm in each animal. d Semi-quantitative staining intensities based upon a scale ranging from 0 (unreactive) to 3 (intensely stained) were multiplied by total number of goblet cells. a

to be sensitive to dietary composition as well as presence of fibre and anti-nutritional factors (More et al., 1987; Sharma and Schumacher, 1995). Indeed, in a very recent experiment, we have demonstrated that dietary supplementation of wheat bran, a stimulant of mucin synthesis, leads to increased ileal losses of threonine which may affect whole-body availability of dietary threonine (Myrie et al., 2002). Therefore, this is probably the reason why the threonine requirement of pigs fed a fibre-free elemental nutrition solution (i.e. our gastrically fed control pigs) was lower than the estimated requirement of pigs fed a corn–soybean meal diet (i.e. NRC recommendations). Indeed, as a percentage of the NRC recommendation, the threonine requirement in parenterally fed pigs was 36% (instead of 45%), which translates to a gut utilization of 64% of dietary threonine. Therefore, the requirement for threonine by the gut versus the whole body depends on dietary composition. 5.3. Sulphur amino acids Although methionine is an indispensable amino acid, cysteine is not because it can be synthesized from methionine. However, increased metabolism of methionine to meet cysteine needs could limit methionine availability for protein synthesis and growth. As a result, dietary cysteine has a “sparing effect” on the amount of methionine required. In a series of experiments, we have recently determined the methionine requirements of piglets fed orally and intravenously, with and without dietary cysteine, using the IAAO technique (Shoveller et al., 2001; table 1). With excess or without dietary cysteine, the methionine requirements in parenterally fed piglets were 72% or 69% of the respective requirements in orally fed piglets. In other words, approximately 30% of dietary methionine is utilized by the gut whether dietary cysteine is present or not. These data are supported by those of Stoll et al. (1998),

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who showed that approximately 30% of dietary methionine disappears in first-pass metabolism by the PDV. Furthermore, the sparing effect of cysteine was similar in pigs fed either intravenously or orally (i.e. excess cysteine reduced the respective methionine requirements by 40% regardless of feeding route) (Shoveller et al., 2001). In addition to demands for protein turnover, this relatively high demand by the gut for both sulphur amino acids can also be attributed to other metabolic functions specific to methionine and cysteine. It is possible that the high nucleic acid turnover of intestinal cells requires a significant amount of methionine, an important methyl donor. Furthermore, cysteine, as a product of methionine metabolism, is incorporated to a large extent into intestinal mucins and the tripeptide antioxidant glutathione; both of these products are critical for the maintenance of the mucosal tissue and protection against pathogens (Martensson et al., 1990). Indeed, the impact of a pathogenic challenge on the methionine requirement may therefore be significant, but has yet to be studied. 5.4. Branched-chain amino acids The aforementioned data regarding substantial first-pass splanchnic metabolism of leucine suggest that the splanchnic tissues extract a surprisingly large amount (i.e. 20−50%) of branched-chain amino acids (BCAA). Recently, using a diet with a fixed ratio of BCAA (1:1.8:1.2, isoleucine:leucine:valine), we have determined that the BCAA requirement in intravenously fed piglets was 56% of that in intragastrically fed piglets (Elango et al., 2002a). The apparent uptake of 44% of enterally fed BCAA by the splanchnic tissues is a significant finding because it is generally accepted that the BCAA are predominantly metabolized by the extrahepatic tissues due to the higher activity of branched-chain aminotransferase (BCAT), the first enzyme in the catabolic pathway of the BCAA, in skeletal muscle compared to the liver. In addition, the pattern of BCAA in the plasma of enterally fed piglets, when compared with parenterally fed piglets, clearly demonstrates that the gut has a high demand for leucine and a clear preference for leucine compared to isoleucine or valine. The observation, during enteral feeding, that plasma valine and isoleucine concentrations increased while leucine concentrations remained low indicates that leucine is being extracted by the gut and therefore may be limiting protein synthesis in the rest of the body. Valine and isoleucine do not appear to be utilized by the gut to the same extent and are therefore being passed to the systemic circulation, but because protein synthesis is limited by leucine, these two amino acids, as well as most of the other indispensable amino acids, increase in concentration in the plasma. The difference in BCAA requirements between routes of feeding is supported by the data in humans (Gelfand et al., 1988; Hoerr et al., 1991, 1993; Biolo et al., 1992; Matthews et al., 1993) and dogs (Yu et al., 1990, 1995), regarding first-pass splanchnic extraction of leucine measured by isotope infusions. In addition, Stoll et al. (1998) reported that the pig PDV extracted 43% of leucine, 39% of valine and 31% of isoleucine. Altogether, the first-pass extraction data compare well with the 44% lower BCAA requirement in parenterally fed piglets observed in our recent study. In a subsequent study, we adapted the IAAO technique to systematically determine which of the BCAA was most limiting (Elango et al., 2002b). In both orally and intravenously fed piglets, diets moderately deficient in BCAA (75% of respective requirement) were fed and indicator amino acid oxidation determined. Piglets were then randomly assigned to receive one of three test diets containing either isoleucine, leucine or valine to meet 100% of requirement, with the remaining two amino acids at 75%.

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Fig. 3. The change in phenylalanine oxidation in parenterally (IV) or enterally (IG) fed pigs after supplementation of individual amino acids (Elango et al., 2002b). Diets were formulated to be slightly deficient in all branched-chain amino acids and indicator oxidation was performed before and after isoleucine, leucine or valine supplementation. * indicates oxidation change was different than zero.

The difference in phenylalanine oxidation between unsupplemented and supplemented diets was used as an indicator of BCAA adequacy (fig. 3). In orally fed piglets, the difference in percent dose oxidized was not significant for any supplemented amino acid. However, in parenterally fed piglets, isoleucine and valine supplementation decreased phenylalanine oxidation; isoleucine had the greatest effect and was first limiting (i.e. oxidation decreased from approximately 22% to 10%) and valine was second limiting (22% to 15%). Leucine, which is the preferred amino acid by the gut according to our previous data, had no effect when supplemented to gut-atrophied, parenterally fed pigs. The optimal ratio of BCAA for orally fed pigs is adequately predicted by requirement estimates (NRC, 1998). However, because the gut does not utilize the BCAA in this proportion, the ideal ratio of BCAA for maintenance of the gut is not the same as that for the whole body and has not yet been determined. 5.5. Tryptophan and lysine The tryptophan requirements of parenterally and orally fed piglets were not different when identical diets were employed (Cvitkovic et al., 2000). This result suggests that the gut’s requirement of tryptophan for protein synthesis and/or for oxidation does not significantly impact whole-body requirements, possibly due to either efficient recycling by an atrophied gut or to this amino acid’s low proportion in protein. We have also determined the lysine requirement of parenterally fed piglets but did not employ a gastrically fed control group (House et al., 1998a). Because this was the first amino acid for which we determined the requirement during TPN feeding, we did not appreciate the large impact of the gut. Because NRC estimates were proportionately similar to gastrically fed control pigs for other amino acids, we therefore compared the parenteral lysine requirement of 0.79 g/kg/d to the NRC (1998) estimate of 0.85 g/kg/d, providing a 7% difference due to gut metabolism. It is important to note that this comparison may not be valid given the lack of empirical data for amino acid requirements in young piglets (NRC, 1998). The net lysine utilization by the gut may still be significant as in previous studies regarding the

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substantial splanchnic extraction of lysine (Hoerr et al., 1993; van Goudoever et al., 2000); however, it appears that in piglets, this utilization is not nearly as profound as that for threonine, the sulphur amino acids or the branched-chain amino acids. The impact of parenteral feeding (i.e. with gut atrophy and lack of first-pass metabolism) on whole-body lysine requirements is probably accounted for by the reduced general protein turnover in the atrophied gut; in other words, there seems to be no disproportionate requirement for lysine by the gut versus the whole body. 5.6. Phenylalanine and tyrosine By employing the direct oxidation approach, we found only moderate differences in the phenylalanine (House et al., 1997b) and tyrosine (House et al., 1997a) requirements of parenterally fed piglets compared to the NRC (1998) estimates of oral requirements. The parenteral tyrosine requirement was estimated at 0.35 g/kg/d; in addition, the parenteral phenylalanine requirement (with excess dietary tyrosine) was only 10% lower than NRC estimates (0.45 vs 0.50 g/kg/d). Combined, the phenylalanine plus tyrosine requirement of parenterally fed piglets (0.80 g/kg/d) was equal to that estimated by NRC (0.80 g/kg/d). Furthermore, we have recently been able to compare these results in piglets to the parenteral tyrosine requirement in parenterally fed human infants (Roberts et al., 2001a); the tyrosine requirement determined for parenterally fed infants was similar to the broad range recommended for orally fed neonates (Snyderman, 1971). Again, this minor effect of gut metabolism on whole-body requirements is in contrast to dual-isotope infusion studies (Biolo et al., 1992; Matthews et al., 1993) where 29–58% of dietary phenylalanine was extracted by splanchnic tissues in adult humans; however, arterial recirculation (van Goudoever et al., 2000) was not estimated in these studies. In addition, we have shown that when the indicator amino acid is delivered orally or intravenously, basal phenylalanine oxidation is significantly increased when first-pass splanchnic metabolism is maintained in adults (Kriengsinyos et al., 2002) or piglets (Cvitkovic et al., 2000). However, an increase in phenylalanine oxidation with first-pass metabolism by the gut does not necessarily translate to a substantial extraction of dietary phenylalanine, as demonstrated with lysine by van Goudoever et al. (2000). Indeed, although phenylalanine oxidation increased by 70% when infused orally versus intravenously in humans, the percent extraction determined by flux rates was only increased by 30% (Kriengsinyos et al., 2002). Furthermore, phenylalanine oxidation amounted to less than 15% of intake during either route of infusion. In piglets, when the indicator phenylalanine was fed at the requirement, oxidation rates were increased from 0.6% to 0.8% of dose, which translated to 0.9% and 1.5% of phenylalanine intake or requirement (Cvitkovic et al., 2000). These latter data also provide a reason why phenylalanine is a good choice for the indicator amino acid. Even if there are large differences between diets or individuals in the level of phenylalanine oxidation, the total amount oxidized is still rather insignificant relative to the quantity used for protein synthesis which is driving the whole-body requirement for the amino acid. As with lysine and tryptophan, it appears that dietary phenylalanine is primarily utilized for non-specific protein synthesis, which does not appear to disproportionately impact wholebody requirements. It has been suggested that the gut may hydroxylate a substantial amount of phenylalanine to tyrosine (Stoll et al., 1998); however, the impact of this capacity on whole-body hydroxylation or requirements has yet to be explored. Estimates of phenylalanine hydroxylation to determine tyrosine requirements have been shown to be unreliable (House et al., 1998b; Thorpe et al., 2000; Roberts et al., 2001a).

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5.7. Arginine and proline Although for most species studied arginine is considered dispensable, arginine has been found to be an indispensable amino acid in some species such as cats (Morris and Rogers, 1978), chicks (Tamir and Ratner, 1963) and ferrets (Deshmukh and Shope, 1983). Furthermore, arginine may be conditionally indispensable in young mammals including the dog (Visek, 1984), rat (Borman et al., 1946) and piglet (Mertz et al., 1952; Ball et al., 1986; Brunton et al., 1999), which means arginine can be synthesized de novo, but not at sufficient rates to maintain required functions (i.e. syntheses of protein, urea cycle intermediates, creatine, nitric oxide, etc.). The neonatal small intestine has been suggested to be the major site of arginine synthesis (Wu et al., 1994; Stoll et al., 1998) and the ontogeny of the necessary enzymes in enterocytes has been well described (Wu, 1998). We initially planned to use the IAAO technique to determine the arginine requirement of piglets during parenteral feeding. An initial pilot experiment was conducted whereby parenterally fed piglets were fed arginine-free diets so that growth and nitrogen balance data could be assessed. Both pigs experienced severe hyperammonemia after only ~16 h without dietary arginine; one pig died and the other was comatose. Because plasma ammonia concentration was found to be a sensitive indicator of arginine deficiency, we used this biological outcome to determine if the arginine synthesis rate of the piglet gut was sufficient to maintain the urea cycle and whether proline, the primary precursor of arginine synthesis in the gut, must be available to maintain synthesis rates. The subsequent study successfully demonstrated that parenterally fed piglets could not synthesize sufficient arginine to maintain the urea cycle, let alone to maintain growth, whether or not proline was present in the diet. This study also demonstrated that orally fed piglets could not synthesize arginine and proline at rates sufficient to maintain plasma concentrations or to prevent hyperammonemia. However, unlike the gut-atrophied parenterally fed piglets, the gut-intact orally fed piglets experienced less severe hyperammonemia when proline was provided in the diet. These data suggested that the conversion of proline to arginine occurs in the piglet, but only during oral feeding. We therefore hypothesized that this conversion occurs almost exclusively in the gut and that parenterally fed piglets could not use proline for arginine synthesis because of gut atrophy and/or gut bypass during feeding. This experiment convincingly demonstrated the essentiality of arginine and proline in continuously fed piglets; we predicted that with voluntary feeding, animals would refuse feed if severely deficient, especially if elevated plasma ammonia levels develop. Indeed, vomiting is a symptom of hyperammonemia in pigs, which functions to lessen the ammonia load by expelling potentially toxic amino acids. This clear demonstration of arginine indispensability in piglets was followed by a multiisotope, dual-route infusion study whereby labelled proline, ornithine and arginine were infused intragastrically or into the portal vein to isolate the in vivo effects of small intestinal first-pass metabolism. This experiment demonstrated that the conversion from proline to arginine is completely dependent on the small intestine, confirming the conclusions from our previous experiment assessing hyperammonemia. Thus, in situations where gut metabolism is bypassed or compromised, such as during TPN feeding or gut disease, arginine synthesis is diminished, increasing the overall requirement compared to normal oral feeding. In addition, this study demonstrated that proline synthesis from arginine is also dependent on gut metabolism and its requirement also would be higher when gut metabolism is bypassed. Although the cumulative evidence clearly indicates that arginine synthesis is dependent on the neonatal small intestine, the impact of this first-pass metabolism on whole-body arginine requirements can only be estimated.

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Most of these data support the hypothesis that the arginine requirement is much higher during parenteral feeding, for both maintenance and growth, due to lower synthetic capacity of an atrophied gut. Indeed, we speculate that for arginine, a separate maintenance requirement can be distinguishable from the growth requirement. Such a hypothesis has enormous implications in neonatal populations in which small intestinal first-pass metabolism is bypassed or compromised by gastrointestinal disease or stress. Indeed, these implications have been demonstrated in parenterally fed infants (Heird et al., 1972) and adult rats with small intestinal resection (Wakabayashi et al., 1995). This latter study is interesting considering that adult rats normally can synthesize adequate amounts of arginine in the kidney, but the citrulline precursor originates in the small intestine (Morris, 1992). So although net intestinal arginine synthesis declines during late suckling as renal synthesis increases (Wu, 1998), the importance of intestinal metabolism in the inter-organ synthesis of arginine is still potentially critical in adult species that normally do not require arginine. Proline has been suggested to be an indispensable amino acid for the piglet (Ball et al., 1986), but subsequent studies indicated that proline indispensability is dependent on availability of precursors such as glutamate (Murphy et al., 1996; Wu, 1998) and arginine (Brunton et al., 1999). The extensive gut metabolism of glutamate and glutamine (Windmueller and Spaeth, 1980; Stoll et al., 1999; Reeds et al., 2000) may limit arginine and proline synthesis in certain conditions. Furthermore, the importance of proline for collagen synthesis probably increases in situations of injury and stress. The obvious interdependence of arginine and proline requirement on gut health as well as availability of precursors makes the quantification of such requirements very complicated. However, there is enough evidence to date to suggest that the impact of gut first-pass metabolism on whole-body requirements must be significant and warrants future investigation.

6. FUTURE PERSPECTIVES Albeit the parenterally fed piglet model has proved useful in estimating the impact of firstpass gut metabolism on whole-body requirements, a more direct determination of gut requirements for amino acids has yet to be developed. With a more direct technique, researchers could then attempt to quantify the effects of gastrointestinal stress, injury, disease or dysfunction on whole-body requirements. In particular, the recent intriguing work on leucine extraction and nematode infection in sheep provides preliminary evidence for the importance of this type of investigation. In addition, Klasing and Calvert (1999) have provided an important advance in this area by estimating that the percent of lysine intake consumed by the chicken immune system increases from 1.2% to 6.7% with an injected immune challenge. This “cost” of an immune challenge must be even more profound with a gastrointestinal pathogenic challenge where the additional costs of gut secretion stimulation, gut tissue repair and compromised dietary absorption of lysine must be considered. Furthermore, this cost mostly relates to lysine requirements for non-specific protein synthetic processes. The costs for threonine, the branched-chain amino acids, methionine and cysteine would be much greater due to their more specialized roles in gut maintenance. In addition, arginine and proline requirements would also be increased due to reduced synthesis as well as increased utilization. The nutritional consequences of gastrointestinal disease and stress require further investigation considering that its importance in the treatment of such conditions cannot be underestimated. Because emerging evidence is demonstrating a substantial impact of gut first-pass metabolism on amino acid requirements, the accommodation of this metabolism will undoubtedly

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generate interest, especially among researchers in animal production and clinical treatment of gut diseases. The role of the native microbial population and subclinical gut infections has an important impact on whole-body requirements; indeed, the higher amino acid utilization efficiency with growth-promoting feed-grade antibiotics may partially be explained by the minimization of subclinical challenges. Considering that the small intestinal capacity to digest and absorb protein and amino acids is substantially greater than possible dietary inputs (Burrin et al., 1999), one is tempted to consider that much of this organ’s demand for amino acids for maintenance may be an unnecessary burden. Considering that the protein component, and especially synthetic amino acids, is the most expensive component of animals feeds, the cost of maintaining the surplus capacity of the gut becomes significant. In addition, recent evidence has demonstrated that a significant proportion of whole-body amino acid catabolism occurs in the gut, presumably for energy. Alternative fuels may be sought to replace this perhaps unnecessary, expensive source of energy. Another compelling solution would be to select animals for lower intestinal metabolism without compromising gut absorptive capacity or protective functions. These animals would have a substantially greater amino acid utilization efficiency. The IAAO technique has proven very useful and versatile in determining amino acid requirements of animals. Its adaptation for vulnerable populations is especially advantageous if amino acid requirements during pathogenic challenges or disease states are to be explored. Furthermore, the development of the technique to determine metabolic availability of amino acids is an important advance in the field of amino acid digestibility, which is of critical importance when providing dietary amino acids to meet requirements. Because the IAAO technique relies on relative differences, its dependence on questionable kinetics assumptions is minimal. This is particularly important given the accumulating evidence that luminal and arterial amino acids are channelled differently intracellularly; the amino acid pools for oxidation and protein synthesis are probably separated to some extent so that respective precursor enrichments may be very different, rendering kinetic equations irrelevant. Further adaptation of the IAAO technique to answer many of these questions is eagerly sought.

REFERENCES Alverdy, J.C., 1995. Amino acids to support gut function and morphology. In: Cynober, L.A. (Ed.), Amino Acid Metabolism and Therapy in Health and Nutritional Disease. CRC Press, New York, pp. 435−440. Baker, D.H., Batal, A.B., Parr, T.M., Augspurger, N.R., Parsons, C.M., 2002. Ideal ratio (relative to lysine) of tryptophan, threonine, isoleucine, and valine for chicks during the second and third weeks posthatch. Poultry Sci. 81, 485−494. Ball, R.O., Atkinson, J.L., Bayley, H.S., 1986. Proline as an essential amino acid for the young pig. Brit. J. Nutr. 55, 659−668. Ball, R.O., Bayley, H.S., 1984. Tryptophan requirement of the 2.5-kg piglet determined by the oxidation of an indicator amino acid. J. Nutr. 114, 1741−1746. Ball, R.O., Bayley, H.S., 1986. Influence of dietary protein concentration on the oxidation of phenylalanine by the young pig. Brit. J. Nutr. 55, 651−658. Ball, R.O., Bertolo, R.F.P., Pencharz, P.B., Moehn, S., 2001. A rapid method to determine “true metabolic availability” of amino acids in feedstuffs for pigs. J. Anim. Sci. 79, Suppl. 1, 66. Ball, R.O., Law, G., Bertolo, R.F.P., Pencharz, P.B., 1999. Adequate oral threonine is critical for mucin production and mucosal growth by the neonatal piglet gut. In: Lobley, G.E., White, A., MacRae, J.C. (Eds.), Protein Metabolism and Nutrition: Book of Abstracts of the VIIIth International Symposium. Wageningen Press, Wageningen, The Netherlands, p. 31. Ballevre, O., Cadenhead, A., Calder, A.G., Rees, W.D., Lobley, G.E., Fuller, M.F., Garlick, P.J., 1990. Quantitative partition of threonine oxidation in pigs: effect of dietary threonine. Amer. J. Physiol. 259, E483−E491.

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Yu, Y.M., Wagner, D.A., Tredget, E.E., Walaszewski, J.A., Burke, J.F., Young, V.R., 1990. Quantitative role of splanchnic region in leucine metabolism: L-[1-13C,15N]leucine and substrate balance studies. Amer. J. Physiol. 259, E36−E51. Yu, Y.M., Young, V.R., Tompkins, R.G., Burke, J.F., 1995. Comparative evaluation of the quantitative utilization of parenterally and enterally administered leucine and L-[1-13C,15N]leucine within the whole-body and the splanchnic region. J. Parent. Enter. Nutr. 19, 209−215. Zello, G.A., Pencharz, P.B., Ball, R.O., 1990. Phenylalanine flux, oxidation, and conversion to tyrosine in humans studied with L-[1-13C]phenylalanine. Amer. J. Physiol. 259, E835−E843. Zello, G.A., Pencharz, P.B., Ball, R.O., 1993. Dietary lysine requirement of young adult males determined by oxidation of L-[1-13C]phenylalanine. Amer. J. Physiol. 264, E677−E685. Zello, G.A., Wykes, L.J., Ball, R.O., Pencharz, P.B., 1995. Recent advances in methods of assessing dietary amino acid requirements for adult humans. J. Nutr. 125, 2907−2915. Zhao, X.H., Wen, Z.M., Meredith, C.N., Matthews, D.E., Bier, D.M., Young, V.R., 1986. Threonine kinetics at graded threonine intakes in young men. Amer. J. Clin. Nutr. 43, 795−802.

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Splanchnic protein and amino acid metabolism in growing animals1 D. G. Burrin and B. Stoll USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, 1100 Bates Street, Houston, TX 77030, USA

The splanchnic tissues, namely liver and gut, play a major role in the regulation of wholebody protein and amino acid metabolism. Given their anatomical design for assimilation of food by the host, these tissues metabolize in first-pass a significant proportion of the dietary amino acids via protein synthesis and oxidation and thereby limit the quantity and alter the pattern of amino acids for systemic availability. This substantial “metabolic cost” incurred by splanchnic tissues is in large part related to the numerous critical physiological functions they perform for the mammalian host, such as digestion, ureagenesis, gluconeogensis, and acutephase protein synthesis. Splanchnic tissues also play a key regulatory role by transmitting endocrine, immune, and neural signals in response to the diet and environment, which in turn determine the rates of peripheral tissue protein metabolism and growth. Splanchnic tissue protein metabolism is regulated by specific amino acids and hormones. Amino acids function not only as substrates, but also as extracellular signals that influence cell functions, such as protein turnover, proliferation, apoptosis, cell volume, and redox status. Many of the intracellular signaling and biochemical pathways involved in amino acid metabolism have been described in the liver, but less is known about the gut tissues. The beneficial health effects of key immunonutrients are mediated by improved cell functions in different splanchnic tissues.

1. INTRODUCTION Numerous studies with growing mammals, ruminant and nonruminant, have established that the splanchnic tissues have a substantial impact on whole-body protein and amino 1

The authors thank Jane Schoppe for her assistance in the preparation of this manuscript. This work is a publication of the USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children’s Hospital, Houston, Texas. The work was supported in part by federal funds from the U.S. Department of Agriculture Agricultural Research Service, Cooperative Agreement No. 58-6258-6001, and by the National Institutes of Health R01 HD33920. The contents of this publication do not necessarily reflect the views or policies of the U.S. Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

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acid metabolism. The liver and portal-drained visceral (PDV) tissues, composed mainly of gastrointestinal tissues, contribute roughly 10% of body protein, yet they account for 30–35% of whole-body protein turnover and energy expenditure (Burrin et al., 1989; Yen et al., 1989; Nieto and Lobley, 1999; Stoll et al., 1999a). Relative to their mass, the splanchnic tissues exert a disproportionate impact on whole-body metabolism due to their relatively high fractional rates of protein synthesis and oxygen consumption. Studies with domestic animals have demonstrated that the fractional protein synthesis rates in the liver and intestinal tissues are several-fold higher than that in peripheral tissues, such as muscle (Lobley et al., 1980, 1992; Attaix et al., 1986; Burrin et al., 1992; Davis et al., 1996). The inherently high metabolic rate of the splanchnic tissues is a function of several factors related to the multitude of biological functions performed by these tissues and the relatively high rate of cell turnover, particularly in the intestine. The gastrointestinal tract, including the stomach, small and large intestine, and pancreas, has a primary function to digest and absorb nutrients from the diet. In these processes, these tissues consume substantial amounts of amino acids for synthesis of structural and secretory proteins and for oxidative energy necessary to actively transport nutrients and replenish a continual loss of epithelial cells and digestive enzymes. However, in addition, these tissues function as a physical and immunological barrier to environmental pathogens and noxious substances, representing one of the largest lymphoid tissues in the body. Compromise of this critical gut barrier can not only lead to a suppression of growth, but jeopardize survival of the organism. The GI tract collectively is also a major endocrine organ secreting dozens of peptide hormones that provide key signals for the metabolism and growth of the organism as a whole. The gastrointestinal (GI) tract is also extensively enervated with its own intrinsic (enteric nervous system) as well as an extrinsic neural network, which allows it to function autonomously or in concert with the central nervous system. Some important examples of how these GI functions affect the host include the regulation of food intake (e.g. peptide YY, ghrelin, glucagon-like peptide 1) and substrate homeostasis (e.g. insulin and glucagon). From a protein metabolic perspective, the GI tract functions to assimilate dietary protein in a chemical form (e.g. amino acids) that can be readily used by all somatic cells and simultaneously communicate the availability of nutrients (e.g. insulin) to enhance their utilization by peripheral tissues (e.g. skeletal muscle). The metabolic function of the liver is closely linked to the GI tract by acting as a buffer and scavenger of the dietary nutrients and byproducts absorbed into the portal venous circulation, such as amino acids, ammonia, and bile acids. The liver also plays a major role in the metabolism of dietary amino acids that serve as carbon precursors for gluconeogenesis and the transfer of nitrogen released from skeletal muscle amino acid catabolism. The liver is a central organ involved in the production of acute-phase proteins in response to inflammation and infection. The intent of this review is to provide an overview of protein and amino acid metabolism in splanchnic tissues and highlight some of the recent advancements in our understanding of how these tissues function in growing mammals.

2. METABOLIC FATE OF AMINO ACIDS 2.1. Anatomical and morphological considerations There have been significant advancements in our understanding of splanchnic amino acid metabolism in the last thirty years. In the early studies, much was learned about amino acid metabolism from in vivo measurements of splanchnic organ balance. These pioneering studies were originally derived based on measurements of the net difference in the concentration of

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amino acids in arterial input and venous drainage and of blood flow (Elwyn et al., 1968; Wolff et al., 1972; Felig, 1975). More detailed kinetic information of unidirectional amino acid fluxes was subsequently obtained by adapting this approach to the use of radioactive, isotopic amino acid tracers (Heitmann and Bergman, 1978) and more recently with stable isotopes of amino acids, in humans, pigs, dogs, mice, sheep, and cattle (Hoerr et al., 1991; Yu et al., 1995; Lobley et al., 1996a; Stoll et al., 1998; Lapierre et al., 1999; Hallemeesch et al., 2001). Other important experimental approaches include in situ organ perfusion, which has been extensively used for liver and intestinal amino acid metabolism (Windmueller and Spaeth, 1980; Haussinger, 1990). However, because there is considerable cellular heterogeneity in the liver and gut tissues, studies with isolated cells have also provided critical evidence for the cellular basis of amino acid metabolism (Haussinger, 1990, 1996; Wu, 1998; van Sluijters et al., 2000; Meijer, 2003). The metabolic fate of amino acids in the splanchnic tissues differs not only between the liver and gastrointestinal tissues, but also among cells within each tissue bed. Even within the liver or gut, the metabolic fate of amino acids is significantly affected by how they are presented to the tissue. For the purposes of this review, the starting point in the metabolic fate of amino acids will be in the intestinal epithelial cell after transport from the gut lumen. However, it is important to recognize that within the gastrointestinal tissues (e.g. PDV), amino acids are presented via both the lumen and arterial circulation. From a quantitative perspective, the rate of input of most amino acids from the arterial circulation is substantially greater (3–5-fold) than that from the diet (fig. 1). However, the fractional PDV utilization (i.e. uptake/input) of dietary amino acids (ranging from 95% to 20%) is generally much greater than amino acids derived from the arterial blood, ranging from approximately 5% to 15%. In the liver, the metabolic fate of amino acids may vary depending on whether the input is from the portal vein or hepatic artery. Studies in piglets suggest that after feeding, portal rather than arterial phenylalanine is preferentially used for the synthesis of constitutive and secretory hepatic proteins (Stoll et al., 1999b). Within a tissue, the morphological localization of a cell can dictate the metabolic fate of amino acids. For example, in the gut, the extent to which epithelial cells derive their amino acids from the luminal or vascular input is affected by their stage of differentiation and physical location along the crypt–villus axis. Studies showed that crypt cells are more highly labeled with isotopic tracers derived from the blood, whereas villus cells are more highly labeled with tracers given luminally (Alpers, 1972).

Fig. 1. Rates of amino acid input into the PDV tissues from the diet and arterial circulation in young piglets. Dietary inputs were based on intake of sow’s milk replacer fed at 12 g protein/kg/day. Arterial inputs were calculated from measurements of arterial amino acid concentration and portal blood flow rate; this assumed total arterial and portal blood flow to be equal. Adapted from Stoll et al. (1998).

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These results implied that crypt cells derive their nutrients predominantly from the arterial circulation, whereas villus cells rely on nutrients absorbed luminally from the diet. Likewise, in the liver, studies have also shown a cellular zonation in the acinus, in which periportal hepatocytes scavenge excess portal ammonia by ureagenesis, whereas perivenous hepatocytes sequester ammonia via glutamine synthetase (Haussinger, 1990). Once taken up by the splanchnic tissues, amino acids have three possible metabolic fates: (1) incorporation into protein; (2) conversion via transamination or deamination into other amino acids, metabolic substrates, and biosynthetic intermediates; and (3) complete oxidation to CO2. In the case of intestinal epithelial cells, a fourth metabolic fate is transport into the portal blood stream. In the first two pathways, amino acids can be deposited and recycled by the body for purposes of growth or other biological functions. This recycling process may be affected by whether an amino acid is incorporated into a constitutive protein or secreted protein. However, from a nutritional perspective, if essential amino acids are irreversibly metabolized or completely oxidized to CO2, this represents a nutritional loss to the animal. If we first consider the metabolic fate of dietary amino acids in the gut, there are some general observations that can be made from estimates of the net portal balance expressed as a proportion of intake (table 1). The net portal balance represents the quantity of dietary amino acid absorption into the portal blood expressed as a percent of the intake. Many of the values Table 1 Summary of portal amino acid balance estimates in young pigs fed liquid milk-replacer under different feeding conditionsa Gastric hourly bolusb (6 h) Lysine Threonine Leucine Isoleucine Valine Methionine Phenylalanine Histidine Arginine Proline Tyrosine Cysteine Alanine Serine Glycine Glutamate Glutamine Aspartate

54 38 60 70 61 48 61 – 138 62 167 – 205 58 52 7 –8 4

Continuous duodenalc (6 h) 51 67 55 84 74 82 81 – 108 43 78 – 110 69 65 5 –10 7

Single oral bolusd (8 h) 49 62 74 78 72 70 61 70 149 88 96 17 190 84 69 29 –29 24

Continuous duodenale (24 h) 64 33 66 30 51 75 49 – 155 43 168 – 112 51 89 10 –18 6

a Values represent net portal balance expressed as percentage of dietary intake. Pigs ranged from 6 to 10 kg body weight and diets were fed to supply the estimated daily NRC protein requirement. b Pigs fed via intragastric hourly boluses for 6 h and average portal balance measured between 4 and 6 h (Stoll et al., 1998). c Pigs fed continuously via intraduodenal infusion for 6 h and average portal balance measured between 4 and 6 h (Burrin et al., 2003a). d Pigs fed single bolus meal orally and cumulative portal balance measured for 8 h (Bos et al., 2003). e Pigs fed continuously via intraduodenal infusion for 12 h and fasted for 12 h; cumulative portal balance measured for total 24 h period (van der Schoor et al., 2002).

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are less than 100%, which implies that a portion of the amino acid is utilized by the gut; this is because the diet fed in these studies was based on milk protein and essentially 95–100% digestible. It is also important to note that the mode of enteral feeding in these four studies varied considerably, between bolus versus continuous and gastric versus duodenal administration of the exact same diet. The first observation is that the net balances of glutamate, glutamine, and aspartate are nearly zero. In other words, the net utilization of these amino acids by the PDV is approximately equal to the dietary intake. In some cases, the net balance of glutamine is negative even in the fed state due to the high rate of metabolism in gut tissues. As will be discussed below, the high fractional metabolism of these amino acids is due to their integral role as oxidative fuels. A second remarkable observation is that the net balance of arginine, alanine, and in some cases tyrosine and proline is greater than 100%, which suggests a net production of these amino acids by the gut. The last observation is that net balance of many essential amino acids is significantly less than 100%, and in some cases less than 50% of the dietary intake. These results indicate that gut metabolism of the dietary amino acids significantly alters both the amount and pattern of amino acids absorbed into the portal circulation. In contrast to the PDV, there are few reports in the literature describing the net amino acid balance by the liver in nonruminants, particularly in the fed state (Elwyn et al., 1968; Barrett et al., 1986; Rerat et al., 1992; de Blaauw et al., 1996). There are numerous reports of hepatic amino acid balance in ruminants (Wolff et al., 1972; Lobley et al., 1996a; Lapierre et al., 1999; Blouin et al., 2002). Studies in pigs and dogs fed enterally demonstrated that the net hepatic uptake of glycine and alanine is substantially greater (150–250%) than the dietary intake. The only amino acids that were significantly released were glutamate and aspartate; the net balance of glutamine was essentially zero. Among the remaining amino acids, hepatic uptake was approximately 50–60% of the dietary intake. Interestingly, the net uptake of the branched-chain amino acids was lower (35–43% of intake) than the other essentials; this latter observation translates into a greater net splanchnic output of BCAA compared to other essential amino acids. In addition to the animals where gut and liver amino acid metabolism have been measured separately, there also have been numerous reports of total splanchnic amino acid uptake in adult humans using stable isotopes (Castillo et al., 1993a; Matthews et al., 1993; Battezzati et al., 1995, 1999; Haisch et al., 2000). These studies demonstrated the substantial first-pass splanchnic extraction (percent enteral input) of amino acids, including glutamate (96%), glutamine (64%), alanine (69%), arginine (38%), leucine (21%), and phenylalanine (29%). A recent report in humans describing the kinetics of ingested 15N-labeled soy protein showed that the splanchnic bed extracted nearly 60% of the dietary N, of which 40% was channeled into protein anabolism and 20% was deaminated (Fouillet et al., 2003). Similar studies examining leucine kinetics found that first-pass splanchnic uptake in preterm infants and elderly men was approximately 2-fold higher than in young adult men (Beaufrere et al., 1992; Boirie et al., 1997). 2.2. Protein synthesis A major metabolic fate of amino acids taken up by the gut and liver is incorporation into cellular protein. Numerous studies have measured the rates of protein synthesis in various tissues of the gastrointestinal tract and the liver. Among the literature reports, various methods have been used to measure tissue protein synthesis in vivo; however, the best established and validated approach has been the flooding dose method, first reported by Garlick and others (see discussion in Chapter 18; Garlick et al., 1980, 1994). Review of these studies reveals some

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Table 2 Summary of tissue fractional protein synthesis rates in various mammalian speciesa Weaned ratb Stomach Reticulo-rumen Abomasum Duodenum Jejunum Ileum Cecum Colon Pancreas Liver Spleen Kidney Brain Skeletal muscle

140 – – – 150 – – 58 440 125 38 44 12 7

Weaned mousec 51 67 55 84 78 79 – 43 – 106 70 38 12 3

Neonatal pigletd 55 – – – 124 60 – – 143 85 55 45 20 30

Preruminant lambe – 30 56 86 93 84 45 38 – 115 – – – 21

a

Values expressed as %/day. Goldspink and Kelly (1984); Goldspink et al. (1984); Preedy et al. (1988); Burrin et al. (1991, 1992). c Bark et al. (1998); Burrin et al. (1999). d Burrin et al. (1995, 1997); Stoll et al. (2000a). e Attaix and Arnal (1987); Attaix et al. (1992). b

general characteristics of protein synthesis (table 2), the first of which is relative rates of protein synthesis within the splanchnic tissue bed. In growing animals, the fractional protein synthesis rates (FSR) are generally highest in the pancreas and progressively decline in small intestine, stomach, and large intestine. In a weanling, 28-day-old rat, FSRs (%/day) in the pancreas, small intestine, and stomach are 440%, 150%, and 140%, respectively (Burrin et al., 1991). In the preruminant, one-week-old lamb, the FSRs also vary considerably among the regions of the gut, including the rumen (30%), abomasum (56%), small intestine (88%), cecum (45%), and colon (38%) (Attaix and Arnal, 1987; Attaix et al., 1992). The pattern of declining proximal to distal gradient in intestinal FSR is also observed in neonatal piglets (Stoll et al., 2000a) and mice (Bark et al., 1998). In comparison to the gastrointestinal tissues, the FSR in the liver is relatively high, being similar to that of the proximal small intestine, but lower than the pancreas. In comparison to the splanchnic tissues, mainly small intestine and liver, the protein synthesis rates in the other visceral organs and peripheral tissues are substantially lower. It is notable that the skeletal muscle generally has the lowest protein synthesis rate among all tissues, yet comprises the largest proportion of whole-body protein mass. If we consider the issue of age or stage of development, the fractional rates of whole-body protein metabolism are highest during fetal life, and decline progressively with advancing postconceptual age, commensurate with fractional growth rates (fig. 2). (Goldspink and Kelly, 1984; Goldspink et al., 1984.) This is also evident in the splanchnic tissues, in which the fractional protein synthesis rates in the small and large intestine (107% to 61%) and in the liver (134% to 48%) decline during the lifespan in rats. Interestingly, in the intestine, after weaning, the decline in intestinal FSR with age is largely due to a decreased synthesis in the muscularis and serosal layers, whereas the mucosal FSR remains constant (Merry et al., 1992). However, in striking contrast to the fractional protein synthesis rate, the protein mass increases approximately 750-fold

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Fig. 2. Ontogeny of whole-body and splanchnic tissue protein synthesis rates in rats. Adapted from Goldspink and Kelly (1984) and Goldspink et al. (1984).

(2.5 to 1893 mg) in the intestine and 600-fold (17 to 3252 mg) in the liver between 18 days gestational age and 105 weeks postpartum age (Goldspink and Kelly, 1984; Goldspink et al., 1984). Despite the overall age-related decline, studies in neonatal rats and mice indicate that the FSRs of the stomach, small intestine, and pancreas increase significantly after weaning (Burrin et al., 1991, 1999a). In domestic animals, there are few, if any, estimates of gastrointestinal FSRs before birth and beyond pubertal ages, yet the changes between birth and weaning in pigs and sheep tend to parallel those in rodents (Seve et al., 1986; Attaix et al., 1992; Davis et al., 1996). In milk-fed animals, the intestinal FSR declines during the neonatal period, but increases markedly (40–50%) after weaning. The sharp increase in gut protein synthesis after weaning in pigs is likely due to the substantial change in the composition of the diet, gut microflora, and resultant stimulation of mucosal cellularity and proliferation (Attaix and Meslin, 1991; Jiang et al., 2000). 2.3. Protein degradation The kinetics of protein degradation are technically difficult to quantify, especially in vivo, and thus there is a limited understanding of how certain metabolic factors affect this in the liver and particularly in gut tissues (see discussion in Chapter 4). With regard to the gut, some reports based on indirect estimates suggest that the fractional rates of protein degradation are quantitatively similar to rates of protein synthesis, because the net balance between these two opposing phenomena, i.e. the fractional protein accretion rate, is relatively low, at least in gut tissues (Burrin et al., 1999b; Stoll et al., 2000a). There is a considerably larger literature on the factors that influence qualitative aspects of protein degradation, based on studies with perfused livers and cultured hepatocytes and colon carcinoma cells (HT-29) (Mortimore et al., 1989; Kadowaki and Kanazawa, 2003; Ogier-Denis and Codogno, 2003). The major class of protein degradation in the liver is lysosomal autophagy via the cathepsin proteases, but also includes the ubiquitin–proteosomal system. In the intestine, three proteolytic systems have been identified – lysosomal–autophagic cathepsins, ubiquitin–proteosomal, and calpains – yet the relative significance of these systems to overall protein degradation is unknown (Baracos et al., 2000). Hepatic autophagy has been shown to be potently inhibited

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by several anabolic factors, including amino acids and insulin, and activated by glucagon (Mortimore and Poso, 1987; van Sluijters et al., 2000). The regulatory actions of amino acids on hepatic autophagy are specific for certain amino acids, particularly leucine. Moreover, the process of autophagy has been linked to cell volume regulation and apoptotic cell death (Schliess and Haussinger, 2002). These extracellular and intracellular signaling pathways will be discussed in more detail in subsequent sections. Studies with perfused rat liver and isolated hepatocytes have shown that autophagy increases to a peak at 6 months of age and then declines with age by a process that can be prevented with calorie restriction (Del Roso et al., 2003). As mentioned, the quantitative significance of protein degradation in intestinal mucosal growth is essentially unknown. However, recent reports have shown that degradation is upregulated by nutrient deprivation and exercise (de Blaauw et al., 1996; Samuels et al., 1996; Halseth et al., 1997), but suppressed by glucagon-like peptide 2 (Burrin et al., 2000a). In the study of de Blaauw et al. (1996), it is of interest to note that the rate of proteolysis derived from 3H-phenylalanine kinetics was markedly higher (5-fold) in the PDV than in the liver of the fasted rat. 2.4.

Endogenous protein secretion and amino acid recycling

Another aspect of protein metabolism in the gut involves the metabolic fate of endogenous protein secreted into the gut lumen. Endogenous proteins include secretions arising from the saliva, gastric mucosa, bile, pancreas, and the exfoliation of epithelial cells. The biochemical characteristics of endogenous proteins include enzymes (amylases, pepsinogen, trypsinogen), glycocalix constituents (mucins), growth factors (epidermal growth factor, insulin-like growth factor), and antioxidants (glutathione). Another significant source of protein in terminal ileum is of microbial origin. Some reports indicate that as much as 25–50% of the nitrogen appearing in ileal output is of microbial origin (Stein and Nyachoti, 2003). Given the long list of endogenous proteins, it is perhaps not surprising that, collectively, these proteins represent a quantitatively significant amount of amino acid released into the gut lumen. Estimated ileal endogenous protein losses range from approximately 10–40 g protein/kg dry matter intake and represent up to 10–25% of the dietary protein intake and 5–10% of the whole-body protein turnover (Fuller and Reeds, 1998; Reeds et al., 1999). Recent studies have also shown that endogenous ileal protein loss increases with protein intake (Hodgkinson et al., 2000). A critical nutritional and metabolic question with respect to endogenous proteins secreted into the gut lumen centers around the extent to which they are recycled and absorbed by the host. It is important to note that measurements of ileal protein losses represent a minimal estimate of total upper gut endogenous protein secretion, because evidence suggests that most (70–80%) of the proteins secreted are hydrolyzed and reabsorbed within the small intestine (Stein and Nyachoti, 2003). A recent study in young pigs demonstrated that, over a 24 h period, 52% of the dietary amino acid intake was absorbed into the portal circulation and one-third of this was derived from recycled intestinal secretions (van der Schoor et al., 2002). In is generally held that the amino acids that pass from the terminal ileum into the cecum and large intestine are catabolized by microbial fermentation. The assumption that the endogenous amino acids are fermented and lost in the large intestine is based on early reports that colonic absorption of amino acid is limited and occurs only during early postnatal development (Fuller and Reeds, 1998). The carbon from colonic microbial amino acid catabolism can be lost as CO2 or reabsorbed into the portal blood in the form of short-chain fatty acids. The nitrogen released by microbial amino acid catabolism may be in the form of ammonia, which can be absorbed and recycled into the body amino acid and urea pools.

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Fig. 3. Model illustrating gut microbial amino acid synthesis. Model depicts microbial lysine synthesis derived from luminal ammonia nitrogen and blood urea nitrogen.

Historically, the colonic microbial catabolism of essential amino acids has been considered a nutritional loss, because by definition, if an amino acid is essential, then this usually means that it cannot be synthesized by mammalian cells. This concept has recently been challenged by a number of elegant studies in pigs and humans, which demonstrated the microbial synthesis of some essential amino acids, particularly lysine, based on labeling with 15N-labeled ammonia and urea (fig. 3) (Metges, 2000; Torrallardona et al., 2003a,b). These studies have revealed two critical phenomena: (1) that microbial lysine synthesis occurs in the upper gastrointestinal tract and thus can be absorbed in the small intestine; and (2) the synthesis of several essential amino acids may be nutritionally significant. These studies raise several intriguing questions, including, (1) What are the carbon and nitrogen precursors for microbial amino acid synthesis?, (2) How does the gut microbial load affect amino acid synthesis?, and (3) How is this phenomenon regulated by dietary nutrient intake? 2.5. Amino acids as oxidative fuels In recent years, it has become increasingly apparent that the splanchnic bed derives a majority of its oxidative energy from the catabolism of amino acids, rather than glucose or fatty acids. The liver is clearly the major site of amino acid metabolism in mammals and has been historically considered a major site of catabolism and oxidation. However, since the classic studies of Windmueller and Spaeth (1974, 1975, 1976, 1978, 1980), it has become readily apparent that the gut, particularly the intestine, is also a major site of catabolism of several amino acids. An important distinction to be made, however, is that while amino acids are catabolized in both the liver and gut tissues, the extent to which they are completely oxidized to CO2 may vary. In a brilliant review of amino acid oxidation, Jungas et al. (1992) systematically characterized the metabolic fate of dietary amino acids in the gut, liver, kidney, and muscle and derived two important conclusions. First, they estimated that a primary metabolic fate of amino acid carbon in the liver is conversion to glucose. They make the argument that

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sufficient ATP is generated from the partial oxidation of dietary amino acids to generate roughly half of the liver’s energy needs, an amount that approximates the energy required for the synthesis of glucose. Thus, while amino acids are consumed in oxidative metabolic pathways in the liver, the complete oxidation of amino acids would far exceed the liver’s energy needs and capacity to handle the end-products. A second important observation from their analysis is that the hepatic oxidation of amino acids to glucose makes nearly two-thirds of the total energy content of dietary amino acids available to peripheral tissues (as glucose). As a result, there is no need for peripheral tissues to synthesize a complex array of enzymes to oxidize amino acids and synthesize urea. As discussed below, the gut is a willing participant in this process by releasing some of the carbon derived from nonessential amino acid catabolism into the portal vein as alanine and lactate, both of which are key precursors for hepatic gluconeogenesis. Glutamate is a key amino acid linking hepatic amino acid catabolism and gluconeogenesis (Brosnan, 2000, 2003). Thus, it is not surprising that the gut releases proline, arginine, and ornithine into the portal vein, all amino acids whose metabolism converges at glutamate in the liver. Therefore, the splanchnic tissues, by design, are situated anatomically and metabolically within the mammalian organism to regulate the flow of dietary amino acids, in such manner as to meet their oxidative energy needs first, while at the same time ensuring the delivery of the primal oxidative fuel for peripheral tissues, namely glucose. The seminal studies of Windmueller and Spaeth (1974, 1975, 1976, 1978, 1980) were the first to show evidence of extensive metabolism of glutamine, glutamate, and aspartate in in situ intestinal perfusions in fasted, anesthetized rats. Results from young piglets fed a highprotein, milk-based formula indicated that more than 95% of the dietary glutamine, glutamate, and aspartate is utilized by the gut (Stoll et al., 1998). The studies of Windmueller and Spaeth focused attention on the role of glutamine as the major oxidative fuel in the gut. However, it is important to note that both glutamate and aspartate are of perhaps equal importance as intestinal oxidative fuels. Recent studies in young pigs and humans confirm the extensive intestinal oxidation of dietary 13C-labeled glutamate and glutamine (Battezati et al., 1995; Stoll et al., 1999a; Haisch et al., 2000). The metabolism of glutamine is accomplished first by the catalysis via phosphate-dependent glutaminase and subsequently by glutamate dehydrogenase (GDH) enzymes, both of which are present in the stomach, small intestine, and colon of the young pig (Madej et al., 1999). Interestingly, the activity of GDH is increased approximately 3-fold in the small intestine after weaning. The resulting ketoacid product of GDH is α-ketoglutarate, which is then metabolized yielding CO2 via the tricarboxylic acid cycle. It is important to note that, although there is extensive uptake and metabolism of these three amino acids, their carbon skeletons are not completely oxidized to CO2 and they do not account for all of the CO2 released by the gut. The in situ studies with perfused rat intestine and those in vivo with piglets and humans indicate that most of the glutamine (55–70%), glutamate (52–64%), and aspartate (52%) are oxidized to CO2 (Windmueller and Spaeth, 1976, 1978; Stoll et al., 1999a). The remaining carbon atoms from these three substrates, which are not oxidized to CO2, are converted to lactate, alanine, proline, citrulline, ornithine, and arginine and then released into the portal circulation (Windmueller and Spaeth, 1975; Stoll et al., 1999a). The metabolic fate of nitrogen from these amino acids is not fully understood. However, evidence suggests that a portion of the nitrogen derived from glutamine and glutamate metabolism is transferred to ammonia and other amino acids, including citrulline, ornithine, proline, and arginine; much of the nitrogen from these products is converted to urea in the liver.

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The rate of glutamine oxidation in isolated enterocytes decreases by roughly 90% in the first 3 weeks of life (Darcy-Vrillon et al., 1994; Wu et al., 1995). Also, after weaning, in intraepithelial lymphocytes, glutamine is mainly metabolized to glutamate and ammonia (92%), with minimal oxidation (4%). The decline in glutamine oxidation with age is paralleled by increased activities of glutamine synthetase, glutaminase, and glutamate dehydrogenase (Hahn et al., 1988; Shenoy et al., 1996; Madej et al., 1999). Studies with isolated enterocytes also have shown that, along with glutamine, glucose is an important intestinal oxidative fuel (Darcy-Vrillon et al., 1994; Kight and Fleming, 1995; Wu et al., 1995). However, glutamine effectively suppresses glucose oxidation in enterocytes, whereas glucose has little impact on glutamine oxidation (Kight and Fleming, 1995; Wu et al., 1995). The oxidation of glutamine and its suppression of glucose oxidation was also found to be nearly twice as high (60%) in the proximal compared to the distal (31%) small intestine (Kight and Fleming, 1995). The relationship is consistent with in vivo studies in piglets demonstrating that, although glucose represents an important oxidative fuel (29%), the proportion of glucose oxidized completely to CO2 is substantially less than that of either glutamate or glutamine. The implication is that glutamine and glutamate are preferentially channeled toward mitochondrial oxidation, while most of the glucose is utilized for other metabolic or biosynthetic purposes. Recent studies based on isotopic PDV tracer kinetics have shown that dietary essential amino acids are also oxidized within the gut. Studies in young pigs showed that intestinal oxidation of dietary lysine accounted for about one-third of whole-body lysine oxidation and was completely suppressed by feeding a low-protein diet (van Goudoever et al., 2000). Interestingly, although arterial lysine was taken up by the PDV, none of this was oxidized, suggesting a preferential oxidation of dietary lysine (van Goudoever et al., 2000). As with lysine, there is significant leucine metabolism by the gut via both transamination to ketoisocaproic acid (KIC) and complete oxidation to CO2. Studies in young pigs and dogs have demonstrated that approximately 5–10% of whole-body leucine flux is oxidized by the PDV (Yu et al., 1995; van der Schoor et al., 2001). Although approximately 40% of the leucine taken up by the gut was converted to KIC, nearly all of this is transaminated back to leucine; thus, the net KIC release is negligible (Yu et al., 1995). Studies in young, grower pigs (15–20 kg) suggest that approximately 40% of the whole-body phenylalanine oxidation occurred in the PDV tissues (Bush et al., 2003a). The oxidation of phenylalanine implies that phenylalanine hydroxylation occurs in the gut and is consistent with previous observations suggesting net portal tyrosine production in excess of dietary intake. Given that hydroxylation rather than complete oxidation to CO2 represents the point of irreversible loss of phenylalanine, further studies are warranted to quantify the proportion of whole-body phenylalanine flux metabolized to tyrosine by the gut. The reports of essential amino acid oxidation by the gut have raised the question of whether this is due to mucosal metabolism or microbial fermentation. The recent evidence of de novo lysine synthesis in the proximal intestine in pigs implicates a metabolically significant microbial flora, which could also catabolize dietary amino acids. To address this issue, emerging studies are beginning to identify the localization of essential amino acid catabolic enzymes within the different mucosal cell phenotypes, i.e. enterocytes and lymphoid cells. Two reports have characterized BCAA and lysine catabolic enzymes in enterocytes isolated from piglets at 0, 3 and 7 days of age (Elango et al., 2003; Pink et al., 2003). Branched-chain amino acid transferase activity (BCAT) was detected in enterocytes and liver of 7-day-old piglets at a level that was 18% and 9%, respectively, of muscle mitochondrial BCAT activity. Enterocyte BCAT and branched-chain dehydrogenase (BCKD) activity also increased between 0 and 7 days-of age.

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BCAT and BCKD activity also was present in small intestinal mucosal tissue in piglets (Burrin et al., 2003a). In the case of lysine, the first catabolic enzyme in the pathway, lysine ketoglutarate reductase, was found in mitochondria from freshly isolated enterocytes at an activity level about 50–60% as high as that measured in liver, whereas the activity of saccharopine dehydrogenase was low. These findings are important for two reasons, the first of which is that they demonstrate the amino acid catabolic capacity of the major mucosal cell type relative to liver. Secondly, the results suggest that the lysine and leucine oxidation reported in vivo in piglets is mediated partially by mucosal metabolism; however, the relative significance of microbial catabolism remains to be determined. 2.6. Amino acids as biosynthetic precursors Besides their incorporation into the bulk protein pool and use as oxidative fuels, amino acids are metabolized by splanchnic tissues into a variety of end-products, which serve a variety of key functions for the cell, specifically, and the host in general (table 3). An especially important phenomenon is the interconversion of arginine, ornithine, and proline and their respective roles in the biosynthesis of polyamines and nitric oxide in the intestinal mucosa. Studies have shown that neonatal small intestine is an important site of arginine and proline synthesis and this interconversion depends on first-pass metabolism (Murphy et al., 1996; Wu, 1998; Stoll et al., 1999a; Bertolo et al., 2003). The milk of humans, pigs, rats, and many other mammals is relatively deficient in arginine (Davis et al., 1994). Moreover, in the suckling pig, the intestinal synthesis of arginine provides only about half of the animal’s needs for growth. Thus, supplementation of dietary arginine is considered to be essential for maximal growth in young piglets. The intestinal synthesis of arginine declines, while arginase activity increases substantially during the late suckling period (Wu and Morris, 1998). Also during this time, the synthesis of citrulline and ornithine increases with age, particularly after weaning, and proline and arginine are major precursors for their synthesis (Wu, 1997). Thus, in adult rats and weaning pigs, the intestinal conversion of glutamine, glutamate, and proline to citrulline provides a critical precursor for arginine synthesis in the kidney (Windmueller and Spaeth, 1980; Dugan et al., 1995). The dependence on intestinal first-pass metabolism for synthesis of either arginine (in neonates) or its immediate renal precursor, citrulline (in adults), results in arginine deficiency when intestinal metabolism is either bypassed during TPN (Brunton et al., 1999) or removed surgically by resection (Wakabayashi et al., 1995). The dependence on the intestine for citrulline synthesis has led to development of plasma citrulline as a marker for enterocyte mass in conditions of disease (Crenn et al., 2003). Studies in cultured intestinal cells have shown that ornithine derived from arginine metabolism is converted to polyamines (Blachier et al., 1995). Polyamines (putrescine, spermidine,

Table 3 Amino acid precursors and functional end-products produced in splanchnic tissues Precursors

• Arginine • Proline

Products

• Nitric oxide • Polyamines • Creatinine

• • • • • •

Glutamine Glutamate Aspartate Nucleotides Glutathione Glucosamine

• Methionine • Cysteine

• Threonine • Serine

• • • •

• Mucins

Cysteine Taurine Glutathione Thioredoxin

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spermine, cadaverine) are ubiquitous cationic amines involved in cell proliferation and differentiation in many tissues, including the gastrointestinal tract. Ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC), converting ornithine to putrescine and putrescine to spermine, respectively, are the rate-limiting enzymes in polyamine synthesis. The synthesis of polyamines from arginine is negligible in enterocytes of newborn and suckling animals (Blachier et al., 1991, 1992), but polyamines are present in mammalian milk (Pollack et al., 1992; Buts et al., 1995). As piglets progress from suckling to weaning, major end-products of intestinal enterocyte arginine metabolism are proline and ornithine. Thus, when the ingestion of milk-borne polyamines by the neonate ceases after weaning, the induction of intestinal polyamine synthesis from ornithine, arginine, and proline becomes physiologically significant for the maintenance of normal intestinal growth and function (Wu et al., 2000a,b). Furthermore, the induction of intestinal polyamine synthesis is dependent on the weaning-induced cortisol surge. Another important end-product of arginine metabolism is nitric oxide. Nitric oxide is a major physiological regulator in the body, particularly by enhancing vascular function, tissue perfusion, and immune function. For this reason, arginine is a key component supplemented to enteral formulas designed for trauma and surgically stressed patients (Huang et al., 2003; McCowen and Bistrian, 2003) and may become limiting under conditions of increased NO production (Hallemeesch et al., 2002). Nitric oxide is produced by conversion of arginine to citrulline by the enzyme nitric oxide synthase (NOS), which is expressed in three isoforms, all of which are found in gastrointestinal and liver tissue. In the whole body, the proportion of arginine that is converted to NO is relatively low (1–10% arginine flux) (Castillo et al., 1996), but is increased in response to stress and trauma (Argaman et al., 2003). However, the first-pass splanchnic utilization of enteral arginine is about 40%, of which metabolism to NO represents 16% of the whole-body nitrate production (Castillo et al., 1993a,b). The rates of liver and PDV NO production have been measured using a similar isotopic approach based on kinetic conversion of 15N-arginine to 15N-citrulline (Luiking and Deutz, 2003). These studies showed that as much as 35% of the arginine utilization is converted to NO in the liver and gut in endotoxemic pigs; NO synthesis also is increased markedly with supplemental arginine (Bruins et al., 2002a,b). The nonessential amino acids, aspartate, glutamine, and glycine, are key precursors for the synthesis of nucleotides. This fact is quantitatively important in the intestinal mucosa given the high rate of cell proliferation coupled with the fact that most of the nucleotides, at least ribonucleotides, are synthesized de novo (Boza et al., 1996). The amide nitrogen from glutamine serves as the nitrogen donor for the synthesis of both purines and pyrimidines, whereas most of the carbon skeleton of nucleotides is derived from aspartate and glycine. The biochemical mechanism whereby glutamine affects intestinal function also may be related to its conversion to glucosamine, which reduces the cellular NADPH and suppresses nitric oxide synthesis (Wu et al., 2001). In immune cells, glutamine and glutamate also provide an important source of NADPH via conversion of malate to pyruvate; NADPH is critical in these cells for production of superoxide and NO and for glutathione reductase activity (Newsholme et al., 2003). Amino acids also serve as precursors for synthesis of compounds involved in support of innate immunity in the gut and antioxidant function in both liver and gut. Dietary threonine and cysteine are considered to be important for mucin synthesis by goblet cells within the stomach and intestinal mucosa. The secretory mucins play a key role in the innate immune defense of the mucosa, and the core protein of the major intestinal mucins contains a large amount of threonine and cysteine (van Klinken et al., 1997; Faure et al., 2002). Studies in pigs

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indicated that as much as 60% of dietary threonine is utilized by the gut in first-pass (Stoll et al., 1998). Consistent with these findings, other studies in piglets (Bertolo et al., 1998) demonstrated that the threonine requirement of piglets maintained by parenteral nutrition was nearly 60% lower than that of piglets receiving enteral feedings. A subsequent report found that feeding threonine-deficient diets to piglets significantly reduces intestinal mass and goblet cell numbers, and this suppression of intestinal growth cannot be fully restored by providing threonine parenterally (Ball et al., 1999). The mucosal synthesis and secretion of mucins are likely to be quantitatively significant, and thus the needs for dietary threonine and cysteine may be increased under conditions of gut hypersensitivity or inflammation. A recent report in rats, using a novel approach to purify mucins, demonstrated that the synthesis rate of mucin glycoproteins was relatively constant along the length of the intestine (range 112–138%/day), but substantially higher than the total mucosal protein synthesis rate, especially in the distal bowel: values for total protein were 77% and 44%/day in the ileum and colon, respectively (Faure et al., 2002). Besides incorporation into mucin and other mucosal proteins, the extent to which threonine is further metabolized within the gut tissues is poorly understood. There is some debate as to which metabolic pathway is most important in the catabolism of threonine in mammals (House et al., 2001). The two predominant pathways of threonine catabolism in the pig are believed to be catalyzed by either threonine aldolase/dehydrogenase or threonine dehydratase (Ballevre et al., 1990; Le Floc’h et al., 1996, 1997). Studies in pigs suggest that conversion to glycine via threonine aldolase/dehydrogenase is the predominant pathway of irreversible threonine catabolism. Moreover, threonine dehydrogenase activity was localized in both the liver and pancreas, but not other gut tissues, implicating the PDV as a possible site of threonine catabolism. In addition to mucins, methionine and cysteine serve as biosynthetic precursors for numerous functional end-products, including glutathione, polyamines, and taurine. In numerous cells within the body, methionine is metabolized via transmethylation to homocysteine and in the process produces S-adenosylmethionine, which donates an aminopropyl moiety in the formation of the polyamines, spermidine and spermine (fig. 4) (Finkelstein, 2000). Homocysteine is converted to cysteine via transsulfuration. Cysteine is one of three constituent amino acids of glutathione (GSH), along with glutamate and glycine. Moreover, cysteine is metabolized to form taurine. Glutathione is a major cellular antioxidant in cells found in the intestinal mucosa and liver (Lash et al., 1986; Martensson et al., 1991). However, cysteine and taurine can also function as cellular antioxidants (Santangelo, 2002; Zafarullah et al., 2003). The quantitative significance of these functional end-products to splanchnic methionine, cysteine, and glutamate utilization is unknown. Early studies in humans suggested that splanchnic tissues are an important site of transsulfuration (Stegink and Den Besten, 1972). Studies in piglets indicate that first-pass utilization of dietary methionine ranged from 30% to 40% (Rerat et al., 1992; Stoll et al., 1998; Bos et al., 2003). A recent study in neonatal piglets showed that the methionine requirement (g/kg/day) was 0.42 and 0.29 in enteral and parenterally fed piglets, respectively, which implies that first-pass splanchnic metabolism accounts for 30% of the dietary methionine requirement (Shoveller et al., 2003). The original studies by Finkelstein (2000) demonstrated that gastrointestinal tissues possess the enzymes necessary to metabolize methionine to cysteine, albeit at significantly lower activities than the liver. However, there are few reports describing the kinetics of methionine metabolism in the gut, either in vivo or in vitro with isolated enterocytes. Recent studies based on enzyme assay and in vivo isotopic tracers in ruminants imply that methionine transmethylation occurs in the ruminant gut and that the activities are comparable to the liver

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Fig. 4. Schematic overview of sulfur amino acid metabolism. Abbreviations: SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine.

(Lobley et al., 1996b, 2003; Lambert et al., 2002). In the case of dietary cysteine, studies in pigs indicate that rate of appearance into the portal blood is very limited (less than 20% dietary, intake), suggesting extensive intestinal utilization of cysteine in first-pass (Rerat et al., 1992; Stoll et al., 1998; Bos et al., 2003). The first step in cysteine catabolism is conversion to cysteinesulfinate via the enzyme cysteine dioxygenase. Cysteinesulfinate is then converted to taurine via cysteinesulfinate decarboxylase or pyruvate via aspartate aminotransferase. Rodent studies with 1-14C-labeled cysteine demonstrated significantly higher oxidation when given via the intragastric (70%) than intraperitoneal (41%) route, suggesting that nearly half of the whole-body cysteine oxidation occurs in splanchnic tissues (Stipanuk and Rotter, 1984). The increased oxidation of intragastric versus systemic cysteine was largely attributed to increased oxidation to pyruvate rather than to taurine. Subsequent work demonstrated that intestinal enterocytes extensively metabolize cysteine via cysteine dioxygenase to cysteinesulfanate (Coloso and Stipanuk, 1989). In vivo rodent studies with intravenous infusion of isotopically labeled 15N-cysteine indicate that an important metabolic fate of cysteine in the gut is incorporation into glutathione (GSH) (Malmezat et al., 2000a). With respect to glutamate, studies in piglets demonstrated that enteral glutamate is preferentially incorporated into mucosal GSH (Reeds et al., 1997).

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3. FACTORS REGULATING PROTEIN AND AMINO ACID METABOLISM 3.1. Nutrition Oral feeding is probably the most potent stimulus of splanchnic protein and amino acid metabolism in growing animals (McNurlan et al., 1979; Burrin et al., 1991, 1992, 1995). Prolonged fasting leads to markedly reduced protein mass in gut and liver tissues via suppressed protein synthesis and increased protein degradation, especially in the small intestine (Samuels et al., 1996). Recent studies have shown that the stimulatory effect of nutrient intake on gut protein metabolism is dependent on enteral administration of nutrients (Dudley et al., 1998; Burrin et al., 2000b, Stoll et al., 2000a). These studies showed that the fractional protein synthesis rate and the net balance between intestinal protein loss and accretion is directly and positively determined by the level of enteral intake. An enteral nutrient intake of 20% of total was found to maintain intestinal protein balance. Similarly, a recent study in preterm infants reported that minimal enteral feeding of approximately 10–15% of total increased splanchnic leucine uptake (Saenz de Pipaon et al., 2003). In contrast to the gut, however, the rate of hepatic protein synthesis decreased with the level of enteral nutrition in neonatal piglets, which is consistent with an enlarged liver mass associated with TPN (fig. 5). The composition of dietary nutrients can also substantially affect splanchnic protein and amino acid metabolism. An important consideration in the neonate is the stimulation of liver and gut function in response to the onset of suckling colostrum at birth (Burrin et al., 1995). Studies in neonatal piglets showed that both liver and gut protein synthesis is rapidly upregulated with the first feeding of colostrum. The relatively high concentrations of colostral growth factors are thought to provide key signals for intestinal and liver development. However, studies comparing colostrum to macronutrient matched formula and those with enteral supplementation of recombinant growth factors (IGF-I) suggest that milk-borne growth factors have limited trophic effects on the intestine (Burrin et al., 1999a, 2001). The impact of these milk-borne growth factors is probably more important for stimulation of the gut immune system. Thus, it appears that the macronutrient intake is the major component of the diet affecting splanchnic tissue protein metabolism in developing mammals. In humans, first-pass splanchnic leucine uptake is nearly 2-fold lower in subjects fed a protein-free diet compared to controls fed complete protein-containing diet (Cayol et al., 1997).

Fig. 5. Role of enteral and parenteral nutrition on intestinal and liver protein synthesis in neonatal piglets. Adapted from Stoll et al. (2000a) and unpublished results.

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Some studies have shown that restriction of dietary protein has limited effects on intestinal protein synthesis (Seve et al., 1986), whereas others have reported a decrease in protein synthesis in both liver and gut (McNurlan and Garlick, 1981; Wykes et al., 1996). Studies in neonatal pigs demonstrated that protein malnutrition significantly reduces whole-body growth and amino acid absorption, but does not affect gut tissue growth (Ebner et al., 1994; van Goudoever et al., 2000). Consistent with this, studies in pigs fed low-protein diets showed that PDV metabolized 75% of enteral 13C-lysine intake, compared to 45% in high-protein-fed pigs (van Goudoever et al., 2000). Protein malnutrition also suppressed the gut oxidation of lysine, leucine, and glutamate, whereas glucose oxidation increased (van Goudoever et al., 2000; van der Schoor et al., 2001). These piglet studies suggest that when dietary protein intake is reduced, the gut amino acid utilization for protein synthesis is maintained, but amino acid oxidation is suppressed. Studies focusing on the impact of dietary macronutrients found that enteral amino acids, but not carbohydrate or lipid, stimulated intestinal protein synthesis, whereas each of these stimulated gut protein accretion, suggesting that carbohydrate and lipid suppressed gut proteolysis (Stoll et al., 2000b). In contrast, in other studies, enteral amino acids rapidly decreased intestinal protein synthesis and proteolysis, whereas parenteral or luminal glucose infusion increased intestinal protein synthesis (Weber et al., 1989; Adegoke et al., 1999, 2003). Amino acids also potently inhibit hepatic autophagic and proteasomal proteolysis (Mortimore and Poso, 1987; Hamel et al., 2003). Feeding a high-fat versus high-carbohydrate diet to young pigs stimulated protein synthesis in the intestine, but not the liver (Ponter et al., 1994). Numerous studies have examined the effect of dietary supplementation with amino acids on intestinal and liver protein metabolism. In young pigs, amino acid deprivation suppresses whereas supplementing amino acids parenterally stimulates hepatic protein and albumin synthesis, but not intestinal protein synthesis (Davis et al., 2002a; Hellstern et al., 2002). Similarly, in humans, parenteral amino acid infusion stimulated splanchnic tissue protein synthesis and suppressed protein degradation (Nygren and Nair, 2003). Among the specific amino acids, glutamine is most extensively studied. Supplementing glutamine has been shown to stimulate protein synthesis and reduce proteolysis in the gut in some cases (Coeffier et al., 2003), but not in others (Garcia-Arumi et al., 1995; Marchini et al., 1999; Bouteloup-Demange et al., 2000). Glutamine also has been shown to stimulate protein synthesis and suppress protein degradation in cultured enterocytes and hepatocytes (Higashiguchi et al., 1993; Le Bacquer et al., 2001, 2003; Haussinger et al., 2001). Studies have shown positive effects of glutamine-supplemented TPN in preventing atrophy, stimulating protein anabolism, and maintaining intestinal permeability (Tamada et al., 1992; Inoue et al., 1993; Platell et al., 1993; Haque et al., 1996; Naka et al., 1997; Khan et al., 1999). In contrast, other studies have shown no effect on gut growth or protein metabolism in animals receiving glutamine supplementation parenterally (Spaeth et al., 1993; Burrin et al., 1994; Marchini et al., 1999; Humbert et al., 2001). Furthermore, parenteral infusion of lipid has been shown to stimulate intestinal protein synthesis more than either glucose or glutamine (Stein et al., 1994). Thus, the intestinal effects of glutamine-supplemented TPN are evident under conditions of compromised gut function, such as sepsis, inflammation, and small-bowel resection, while in healthy animals glutamine has limited effect. Dietary leucine stimulates liver protein synthesis (Kimball and Jefferson, 2001; Lynch et al., 2002) and suppresses proteolysis (Mortimore and Poso, 1987). Dietary tryptophan increased (Ponter et al., 1994) whereas arginine supplementation reduced (Bruins et al., 2002b) hepatic protein synthesis in pigs. Other dietary components that stimulate mucosal proliferation and cell turnover also tend to increase protein synthesis. Feeding fiber and lectins has been shown

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to stimulate intestinal protein synthesis in some cases (Southon et al., 1985; Palmer et al., 1987), but not in others (Nyachoti et al., 2000). Studies show that neonatal pigs fed elemental diets have higher rates of protein synthesis than those fed a polymeric, cow’s milk formula (Stoll et al., 2000c). 3.2.

Anabolic and catabolic hormones

3.2.1. Insulin Insulin is generally considered to be one of the most anabolic hormones in the body, affecting multiple metabolic pathways, including those involving protein and amino acid metabolism. However, with respect to protein and amino acid metabolism, the effects of insulin on splanchnic tissues (i.e. liver and gut) are somewhat different from other major tissues, such as muscle. Early studies in vivo and with perfused livers and isolated hepatocytes from normal and streptozotocin-induced diabetic rats indicated that insulin increased protein synthesis and suppressed proteolysis (McNurlan and Garlick, 1981; Mortimore and Poso, 1987; Kimball and Jefferson, 1994). The effects of insulin on hepatic protein synthesis are most pronounced for albumin. In the intestine, however, diabetes has no effect on protein synthesis despite the induction of significant hyperphagia. More recently, several in vivo studies in pigs, mice, and humans suggest that insulin has either no effect or suppresses hepatic protein synthesis (Mosoni et al., 1993; Nair et al., 1995; Bark et al., 1998; Meek et al., 1998; Ahlman et al., 2001, Boirie et al., 2001; Davis et al., 2001, 2002a; Nygren and Nair, 2003). Similarly, studies in pigs reported no effect of insulin on intestinal protein synthesis (Davis et al., 2001, 2002a), whereas insulin administration to adult, type I diabetic subjects only modestly (~15%) increased intestinal protein synthesis (Charlton et al., 2000). Thus, with respect to protein synthesis, the splanchnic tissues appear to be relatively insulininsensitive. This finding is consistent with the idea that protein synthesis in the splanchnic tissues is more tightly regulated by nutrient availability, especially amino acids, than by hormones. The early observations that insulin suppresses proteolysis in perfused liver and hepatocytes have been supported by recent in vivo studies in humans (Nair et al., 1995; Nygren and Nair, 2003). Moreover, the studies have established several cellular signaling pathways that appear to mediate the insulin-induced suppression of hepatic proteolysis (Kadowaki and Kanazawa, 2003; Schliess and Haussinger, 2003). In isolated hepatocytes, insulin activates its membrane receptor, which triggers activation of downstream factors including PI3-kinase, protein kinase B, and p70S6-kinase (fig. 6) (Krause et al., 2002). The key amino acid-sensing and insulininducible signaling component involved in cell protein metabolism is mTOR, yet it is unclear whether mTOR mediates the insulin-induced suppression of proteolysis in hepatocytes. With respect to protein metabolism, the explanation for the observed insulin-insensitivity of intestinal tissue is largely unexplored, yet intestinal epithelial cells express insulin receptors and express all of the signaling pathways known to be responsive to insulin. Cell volume or hydration state is another key cellular signaling process found to be involved in insulin-mediated suppression of hepatic proteolysis. Studies in hepatocytes indicate that increased cell volume is critical for many of the insulin-mediated effects. Insulin produces cell swelling by induction of sodium and potassium accumulation. The signaling mechanisms involved with insulin-induced cell swelling are not fully established, although cell swelling has been shown to activate three major mitogen-activated protein (MAP)-kinase pathways (ERK, JNK, p38MAPK) (Haussinger and Schliess, 1999).

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Fig. 6. Intracellular signaling pathways for selected hormones and nutrients that alter tissue protein metabolism. Abbreviations: cAMP, cyclic AMP; PKA, protein kinase A; AMPK, adenosine monophosphate-activated protein kinase; ATP, adenosine triphosphate; GLP-2, glucagon-like peptide 2: GCN2, general control nondepressing kinase-2 amino acid-regulated eukaryotic initiation factor kinase; mTOR, mammalian target of rapamycin; eIF2B, eukaryotic initiation factor (eIF) 2B; PKB, protein kinase B; IRS, insulin receptor substrate; GSK3, glycogen synthase kinase-3; 4E-BP1, eukaryotic initiation factor 4E binding protein-1; p70S6K, 70 kDa ribosomal protein S6 protein kinase; PI3K, phosphatidylinositol 3-kinase; IGF-I, insulin-like growth factor I.

3.2.2. Growth hormone–insulin-like growth factor I axis Another major anabolic influence on protein metabolism in developing mammals is the somatotropic axis, which involves pituitary growth hormone (GH) secretion and local expression and secretion of insulin-like growth factors (IGF), mainly IGF-I. The somatomedin theory originally proposed was based on the idea that GH-induced secretion of IGF-I in the liver is a key endocrine signal for somatic growth and metabolism during postnatal development (Butler et al., 2002). However, recent use of tissue-specific gene deletion of hepatic IGF-I has challenged this idea and suggestes that the endocrine role of circulating IGF-I is not essential for normal postnatal growth. Both GH and IGF-I appear to have trophic effects in splanchnic tissues that appear to be mediated by stimulation of protein and amino acid metabolism; however, there is considerably less information than for skeletal muscle. The receptors for both GH and the type I IGF receptor are expressed in liver and intestine tissues. Systemic administration of either GH or IGF-I has been shown to stimulate liver and intestinal growth in growing animals, yet the response is dependent on stage of development, being less responsive during early postnatal life (Etherton and Bauman, 1998; Wester et al., 1998). The diminished effect of GH in neonatal animals is due to lower expression of the GH receptor. Early studies with rats in vivo and perfused livers indicated that hepatic protein synthesis, particularly albumin, and amino acid transport were suppressed by hypophysectomy and restored with growth hormone treatment (Jefferson et al., 1975; Feldhoff et al., 1977). These findings have been confirmed by more recent studies in pigs, rats, and humans showing GH-mediated increases in liver protein synthesis (Pell and Bates, 1992; Wester et al., 1998; Barle et al., 1999, 2001; O’Leary et al., 2003; Bush et al., 2003b). In pigs, the stimulation of liver protein synthesis occurred in both fasted and fed animals and was associated with increased ribosome number rather than translational mechanisms (Bush et al., 2003a). Evidence of GH

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action in the gut is limited, with two studies reporting a stimulation of protein synthesis in intestinal (Lo and Ney, 1996) and PDV tissues (Bush et al., 2003b). An important consideration with respect to the mechanism of GH action is whether the effects are mediated directly via the GH receptor or indirectly via increased local expression of IGF-I. Interpretation of this issue has been complicated by studies in which IGF-I infusion was found to stimulate hepatic (Douglas et al., 1991; Koea et al., 1992) and intestinal (Lo and Ney, 1996; Tashiro et al., 1999) protein synthesis in some cases, but not in others (Pell and Bates, 1992; Ling et al., 1995; Lo and Ney, 1996; Bark et al., 1998; Davis et al., 2002b). However, enteral administration of IGF-I in neonatal piglets and mice has no effect on liver or intestinal protein synthesis (Burrin et al., 1999a, 2001). The observation of consistent responses to GH, but not IGF-I infusion alone, suggests a direct effect of GH on hepatic protein synthesis. However, there is virtually no information describing the intracellular signaling pathways, which link GH receptor signals (i.e. Janus family kinases [JAK] and signal transducers and activators of transcription [STAT]) to the cellular protein synthesis machinery. To the extent that the anabolic effects of GH are driven by IGF-I production, it is of interest that studies in vivo and in cultured hepatocytes indicate that the hepatic response to GH is reduced by limiting dietary protein intake or amino acid availability (Harp et al., 1991; Brameld, 1997; Brameld et al., 1999). Recent studies in hepatocytes suggest that the availability of amino acids and glucose directly suppresses IGF-I and GH receptor expression, respectively (Brameld et al., 1999; Stubbs et al., 2002). Moreover, some specific amino acids (methionine, lysine, leucine, tryptophan) appear to be essential for the GH-mediated induction of IGF-I expression in hepatocytes. A further contributing factor to the positive interaction between dietary protein and GH-induced IGF-I expression is insulin. Studies indicate that insulin stimulates hepatic IGF-I expression, thus increased insulin secretion in response to higher dietary protein may contribute to the stimulation of IGF-I expression (Boni-Schnetzler et al., 1991; Brameld, 1997). Other hormones, namely thyroxine and glucocorticoids, have been shown to increase hepatic GH receptor and IGF-I expression when given in combination with GH, in some cases (Brameld, 1997) but not others (Beauloye et al., 1999). The suppression of hepatic amino acid catabolism and urea synthesis also contributes to the protein anabolic effect of GH. A number of studies in rodents, pigs, and humans have shown that GH reduces the in vivo synthesis of urea, activity of urea cycle enzymes, and catabolism of essential amino acids (Dahms et al., 1989; Blemings et al., 1996; Grofte et al., 1997; Gahl et al., 1998; Bush et al., 2002). GH has also been reported to increase the expression of hepatic glutamine synthetase (Nolan et al., 1990). Whether these effects are mediated directly via GH receptor signaling or indirectly via IGF-I expression is unknown and warrants further study. In addition, it is unknown how the intracellular signaling pathways associated with GH and IGF-I are linked to cytosolic and mitochondrial amino acid catabolic and urea cycle enzymes. Thus, the general metabolic effect of GH is to reduce amino acid catabolism and increase scavenging of ammonia in the liver, thereby channeling amino acids into protein synthesis in both the liver and their release to peripheral tissues, such as muscle. 3.2.3. Glucocorticoids and glucagon Glucocorticoids play a critical role in the induction of differentiation and development of many organ systems in mammals. However, glucocorticoids appear to have tissue-specific actions on protein metabolism, being catabolic in skeletal muscle and intestine and anabolic in the liver. Pharmacological doses of glucocorticoids (e.g. dexamethasone) usually increase liver protein mass, thus its classification as a catabolic hormone is not strictly correct.

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Early studies in rodents in vivo and in cultured hepatocytes showed that corticosterone or dexamethasone treatment induced a significant stimulation of constitutive and secretory hepatic protein synthesis (Odedra et al., 1983; Hutson et al., 1987; Southorn et al., 1990). Glucocorticoids also stimulate proteolysis in hepatocytes isolated from weanling but not fetal or neonatal rats, which may in part explain the generally higher state of liver protein anabolism in early development (Hopgood et al., 1981; Blommaart et al., 1993). However, under conditions where plasma glucocorticoids are physiologically elevated, as in starvation and stress, hepatic protein mass declines and gluconeogenic enzyme activity is increased. Consistent with this, dexamethasone has been shown to increase the hepatic activity of amino acid catabolic enzymes involved in transsulfuration and branched-chain amino acid metabolism (Huang and Chuang, 1999; Ratnam et al., 2002). These findings in the liver contrast with the suppression of protein synthesis by glucocorticoids in intestine (Burrin et al., 1999c; Boza et al., 2001) and muscle (Southorn et al., 1990). Dexamethasone results in a catabolic suppression of intestinal growth, as well as a stimulation of the transport and metabolism of amino acids, especially glutamine (Souba et al., 1985; Salleh et al., 1988; Iannoli et al., 1998). The dexamethasone induction of glutamine metabolism is associated with an increase in glutaminase activity and gene expression. Glucagon is generally considered as a catabolic hormone associated with starvation, but also is significantly increased in the circulation during high-protein feeding. The available literature describing the effects of glucagon on splanchnic tissue protein metabolism is confined largely to the liver. Studies in vivo and in isolated hepatocytes showed that glucagon stimulates hepatic amino acid catabolism, urea cycle enzyme activities, and urea synthesis (Morris, 2002). Most of the effects of glucagon are considered to be mediated by induction of cellular cAMP production. These studies have reported a 2-fold stimulation of methionine uptake and a 5-fold stimulation of transsulfuration via cystathionine β-synthase in glucagontreated hepatocytes (Jacobs et al., 2001). Glucagon treatment also increased threonine uptake and oxidation via activation of threonine dehydratase activity in hepatocytes (House et al., 2001). Oxidation of arginine, but not ornithine, in hepatocytes is increased by glucagon treatment (O’Sullivan et al., 2000). Studies with perfused livers have shown an increased incorporation of glutamine nitrogen into urea in association with increased glutaminase activity after glucagon treatment (Brosnan et al., 1995; Nissim et al., 1999). With respect to protein turnover, studies with hepatocytes and perfused livers indicate that glucagon stimulates both albumin synthesis (Kimball et al., 1995) and autophagic proteolysis (Mortimore and Poso, 1987). There is little known about the effect of glucagon, per se, on the intestinal protein metabolism. However, there are numerous studies that have examined the effects of glucagonlike peptides (GLP-1 and GLP-2) on the intestine and pancreas (Drucker, 2002; Burrin et al., 2003b); yet few of these have examined aspects of protein and amino acid metabolism. GLP-1 and GLP-2 have anabolic effects on the pancreatic beta cells and small intestinal mucosa, respectively. Recent studies in TPN-fed neonatal piglets have demonstrated that chronic GLP-2 treatment prevents mucosal atrophy by suppressing apoptosis and proteolysis (Burrin et al., 2000a), whereas acute GLP-2 infusion upregulates intestinal amino acid uptake and protein synthesis (Guan et al., 2003). 3.3. Infection, inflammation, and commensal microflora 3.3.1. Pathogenic infection In the past twenty years, it has become increasingly evident that the relationship between the host and the ecology of resident microbes plays an integral role in the normal development, metabolism, and survival of mammalian species (Hooper et al., 2002). The infestation with

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pathogenic microbial and viral organisms, or exposure to the toxins they produce, has a potent protein anabolic effect on splanchnic tissues, whereas protein catabolism is increased in skeletal muscle in response to infection and inflammation (MacRae, 1993; Grimble, 2001). Proinflammatory cytokines, (i.e. tumor necrosis factor, interleukin-1, and interleukin-6) are the key signals that transmit the presence of pathogenic insult to the organism resulting in activation of the immune system and acute-phase response and suppression of feeding behavior (Johnson, 1997, 2002). Splanchnic tissues play a central role in the proinflammatory response since the liver, spleen, and intestine are the major lymphoid tissues and primary sites of acutephase protein synthesis in the body. The acute-phase response functions to mobilize endogenous amino acids from muscle, which are used for support of acute-phase protein synthesis and cell-mediated immune response. Numerous studies have demonstrated that treatment with proinflammatory stimuli, including live bacteria, enteric parasites, endotoxin, and specific cytokines, significantly increases acute-phase protein synthesis in visceral tissues, especially the liver, gut, and spleen (Jepson et al., 1986; Vary and Kimball, 1992; von Allmen et al., 1992; Higashiguchi et al., 1994; Breuille et al., 1998; Wang et al., 1998; Breuille et al., 1999; Mack et al., 1999). Conversely, these factors increase catabolism and net loss of muscle protein mass. In growing animals, the amino acids required to maintain the proinflammatory, acute-phase protein synthesis in splanchnic tissues impart a metabolic cost, which results in suppression of muscle protein synthesis and increased catabolism and loss of skeletal muscle mass. In addition to suppressed growth, the loss of lean body mass coupled with disruption of organ function contributes to the increased morbidity and mortality associated with infection. 3.3.2. Commensal microflora The phenomenon described above illustrates an acute mechanism whereby the pathogenic microflora activate the immune system, suppress the growth rate, induce fever and diarrhea, and in some cases cause death in domestic animals. In the long term, however, over the lifespan of all mammals beginning at birth, animals are naturally colonized with commensal microbes and these mostly bacterial species coexist with the host organism. This symbiosis serves to activate the maturation of the immune system and enables nonruminant animals to utilize dietary carbohydrate fermented to short-chain fatty acids from otherwise indigestible plant polysaccharides and recycle body nitrogen when dietary protein availability is limited (Fuller and Reeds, 1998; Hooper et al., 2002). Despite the general mutually beneficial relationship between commensal microbes and the host, there remains to a lesser degree some activation of the immune system. The manifestations of commensal microbes have been demonstrated most clearly in animals reared under germ-free compared to conventional environments. These studies indicate that commensal microbes modestly reduced food intake, increased metabolic rate, and increased mass and metabolic activity of gastrointestinal tissues (Gaskins et al., 2002). The increase in gastrointestinal mass is associated with increased numbers and activity of lymphoid cells and increased proliferation of epithelial crypt cells. As with more severe, pathogenic infection, the presence of commensal bacteria results in suppression of growth, albeit of lesser magnitude. In the past fifty years, antimicrobial compounds have been fed to domestic animals in order to suppress the activity of the gut microflora and enhance growth. Despite the widespread use and success of antimicrobials, however, their exact mechanism of action remains poorly defined. It has been shown that by suppressing microbial activity, antimicrobials reduce the luminal concentration and associated toxic insult of ammonia, and thereby diminish the thickness and

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mass of the intestinal mucosa and associated lymphoid tissue (Visek, 1978). Studies in pigs and chickens have shown that feeding antimicrobial compounds significantly reduces liver and small intestinal mass, cell proliferation, protein synthesis, and intestinal ammonia absorption (Muramatsu et al., 1983; Yen et al., 1987; Yen and Pond, 1990; Krinke and Jamroz, 1996). Additional evidence indicates that much of the luminal ammonia originates from bacterial hydrolysis of urea and deamination of dietary amino acids. Thus, it appears that part of the protein anabolic response of antimicrobials is associated with three phenomena: (1) reduced microbial degradation of dietary essential amino acids, (2) increased intestinal absorption of dietary amino acids, and (3) reduced utilization of dietary amino acids by splanchnic tissues for maintenance of immune-associated lymphoid cells and acute-phase protein synthesis. 3.3.3. Immunonutrients During the proinflammatory, acute-phase response, the metabolic basis for increased splanchnic amino acid utilization has been linked to several cellular immune functions, including synthesis of specific acute-phase proteins, glutathione synthesis and antioxidant function, lymphocyte and mucosal crypt cell proliferation, and nitric oxide production. Researchers have determined that a particular group of nutrients, termed “immunonutrients”, play a key supportive role in these immune-related processes, serving to either act as biosynthetic substrates or alter cellular function (Grimble, 2001; Huang et al., 2003). Among those considered as immunonutrients are the sulfur amino acids, glutamine, arginine, nucleotides, and omega3 fatty acids. There are numerous acute-phase proteins synthesized by the liver, whose plasma concentration increases significantly during infection and trauma, including C-reactive protein, serum amyloid A, fibrinogen, and haptoglobin. Cysteine is considered to be one of the limiting amino acids for acute-phase protein synthesis, based on the estimated balance of amino acids released from muscle proteolysis (Reeds et al., 1994). Cysteine is also a constituent amino acid of glutathione and thioredoxin, both major cellular antioxidants (see section below). Studies in infected and stressed rats indicate that cysteine utilization and GSH synthesis and concentration in splanchnic tissues increased markedly (Malmezat et al., 1998, 2000a; Mercier et al., 2002). In protein-malnourished children and pigs, GSH concentrations and synthesis rates in response to infection and stress are compromised, but can be restored with cysteine supplementation (Jahoor et al., 1995; Reid et al., 2000; Badaloo et al., 2002). The increased demand for cysteine during infection also markedly stimulated methionine utilization via the transsulfuration pathway (Malmezat et al., 2000b). Recent studies provide evidence that the proinflammatory cytokine, tumor necrosis factor-α, directly activates cystathionine β-synthase activity, the enzyme catalyzing transsulfuration (Zou and Banerjee, 2003). In addition, methionine and cysteine are precursors for taurine, which also functions as an important antioxidant in phagocytic cells (e.g. neutrophils) via stable neutralization of intracellular hypochlorous acid (Santangelo, 2002). Hepatic taurine synthesis also increased 3-fold in infected rats (Malmezat et al., 2000a). Glutamine and arginine also are considered immunonutrients because of their ability to stimulate lymphoid and epithelial cell function and serve as precursors for nitric oxide, glutathione, and nucleotide synthesis. Glutamine increased proliferation of lymphocytes and intestinal crypt cells, increased macrophage phagocytic activity, reduced mucosal inflammatory cytokine production, improved intestinal epithelial tight junction function, and increased survival in bacterial-infected mice (Wilmore and Shabert, 1998; Huang et al., 2003). Glutamine can be readily deaminated to glutamate in gut, liver, and lymphoid cells and can

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also serve as a precursor for glutathione. Glutamine and glutamate nitrogen moieties are used for nucleotide synthesis and nucleotides have been shown to enhance lymphoid cell function. Sepsis has been shown to significantly increase uptake of glutamine by the liver, but not the intestine (Karinch et al., 2001). Arginine deficiency induced by overexpression of intestinal arginase I compromised the development of B-cell-related gut-associated lymphoid tissue (de Jonge et al., 2002). Studies in endotoxemic pigs indicate that the liver and gut nitric oxide production is increased in parallel with arginine utilization, whereas arginine is released from muscle (Bruins et al., 2002a).

4. AMINO ACIDS AS EXTRACELLULAR SIGNALS 4.1. Glutamine There is a clear recognition that extracellular amino acid availability has profound effects on many aspects of cell function, including the control of cell signaling, gene expression, cell volume, cell proliferation, apoptosis, and protein turnover. The precise cellular mechanisms by which amino acids are able to elicit control over such diverse processes have become the focus of intense investigation recently (fig. 6) (McDaniel et al., 2002; Averous et al., 2003; Jefferson and Kimball, 2003; Kadowaki and Kanazawa, 2003; Meijer, 2003). Many of the new developments in this area stem from the discovery of nutrient responsive genes and their function in Drosophila and yeast systems. The effects of glutamine have been of particular interest, since it has been shown to have pluripotent actions, including stimulation of cell proliferation, protein synthesis, differentiation, ornithine decarboxylase (ODC) and immediate early gene (c-jun) expression, and polyamine synthesis (Higashiguchi et al., 1993; Kandil et al., 1995; Wang et al., 1996). In a recent review, Rhoads (1999) postulated that glutamine stimulates cell proliferation by a signaling mechanism that involves the activation of two related, but distinct, classes of mitogen-activated protein kinases (MAPK). Glutamine also suppresses apoptosis and protein degradation in intestinal and liver tissue, which suggests that it may be an important survival factor (Mortimore and Poso, 1987; Papaconstantinou et al., 1998; Coeffier et al., 2003). Glutamine has been shown to suppress proteolysis in hepatocytes in association with increased cell hydration and swelling, a process that involves the MAPK pathways, extracellular-regulated kinases (ERK), and p38MAPK (Haussinger et al., 2001). Recent studies also indicate that the antiapoptotic effect of glutamine involves an inhibitory interaction between glutaminyl-tRNA synthetase and apoptosis signal-regulating kinase 1 (Ko et al., 2001). 4.2. Leucine Leucine is another amino acid that has been shown to have important cell regulatory effects in various tissues, including the liver and pancreatic beta-cells, yet there is limited information of its effects in intestinal cells. Studies in perfused livers and hepatocytes demonstrated that leucine was capable of suppressing proteolysis and stimulating protein synthesis (Mortimore and Poso et al., 1987; Anthony et al., 2001a). In vivo studies also have shown that feeding a leucine-deficient diet or leucine alone affects the expression of ribosomal protein mRNA and activation of intracellular signaling molecules involved in protein synthesis (Anthony et al., 2001b). In vivo studies in diabetic rats indicate that the effects of leucine appear to be mediated, in part, via an insulin-independent pathway. The specific action of leucine may be mediated by an interaction with a cell-membrane binding protein, since

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cell-impermeant and nonmetabolizable analogs of leucine also suppress hepatic proteolysis and stimulate protein synthesis (van Sluijters et al., 2000; Lynch, 2001; Lynch et al., 2002). Others have suggested that leucine metabolism is important for cellular activation, based on reports that BCAA ketoacids activate downstream signaling elements involved in protein synthesis. The intracellular signaling pathway that mediates the effects of leucine on protein turnover has been studied intensively in recent years. Amino acid deprivation, including leucine, activates the general control nonderepressing kinase 2 (GCN2), resulting in phosphorylation of eukaryotic initiation factor-2 (eIF2), which leads to suppression of global protein synthesis (Jefferson and Kimball, 2003). Specific deprivation of BCAA in cultured lymphocytes activates the branched-chain α-ketoacid dehydrogenase (BCKD) kinase, which translates into reduced BCKD activity and BCAA catabolism (Doering and Danner, 2000). Other players in the amino acid signaling pathway include those involved in assembly of initiation factors (eIF4) and phosphorylation of ribosomal S6. A key intermediate in this pathway is the mammalian target of rapamycin (mTOR), which is a serine–threonine kinase involved in coordinating nutrient availability with cell growth and proliferation. mTOR represents a point of convergence in the pathways that mediate the stimulation of protein synthesis by amino acids and insulin. Studies in hepatocytes show that leucine, glutamine, and insulin activate p70 ribosomal S6 kinase, which is downstream of mTOR (Krause et al., 2002). However, insulin also activates signals upstream from mTOR, namely phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB), whereas leucine and glutamine do not affect these intermediates. The factors that transduce the signal between leucine (or other effector amino acids) and mTOR is the focus of intense interest. One such mechanism may involve adenosine monophosphate-activated protein kinase (AMPK), which senses cellular AMP levels and is activated under conditions of nutrient deprivation and ATP depletion. Activation of AMPK leads to suppression of protein synthesis and mTOR phosphorylation and stimulation of autophagic proteolysis (Kimura et al., 2003; Meijer, 2003). The role of AMPK is intriguing in light of evidence that mTOR seems to function as a cellular ATP-sensing molecule (Dennis et al., 2001). Another potential upstream activator of mTOR may be phosphatidic acid, which is produced by the action of phospholipase D (Chen and Fang, 2002). This model of mTOR activation by phosphatidic acid could form a link to other cellular signals such as the Rho family of G proteins, protein kinase C, and intracellular calcium. Intracellular aminoacyl-tRNA synthetases also have been implicated in the regulation of mTOR. 4.3. Sulfur amino acids Sulfur amino acids (SAA), especially methionine and cysteine, play a key role in antioxidant status and cellular function (Sen, 1998; Aw, 1999; Deplancke and Gaskins, 2002). Glutathione and thioredoxin are the most important cellular antioxidants in mammals and have a critical function in reacting with reactive oxygen species and maintaining cellular redox status. Reduced glutathione (GSH) is an ubiquitous tripeptide (Glu-Cys-Gly) present throughout the body at relatively high intracellular concentrations, especially in the small intestine. Cellular GSH homeostasis is maintained through de novo synthesis from precursor SAA methionine and cysteine, regeneration from its oxidized form glutathione disulfide (GSSG), and uptake of extracellular intact GSH. Thioredoxin is another major cellular antioxidant; it is a larger protein (12 kDa) than GSH, but also contains key cysteine residues in the catalytic active site. Mediating oxidant stress and maintaining normal redox status is especially important in intestinal and liver cells. Studies with intestinal epithelial cells indicate

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that increased oxidant stress and redox imbalance suppress cell proliferation and induce apoptosis and that this is closely correlated with a higher oxidized glutathione state, as measured by the ratio of GSH : GSSG (Noda et al., 2001; Jonas et al., 2002; Pias and Aw, 2002). Culture studies also show that cells grown in cysteine-deficient media have suppressed GSH concentrations and cell proliferation rates, both of which are stimulated with increased cysteine supplementation (Miller et al., 2002; Noda et al., 2002). Other studies with human colonic epithelial cells (Caco-2) indicate that, as differentiation proceeds, cell GSH concentration and proliferation rate decrease, whereas apoptosis rate increases (Nkabyo et al., 2002). Collectively, these studies suggest that cysteine availability and local GSH concentration have a direct influence on epithelial cell proliferation and survival and are inversely proportional to cellular differentiation state. Given evidence that cysteine availability is important for maintenance of epithelial cell GSH level and cell redox status, the question has been raised as to whether methionine can, via transsulfuration, affect the cysteine availability. Evidence in support of this idea is the finding that, in HepG2 cells, oxidant stress increased transsulfuration measured by cystathionine synthesis and 35S-methionine incorporation into glutathione (Mosharov et al., 2000). Moreover, studies in HepG2 cells also show that cystathionine synthase activity is coordinately regulated with proliferation via a redox-sensitive mechanism (MacLean et al., 2002). These results imply that cells exposed to oxidant stress may meet the increased cysteine requirement for GSH synthesis via activation of methionine transsulfuration. A broader implication of these results is that methionine availability and its conversion to cysteine and GSH via transsulfuration may be important for maintenance of normal intestinal and hepatic cell proliferation and survival.

5. FUTURE PERSPECTIVES The splanchnic tissues represent a quantitatively and functionally significant component of whole-body protein and amino acid metabolism of growing animals. The gastrointestinal and liver tissues consume a substantial fraction of the dietary amino acid intake for the purposes of oxidative metabolism, protein synthesis, and gluconeogenesis. Relatively few nonessential amino acids (glutamine, glutamate, aspartate) appear to be major oxidative fuels in the intestine and this metabolism occurs in both epithelial and lymphoid cells. Although ammonia appears to be the main fate of nitrogen derived from glutamine and glutamate catabolism, incorporation of these nitrogen moieties into nucleotides and glucosamines also may be functionally significant. However, some essential amino acids are also oxidized in the intestine, yet the biochemical and cellular bases for this metabolism are poorly understood. Preliminary studies suggest that essential amino acid catabolism occurs in epithelial cells, but further studies are warranted to examine the impact of the gut microflora on this process, especially in the small intestine. Whether this phenomenon is a central mechanism explaining the growthpromoting action for antimicrobials has yet to be proven. However, there is a compelling need to address this question given the increasing concern about the impact of antimicrobials on the environment and human health. Among the factors that regulate splanchnic tissue protein and amino acid metabolism in healthy animals, nutrition plays a major role and it is apparent that these tissues may be more responsive to extracellular nutrient availability than endocrine signals. However, under conditions of infection or stress, proinflammatory cytokines are key activators of hepatic acute-phase protein synthesis, but stimulate protein catabolism in muscle. This phenomenon serves to shuttle amino acids from muscle into hepatic protein synthesis, yet it is not clear how the same cytokines differentially affect protein turnover in these two tissues.

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The answers to many of these questions will require a greater understanding of the intracellular signaling pathways that link both extracellular nutrient availability and endocrine signals to cellular protein and amino acid metabolism. Despite the explosion of knowledge of cellular signaling mechanisms in the past twenty years, there have been few efforts to explore their relevance to protein and amino acid metabolism in splanchnic tissues, especially the intestine. In the past, animal science has legitimately focused considerable attention on the growth and metabolism of skeletal muscle and adipose tissue, given its relevance to lean tissue growth. Yet, the splanchnic tissues act as key metabolic regulators of lean tissue growth, serving to alter the peripheral availability of dietary nutrients and transmit nutrient-dependent endocrine signals that determine peripheral tissue metabolism. Previous studies of physiology and whole animal metabolism combined with clinical trials have shown that some nutrients appear to be unique in their ability to ameliorate the metabolic effects of diseases. Moreover, these nutrients seem to affect splanchnic tissue function, especially the immune system. Thus, further study with these immunonutrients is necessary to establish their mechanism of action and whether they can be used to improve growth and the health of domestic animals. An expanding range of analytical tools, from the molecular to the whole-organ level, is now becoming available to explore these issues in growing animals. The use of genomic and proteomic analysis of individual cells and tissues coupled with genetic manipulation of animals will aid in determining the essentiality and function of specific regulators of protein and amino acid metabolism. In addition, the novel application of techniques such as germ-free environments, mass isotopomer analysis, laser capture microdissection, and transorgan balance approaches should provide useful information on the localization of amino acid metabolism in specific tissues and cells and how this is altered by the host environment.

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Southorn, B.G., Palmer, R.M., Garlick, P.J., 1990. Acute effects of corticosterone on tissue protein synthesis and insulin-sensitivity in rats in vivo. Biochem. J. 272, 187–191. Spaeth, G., Gottwald, T., Haas, W., Holmer, M., 1993. Glutamine peptide does not improve gut barrier function and mucosal immunity in total parenteral nutrition. J. Parent. Enter. Nutr. 17, 317–323. Stegink, L.D., Den Besten, L., 1972. Synthesis of cysteine from methionine in normal adult subjects: effect of route of alimentation. Science 178, 514–516. Stein, H.H., Nyachoti, M., 2003. Animal effects on ileal amino acid digestibility. In: Ball, R. (Ed.), Proceedings of the 9th International Symposium on Digestive Physiology in Pigs. University of Alberta, Department of Agriculture, Food, and Nutritional Science, Edmonton, Alberta. Vol. 2, p. 223. Stein, T.P., Yoshida, S., Schluter, M.D., Drews, D., Assimon, S.A., Leskiw, M.D., 1994. Comparison of intravenous nutrients on gut mucosal protein synthesis. J. Parent. Enter. Nutr. 18, 447–452. Stipanuk, M.H., Rotter, M.A., 1984. Metabolism of cysteine, cysteinesulfinate and cysteinesulfonate in rats fed adequate and excess levels of sulfur-containing amino acids. J. Nutr. 114, 1426–1437. Stoll, B., Henry, J., Reeds, P.J., Yu, H., Jahoor, F., Burrin, D.G., 1998. Catabolism dominates the first-pass intestinal metabolism of dietary essential amino acids in milk protein-fed piglets. J. Nutr. 128, 606–614. Stoll, B., Burrin, D.G., Henry, J., Yu, H., Jahoor, F., Reeds, P.J., 1999a. Substrate oxidation by the portal drained viscera of fed piglets. Amer. J. Physiol. 277, E168–E175. Stoll, B., Burrin, D.G., Henry, J., Jahoor, F., Reeds, P.J., 1999b. Dietary and systemic phenylalanine utilization for mucosal and hepatic constitutive protein synthesis in pigs. Amer. J. Physiol. 276, G49–G57. Stoll, B., Chang, X., Fan, M.Z., Reeds, P.J., Burrin, D.G., 2000a. Enteral nutrient intake level determines intestinal protein synthesis and accretion rates in neonatal pigs. Amer. J. Physiol. 279, G288–G294. Stoll, B., Chang, X., Jiang, R., van Goudoever, J.B., Reeds, P.J., Burrin, D.G., 2000b. Enteral carbohydrate and lipid inhibit small intestinal proteolysis in neonatal pigs. FASEB J. 14, A558. Stoll, B., Price, P., Reeds, P.J., Henry, J., Burrin, D.G., 2000c. Feeding an elemental diet versus a milkbased formula does not decrease intestinal mucosal growth in infant pigs. J. Pediat. Gastroenterol. Nutr. 31, S169. Stubbs, A.K., Wheelhouse, N.M., Lomax, M.A., Hazlerigg, D.G., 2002. Nutrient-hormone interaction in the ovine liver: methionine supply selectively modulates growth hormone-induced IGF-I gene expression. J. Endocrinol. 174, 335–341. Tamada, H., Nezu, R., Imamura, I., Matsuo, Y., Takagi, Y., Kamata, S., Okada, A., 1992. The dipeptide alanyl-glutamine prevents intestinal mucosal atrophy in parenterally fed rats. J. Parent. Enter. Nutr. 16, 110–116. Tashiro, T., Sugiura, T., Morishima, Y., Shimoda, N., Yamamori, H., Takagi, K., Nakajima, N., 1999. Effect of IGF-1 on protein metabolism in burned rats. J. Parent. Enter. Nutr. 23, 5 Suppl., S93. Torrallardona, D., Harris, C.I., Fuller, M.F., 2003a. Lysine synthesized by the gastrointestinal microflora of pigs is absorbed, mostly in the small intestine. Amer. J. Physiol. 284, E1177–E1180. Torrallardona, D., Harris, C.I., Fuller, M.F., 2003b. Pigs’ gastrointestinal microflora provide them with essential amino acids. J. Nutr. 133, 1127–1131. van der Schoor, S.R.D., van Goudeoever, J.B., Stoll, B., Henry, J.F., Rosenberger, J.R., Burrin, D.G., Reeds, P.J., 2001. The pattern of intestinal substrate oxidation is altered by protein restriction in pigs. Gastroenterology 121, 1167–1175. van der Schoor, S.R., Reeds, P.J., Stoll, B., Henry, J.F., Rosenberger, J.R., Burrin, D.G., van Goudoever, J.B., 2002. The high metabolic cost of a functional gut. Gastroenterology 123, 1931–1940. van Goudoever, J.B., Stoll, B., Henry, J.F., Burrin, D.G., Reeds, P.J., 2000. Adaptive regulation of intestinal lysine metabolism. Proc. Natl. Acad. Sci. 97, 11620–11625. van Klinken, B.J., Dekker, J., Büller, H.A., de Bols, C., Einerhand, A.W., 1997. Biosynthesis of mucins (MUC2-6) along the longitudinal axis of the human gastrointestinal tract. Amer. J. Physiol. 273, G296–G302. van Sluijters, D.A., Dubbelhuis, P.F., Blommaart, E.F., Meijer, A.J., 2000. Amino-acid-dependent signal transduction. Biochem. J. 351, 545–550. Vary, T.C., Kimball, S.R., 1992. Regulation of hepatic protein synthesis in chronic inflammation and sepsis. Amer. J. Physiol. 262, C445–C452.

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Yu, Y.M., Young, V.R., Tompkins, R.G., Burke, J.F., 1995. Comparative evaluation of the quantitative utilization of parenterally and enterally administered leucine and L-[1-13C,15N]leucine within the whole body and the splanchnic region. J. Parent. Enter. Nutr. 19, 209–215. Zafarullah, M., Li, W.Q., Sylvester, J., Ahmad, M., 2003. Molecular mechanisms of N-acetylcysteine actions. Cell. Mol. Life Sci. 60, 6–20. Zou, C.G., Banerjee, R., 2003. Tumor necrosis factor-α-induced targeted proteolysis of cystathionine β-synthase modulates redox homeostasis. J. Biol. Chem. 278, 16802–16808.

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Nitrogen metabolism by splanchnic tissues of ruminants C. K. Reynolds Department of Animal Sciences, The Ohio State University, OARDC, 1680 Madison Avenue, Wooster, OH 44691-4096, USA

Ruminant amino acid metabolism is differentiated from the nonruminant by the extensive development of the stomach and dietary fermentation that occurs there. These tissues are extremely active metabolically, and their metabolism reduces the net availability of absorbed amino acids. However, for essential amino acids, this metabolism largely reflects the sequestration of arterial amino acids, and not the utilization of amino acids during their absorption, which represents a relatively small portion of total use by the PDV. The use of essential amino acids by the PDV largely represents synthesis of constitutive and secreted proteins, and endogenous losses to the gut, as well as oxidation of branched-chain amino acids. Absorptive use is more extensive for nonessential amino acids, particularly those used as oxidative fuels. Fermentation in the rumen results in a substantial recycling of urea to the gut lumen and reabsorption as ammonia, which is subsequently converted to urea in the liver. The costs of urea synthesis from ammonia appear to be relatively small in terms of oxidative metabolism, and contrary to earlier suggestions it does not appear to require additional amino acid nitrogen. In contrast, consumption of excess protein can increase heat production in both the PDV and liver, perhaps as a consequence of surplus amino acid oxidation. Liver removal of amino acids reflects liver requirements and supply relative to body requirements, but in ruminants a net release of branched-chain amino acids is often observed, and leucine is oxidized in the PDV and other peripheral tissues. Finally, amino acids also make important contributions to liver glucose production in ruminants, but apart from alanine, this appears to reflect the availability of excess amino acid carbon, and not a metabolic priority, even in very early lactation.

1. INTRODUCTION The ability of ruminants to derive their nutritional requirements from forages and byproduct feeds is a consequence of their extensive fore-stomach development and fermentative capacity. This pregastric microbial symbiosis characterizes ruminant digestion and has unique effects on their protein and amino acid metabolism compared to nonruminants. Microbial fermentation

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provides essential amino acids, even in the absence of protein from their diet, allows the utilization of nonprotein nitrogen from blood urea for microbial amino acid synthesis and absorption by the host, and thus enables extensive recycling of nitrogenous compounds between the gut lumen, blood, and body tissues (Lapierre and Lobley, 2001). This enables the ruminant to survive, and reproduce, under conditions where forage quality and thus food protein are scarce. However, the extensive development, metabolism, and fermentation within the stomach are not without consequences for the ruminant animal. Microbial protein digestion and urea catabolism give rise to extensive absorption of ammonia, which must be detoxified through urea synthesis and amination reactions, largely occurring in the liver. In addition, the extensive development of the musculature and epithelium of the reticulo-rumen and omasum, and their functional activity, give rise to a high rate of protein turnover and amino acid requirement (MacRae et al., 1997a). Other considerations for the nitrogen economy of the ruminant include the effects of the fermentative process on the form and pattern in which energy is absorbed. Extensive carbohydrate fermentation in the rumen provides a more continuous pattern of energy absorption, primarily in the form of volatile fatty acids, as typically little starch escapes rumen fermentation and is available for digestion to glucose absorbed from the small intestine. As a consequence, the ruminant relies primarily on liver gluconeogensis to meet glucose requirements, and glucose is continually released from the liver, with little apparent diurnal fluctuation in liver glycogen flux (Bergman, 1975). The constant requirement for glucose precursors is met to a large extent by propionate absorbed from the rumen and hindgut, as well as amino acid carbon. In addition, the flow of protein to the small intestine, and amino acid absorption, exhibit less variation than in nonruminants. Most current feeding standards and models of nutrient utilization used for rationing protein in beef and dairy cattle are based on estimates of dietary protein degradation in the rumen, the synthesis of microbial protein using available energy and nitrogenous compounds, and the net flow of feed and microbial and endogenous protein to the abomasum. After reaching the small intestine, protein digestion and amino acid absorption proceed, much as in nonruminants. While far from perfect, dynamic, mechanistic models of rumen digestion are available for the prediction of metabolizable protein flow to the small intestine (Russell et al., 1992; Dijkstra et al., 2002). However, most of these approaches rely on empirical relationships between estimates of total or individual amino acid flow to the small intestine, assumptions about the basal flow of endogenous amino acid secretions into the small intestine, and a fixed efficiency of transfer from the lumen of the small intestine to their appearance in a product (Sniffen et al., 1992; NRC, 2001). Extensive metabolism of absorbed amino acids occurs in the tissues of the gut and liver, and the extent to which this metabolism determines the quantity and pattern of amino acids reaching peripheral tissues, and the effects of diet composition and intake level on fractional amino acid utilization, are not certain. Therefore, a greater understanding of these processes, and the contributions of endogenous recycling to amino acid supply, is needed to refine current empirical models of the efficiency of utilization of amino acids reaching the small intestine in ruminants, and the development of more enlightened, mechanistic predictions of nutrient utilization for production. Ultimately, these models will enable more precise rationing of amino acids for productive purposes and thus reductions in the total amounts of protein fed, and subsequent environmental losses arising from the production of beef and milk.

2. SPLANCHNIC TISSUE METABOLISM While the word “splanchnic” generally applies to the viscera, the term “total splanchnic” has historically been used by physiologists as a collective term for the tissues of the portal-drained

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viscera (PDV) and liver combined (e.g. Elwyn, 1970). The PDV are those tissues drained by the hepatic portal vein, and in ruminants include the gastrointestinal tract (reticulo-rumen, omasum, abomasum, small intestine, cecum, and large intestine), pancreas, spleen, and associated omental, mesenteric, and other adipose tissue. The PDV and liver are tissues in vascular series, which are integrated anatomically and functionally through their vascular and neural connections. This integrated metabolism determines the flow of nutrients from food to other body tissues, through the gastrointestinal tract’s role in digestion and absorption, and the liver’s metabolic role as integrator of nutrient supply with requirement. In addition, the splanchnic tissues are extremely active metabolically, accounting for as much as 50% of total body oxygen consumption, but a considerably lower proportion of body mass (Reynolds, 1995). This high rate of metabolism requires a high rate of blood flow, and PDV and liver blood flow (fig. 1) and oxygen consumption (Reynolds, 2002) in cattle increase with diet dry matter intake (DMI). The high rate of oxygen consumption by the splanchnic tissues is in part a consequence of a high rate of protein turnover and secretion, and PDV and liver mass, and thus amino acid requirements, increase with greater DMI and metabolizable energy (ME) supply (Burrin et al., 1990; Lobley, 1994). In cattle, daily PDV and liver heat production, estimated from oxygen consumption, increases with greater ME both on a total (fig. 2) or metabolic body size (body weight 0.75) basis (fig. 3). As much as 38% of cardiac output is distributed to the liver (Huntington et al., 1990), thus while the splanchnic tissues produce a disproportionate amount of the body’s oxidative metabolism and heat, they have access to a disproportionate amount of available nutrients, both during absorption into blood and transfer to the vena cava, as well as from the arterial blood pool (Reynolds, 2002).

Fig. 1. Portal vein and liver blood flow in growing, lactating, and dry mature cattle. Data are averages of hourly measurements for a given animal and treatment (n = 335). Data sources described by Reynolds (1995, 2002), Maltby et al. (1993), and Benson et al. (2002).

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Fig. 2. Portal-drained visceral (PDV) and liver blood heat production and metabolizable energy (ME) intake in growing, lactating, and dry mature cattle. Data are daily averages of hourly measurements for a given animal and treatment (n = 335). Data sources described by Reynolds (1995, 2002), Maltby et al. (1993), and Benson et al. (2002).

Fig. 3. Portal-drained visceral (PDV) and liver blood heat production and metabolizable energy (ME) intake in growing, lactating, and dry mature cattle. Data are daily averages of hourly measurements for a given animal and treatment (n = 335) scaled to metabolic body size (body weight0.75). Data sources described by Reynolds (1995, 2002), Maltby et al. (1993), and Benson et al. (2002).

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2.1. Measurement of net splanchnic metabolism The use of multicatheterization procedures to measure the quantitative metabolism of nitrogenous compounds by the splanchnic tissues of ruminants in vivo has been previously described in detail (Katz and Bergman, 1969; Bergman, 1975; Huntington et al., 1989; Seal and Reynolds, 1993; Reynolds, 1995). Based on initial studies in dogs (Shoemaker, 1964), pioneering work in sheep by E.N. Bergman of Cornell University elegantly described basic aspects of the interorgan flow of amino acids and other metabolites between the ruminant PDV, liver, kidneys, and hindlimbs (Bergman and Heitmann, 1980; Bergman, 1986). The basic procedure is relatively simple: permanent indwelling catheters are surgically established in appropriate blood vessels which enable the measurement of venous–arterial concentration difference (VA) and blood flow across the tissue of interest, which are multiplied mathematically to obtain a measurement of net nutrient appearance in venous blood (a positive VA) or removal from arterial blood (a negative VA). Blood flow is typically obtained by measuring the downstream dilution of a dye (usually ρ-aminohippurate) infused into a distal mesenteric vein, but can also be measured electronically using electromagnetic or ultrasound probes. Historically, the usefulness of electronic probes for measurement of portal vein blood flow was limited by their longevity and requirement for a round vessel (i.e. an artery). The more recent development of “transit-time” ultrasound probes (Transonics®, Ithaca, NY) has enabled measurement of total blood flow in blood vessels with irregular shape using long-term (months) in vivo preparations. However, until the recent development of a slimmer probe, the bulky size of the probe body and anatomy of the portal vein have limited the usefulness of transit-time probes for measuring portal vein blood flow in cattle (Lindsay and Reynolds, 2003). While simple in concept, the obtaining of valid, statistically sound measurements of dietary effects or physiological state on splanchnic nutrient flux goes far beyond the maintenance of catheter patency. Considerations include laminar flow in the portal vein, heterogeneous portal blood distribution to the liver, the frequency of sampling, postprandial fluctuations, the measurement of small VA coupled with large blood flow rates, etc. The PDV represents a diverse, heterogeneous, collection of tissue types, and the small intestinal enterocytes where amino acid absorption occurs represent a small fraction of the total PDV tissues. Therefore, as a consequence of their anatomical location, the majority of the PDV tissues must to a large extent rely on the supply of amino acids in arterial blood for their requirements (MacRae et al., 1997a; Reynolds, 2002). Measurements of nutrient flux across sections of the PDV can also be obtained by placing catheters in vessels draining specific tissue beds, such as the mesenteric-drained viscera (MDV), but depending on the species of interest and the location of the sampling tip relative to the ileocecal vein, the measurement may or may not include the cecum and large intestine (Seal and Reynolds, 1993). As cattle, but not sheep, have a collateral branch of the mesenteric vein draining the ileum (Habel, 1992), measurements of total small intestinal flux, excluding the contributions of the hindgut, are exceedingly difficult to obtain in cattle without ligating the collateral branch. Precise placement of the mesenteric sampling catheter between the convergence of the collateral and jejunal branches and the ileocecal branch is required, and complete mixing of PAH and venous blood in such a short distance makes measurements of blood flow difficult. 2.2. Isotopic labeling approaches Measurements of net amino acid flux across the PDV reflect the mathematical summation of a number of metabolic processes, including absorption from the lumen of the small intestine into the mesenteric veins, utilization during absorption by the enterocytes, release from protein turnover, or synthesis, by a variety of PDV tissues, and removal from arterial blood.

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Similarly, liver is not a homogeneous tissue, and simultaneously removes individual amino acids from the portal vein and hepatic artery and releases them into the hepatic veins. To obtain the true or “gross” rate of metabolite release and removal by a tissue (often called “unidirectional flux”), isotopic labeling of the arterial blood pool is used to obtain an estimate of gross nutrient removal from blood (fractional extraction times total arterial supply, or “arterial use”), which is added to net flux to calculate the gross release into venous blood (Bergman, 1975). This still underestimates utilization during absorption from the gut lumen, before reaching venous blood. This “absorptive use” can be measured by infusing labeled metabolite into the small intestine (usually the duodenum or jejunum) and measuring its quantitative appearance in the portal (or mesenteric) vein (MacRae et al., 1997a; Yu et al., 2000; Lindsay and Reynolds, 2003). However, this measurement must be corrected for the extraction of absorbed label from arterial blood, which is accomplished by simultaneously, or on a separate occasion, labeling the arterial blood pool (MacRae et al., 1997a). Otherwise, measurements of absorptive use or “first-pass” metabolism by the small intestine will be overestimated. Isotopic labeling of the carbon in individual amino acids also allows the measurement of oxidation by specific tissues (Lobley et al., 1995, 2003; Lapierre et al., 1999, 2002), depending on the amino acid labeled, the specific carbon labeled, and the metabolic route of oxidation. However, when 13C labeling is used, consideration must be given to the amount of tracer required to label CO2 relative to tracee turnover, especially under nutritional conditions that reduce oxidation. In addition, the PDV release of fermentative and salivary CO2, as well as the removal of arterial blood CO2 (Lobley et al., 2003), complicate the interpretation of CO2 flux across the PDV (Lindsay and Reynolds, 2003). In addition to measurements of amino acid oxidation, the use of multiple labeled amino acids, on separate occasions or differentially labeled, allows estimation of the transfer of carbon among individual amino acids and metabolites within specific tissues (Wolff and Bergman, 1972a; Bergman, 1975; Lindsay and Reynolds, 2003).

2.3. Interorgan amino acid exchanges in maintenance-fed sheep Using a combination of multicatheterization and isotopic labeling procedures, as well as multiple gut cannulation, Bergman and his colleagues conducted a detailed series of studies describing the metabolism of amino acids by the visceral tissues and hind limbs of sheep (for reviews see Bergman and Heitmann, 1980; Bergman, 1986). The data provide a picture of the basic patterns of interorgan amino acid exchange in maintenance-fed or 3-day fasted sheep fed alfalfa pellets, and provided a basis for subsequent studies of the effects of diet composition and physiological state on amino acid metabolism in sheep and cattle. However, the data were often obtained from a limited number of animals sampled multiple times to increase replication (see Wolff et al., 1972), and measurements appear to have been made with limited time for recovery from surgery. Regardless, these concerns do not diminish the contribution that this research represents. The data represent a pioneering effort, and have stood the test of time. As emphasized in their reviews, the work of Bergman and his colleagues highlighted a number of basic concepts, which will be explored in the sections that follow in light of more recent observations.

3. AMINO ACID UTILIZATION BY THE PORTAL-DRAINED VISCERA 3.1. Absorptive use of amino acids On a net basis, the disappearance of many amino acids from the lumen of the small intestine of 2 sheep was considerably greater than their simultaneous net appearance in the portal vein

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Table 1 Apparent disappearance of amino acids from the small intestine (SI, g/d) in ruminants and their net appearance across the portal-drained viscera (PDV) or mesenteric-drained viscera (MDV) as a fraction of disappearance from the SIa Sheep (n = 2)b Amino acid Leucine Valine Lysine Threonine Isoleucine Phenylalanine Histidine Methionine Arginine Alanine Aspartate Asparagine Cysteine Glutamate Glutamine Glycine Proline Serine

Sheep (n = 3)c

Lactating dairy cowsd

SI, g/d

PDV/SI

SI, g/d

PDV/SI

MDV/SI

SI, g/d

5.02 3.73 3.60 2.82 3.38 3.22 1.49 1.44 2.84 3.59 5.97 – 0.61 6.23 – 3.61 2.10 2.10

0.21 0.33 0.66 0.38 0.43 0.64 0.11 0.60 0.48 0.85 0.02 0.12 0.48 (0.01) (0.68) 0.52 0.39 0.83

7.77 5.47 5.78 4.67 5.08 5.06

0.65 0.57 0.64 0.73 0.58 0.76

1.02 0.95 1.09 0.99 1.13 1.11

127.7 77.9 107.9 65.2 81.7 69.6 23.6 36.3 79.6 88 153.4 – 15.1 191.1 – 88.8 64.5 58.2

PDV/SI 0.62 0.51 0.55 0.43 0.62 0.76 0.95 0.67 0.63 0.80 0.08 0.19 (1.62) 0.08 (0.19) 0.42 0.09 0.75

MDV/SI 0.92 1.11 0.76 1.15 1.02 1.00 1.27 1.01 1.03 1.16 0.03 0.37 0.28 0.11 0.20 0.57 0.49 1.23

a Disappearance calculated as duodenum minus ileal flow, uncorrected for endogenous amino acid flow in the ileum. Recoveries of asparagine and glutamine calculated relative to aspartate and glutamate disappearance, respectively. Negative recoveries (net removal from arterial blood) given in parentheses. b Tagari and Bergman (1978). Averages for measurements in 2 sheep fed high- and medium-protein diets. c MacRae et al. (1997b). Averages for measurements in 3 sheep fed 800 or 1200 g alfalfa pellets per day. d Berthiaume et al. (2001). Unlike the data for sheep, measurements of SI, PDV, and MDV were not obtained simultaneously in the same animals. Data for SI are from 2 cows, whilst data for PDV are apparently from 3 separate cows, 2 of which were also sampled for MDV.

(table 1; Tagari and Bergman, 1978). This was especially true for some of the nonessential amino acids, such as glutamate and aspartate, which are present in large amounts in duodenal digesta, as well as some of the essential amino acids. The data suggested that there was a considerable absorptive use of amino acids by the enterocytes of the small intestine, as observed previously for volatile fatty acid absorption by rumen epithelia (Bergman, 1975). This interpretation was supported by the work of Windmeuller and Spaeth (1980), which showed that glutamate, glutamine, and aspartate are important energy substrates for the small intestinal enterocyte of rats. More recent studies in pigs have confirmed that there is an extensive use of glutamate during its absorption from the small intestine (Stoll et al., 1999). For alanine and serine, the apparent recovery in the portal vein was much higher, but this in part reflects synthesis by the PDV from products of glycolysis. Alanine, which is synthesized by the PDV and peripheral muscle from pyruvate, is often the amino acid released by the PDV in the largest amount, across a variety of nutritional and physiological states (Wolff et al., 1972; Bergman and Heitmann, 1980). This in part reflects a transfer of N from the catabolism of other amino acids in the PDV to the liver for ureagenesis, and alanine is typically removed by the liver at rates equal to net PDV release or greater, on a net basis (Bergman, 1986; Reynolds, 1995; Lobley et al., 2001).

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The extensive absorptive use of amino acids suggested by Tagari and Bergman (1978), and other estimates of “first-pass absorptive use” of amino acids, are in part attributable to anatomical and technical considerations for the measurements reported. As discussed previously (Reynolds, 2002), the high rates of glutamate and aspartate disappearance from the small intestine in part reflects the deamination of glutamine and asparagine during the acid hydrolysis of digesta prior to amino acid analysis, and resulting overestimate of their flow to the duodenum. For other amino acids, their low net recovery in the portal vein largely reflects their arterial use by other PDV tissues, such as the stomach. In sheep (MacRae et al., 1997b) and cattle (Berthiaume et al., 2001), measurements of net amino acid release by the MDV were considerably higher (30–50%) than their net release by the PDV (table 1). This is a consequence of the utilization of amino acids from arterial blood by those tissues not drained by the mesenteric vein. These data strongly suggests that the low recovery of absorbed amino acids across the PDV, on a net basis, is due to sequestration of amino acids from the arterial blood pool. In these studies (MacRae et al., 1997b; Berthiaume et al., 2001), comparison of small intestinal disappearance and net MDV appearance of essential amino acids showed nearly equal, or lower, rates of disappearance from the small intestine compared to appearance in the mesenteric vein, on a net basis, but these estimates underestimate true rates of disappearance to the extent that endogenous secretions appear in the ileum. For nonessential amino acids (Berthiaume et al., 2001), the same comparisons of net intestinal disappearance and MDV appearance (table 1) compare more favorably with the findings of Tagari and Bergman (1978), with a low net recovery of aspartate, glutamate, cysteine, proline, and glycine, and a greater appearance of alanine and serine suggesting the synthesis of the latter two amino acids by tissues of the MDV. 3.2. Comparison of absorptive and arterial essential amino acid use Direct measurements of the absorptive use of a mixture of essential amino acids were obtained by MacRae et al. (1997a) in sheep equipped with multiple intestinal cannulas and catheters enabling measurement of small intestinal disappearance and PDV flux of amino acids, along with dual-site isotope infusions to allow simultaneous (consecutive samplings) measurements of absorptive and arterial use of a mixture of 13C-labeled essential amino acids. The measurements confirmed that across the total PDV, arterial use of most essential amino acids (leucine, valine, lysine, threonine, isoleucine, and histidine) accounts for the majority (75–87%) of total PDV use or “sequestration”. For phenylalanine, a greater proportion (51%) of total PDV utilization was accounted for by absorptive use. Using a similar approach to study both PDV and MDV utilization of dual-labeled leucine in sheep (Yu et al., 2000), the MDV accounted for only a small proportion (12%) of total arterial use of leucine by the PDV. Of the leucine sequestered during absorption, oxidation accounted for only 2%, thus most leucine sequestered during absorption is used for synthesis of constitutive and secreted proteins. Using a similar approach, measurements of arterial and absorptive use of leucine and phenylalanine were obtained in late-lactation dairy cows fed the same ration at two levels of DMI (16 or 20 kg/day) and two different levels of DMI in the subsequent dry period (8 or 12 kg/day; Reynolds et al., 2001a). These data confirmed that arterial use of these amino acids accounted for the majority of total PDV use, but absorptive use accounted for a greater proportion of total PDV phenylalanine use than measured for leucine. Absorptive use was negligible at lower DMI within each physiological state, and increased with greater intake, perhaps reflecting an increase in gut mass relative to body requirements. A low absorptive use of phenylalanine was also evident in other studies in lactating dairy cows at restricted intakes

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(Reynolds et al., 2000), where the increase in PDV release of phenylalanine at the end of a 6-day abomasal infusion of a mixture of essential amino acids equivalent to 800 g of milk protein was 77% of the phenylalanine infused on a net basis, and 99% after correction for arterial use. Taken together, these data confirm that there is substantial utilization of amino acids by the tissues of the PDV. For many nonessential amino acids, with some notable exceptions, their utilization during absorption is substantial, and they make important contributions to the energy and synthetic process of the enterocytes of the small intestine. Those contributions, in the nonruminant small intestine, are discussed in more detail in other chapters. For most of the essential amino acids, absorptive use accounts for 25%, or less, of total PDV sequestration. Therefore, metabolism by the PDV tissues largely reflects use of amino acids from the arterial pool, and absorptive use has less effect on the quantity and profile of amino acids reaching the portal vein during the “first pass” of absorption than suggested by simple net flux measurements. For phenylalanine, absorptive use accounted for a greater proportion of total PDV sequestration, but this reflected less quantitative arterial use relative to the other essential amino acids, and not more absorptive use per se (MacRae et al., 1997a; Reynolds et al., 2001a). 3.3. Arterial use of essential amino acids by the PDV In sheep, total PDV sequestration of essential amino acids varied from 32% (histidine) to 67% (valine) of whole-body irreversible loss (IRL), indicating a substantial contribution of PDV tissues to whole-body protein synthesis (MacRae et al., 1997a). In dairy cows, total PDV sequestration of leucine (27–40%) and phenylalanine (22–37%) represented a smaller proportion of body IRL (table 2), but the proportion was increased by greater DMI in both dry and lactating cows, yet was not affected by stage of lactation and associated differences in average DMI (Reynolds et al., 2001a). This suggests that PDV sequestration of these amino acids, as a fraction of total body IRL, is affected by their supply from the diet relative to requirement. Although these measurements were not corrected for oxidation, or release of 4-methyl-2-oxopentanoate (MOP), it is likely that a large portion of the leucine sequestered by the PDV is used for protein synthesis. Numerous studies have shown that the PDV of sheep and cattle accounts for a major portion of body protein synthesis (24–35%), depending on the technology used and interpretation of the results obtained (Lobley, 1994; Lobley et al., 1995, 1996; Lapierre et al., 1999, 2002). Therefore, the tissues of the PDV have a substantial requirement for essential amino acids, which must to a large extent be met by extraction from the arterial supply. The relationship between PDV protein turnover and essential amino acid use is illustrated by the close agreement between the proportions of essential amino acids sequestered by the PDV and their relative concentration in constitutive proteins (MacRae et al., 1997a). In addition, branched-chain amino acids are to a large extent oxidized by extrahepatic tissues, but it is apparent that leucine, and perhaps other branched-chain amino acids, are oxidized by the PDV (Lapierre et al., 1999, 2002; Lobley et al., 2003). This oxidation appears to be sensitive to leucine supply relative to requirement (van der Schoor et al., 2001). In dairy cows, PDV oxidation of leucine tended to increase when a higher protein diet was fed (Lapierre et al., 2002), but the effect of protein status on PDV oxidation of other amino acids has not been determined in ruminants, to my knowledge, and deserves further research. For phenylalanine, little oxidation occurs in the PDV (Reynolds et al., 2000; Lobley et al., 2003), but liver clearance and hydroxylation is extensive relative to other essential amino acids (Reynolds et al., 2000; Lobley et al, 2001). This in part reflects the conversion of phenylalanine to tyrosine, as

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Table 2 Effects of intake level during lactation and the subsequent dry period on leucine and phenylalanine kinetics and endogenous leucine flow in the duodenum and ileum of 3 dairy cows (Reynolds et al., 2001b) Dry

DMI, kg/d Milk yield, kg/d Leucine metabolism, mmol/h Body irreversible loss rate Total PDV sequestrationb Duodenal flow Endogenous duodenal flow Ileal flow Endogenous ileal flow Phenylalanine metabolism, mmol/h Body irreversible loss rate Total PDV sequestrationb

Lactating

P< Stage

DMI

Inter a

0.4 2.9

– –

– 0.200

– –

94.4 40.3 106.9 8.9 26.2 3.6

5.0 3.2 3.3 0.5 1.1 0.5

0.905 0.153 0.030 0.040 0.307 0.693

0.015 0.021 0.010 0.025 0.003 0.129

0.393 0.884 0.204 0.042 0.611 0.848

33.6 17.7

1.5 1.7

0.898 0.070

0.013 0.017

0.832 0.163

Low

High

Low

High

SEM

8.0 –

12.0 –

14.9 14.3

19.4 16.5

51.5 14.8 35.4 3.2 9.8 1.5

63.2 29.3 46.6 3.4 15.2 2.2

73.0 25.3 82.0 6.4 21.1 3.0

20.4 6.4

25.4 10.4

28.5 6.3

a Interaction b Sum

of DMI and stage of lactation. of arterial and absorptive use.

well as its use for protein synthesis, but largely represents oxidative metabolism. In lactating dairy cows in mid-lactation, gross liver removal was equal to 70–80% of total body IRL (Reynolds et al., 2000). Of the phenylalanine removed, 33–57% was oxidized, while only 4–8% was converted to tyrosine which was subsequently released into blood. 3.4. Endogenous amino acid secretions In addition to constitutive protein synthesis and oxidation, essential amino acid sequestration by the PDV must also support the synthesis of protein appearing in the lumen of the gut as endogenous secretions (or sloughage of constitutive proteins). Depending on their site of entry into the gut lumen, these endogenous amino acids are reabsorbed or lost in the faeces. Tagari and Bergman (1978) did not account for endogenous amino acid flow in the ileum in their study, and thus underestimated total amino acid disappearance. Measurements of endogenous amino acid flow within the lumen of the gut are difficult to obtain and are often assumed to be a fixed, basal amount from the regression of amino acid flow on intake (MacRae et al., 1997a). Other approaches for estimating endogenous protein or amino acid flow in the gut are based on statistical interpretation of digesta amino acid composition (Larsen et al., 2000), or the isotopic labeling of precursor pools and gut contents, often using 15N- or 13C-labeled leucine (see Ouellet et al., 2002). Using long-term intravenous 15N-labeled leucine infusion to estimate total endogenous N flow into specific components of duodenal and fecal nitrogen flow, and mathematical modeling of N exchanges, Ouellet et al. (2002) recently estimated that total endogenous N in the duodenum, arising from urea and amino acid N transfer to bacterial N, or appearing directly as free endogenous N, accounted for 24% of total N flow into the duodenum, and 4% of total N loss in the feces. Using 13C-labeled leucine infused into the jugular vein for 7 days, endogenous leucine represented 8% of duodenal leucine flow to the duodenum (table 2; Reynolds et al., 2001a),

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a number that is in agreement with measurements based on 14C-labeled leucine in sheep (Marsden et al., 1988). Endogenous leucine flow in the ileum accounted for 14.5% of total leucine flow, and the fraction of duodenal and ileal leucine flow of endogenous origin was unaffected by stage of lactation or DMI, thus endogenous leucine flow increased linearly with increased DMI (table 2). Larsen et al. (2000) also estimated an increase in endogenous amino acid flow into the duodenum with increased DMI. These data show that, rather than comprising a basal amount equal to flow in fasted animals, endogenous amino acid flow increases with intake and digesta flow. While the values obtained through the use of 13C- or 14C-leucine labeling are considerably lower than estimates of the total endogenous contribution to N flow in the duodenum of dairy cows (Ouellet et al., 2002), they are similar to the estimate of the contribution of free endogenous sources (Ouellet et al., 2002), and thus the differences surely reflect technical differences between the two methods for estimating endogenous amino acid flow. The appearance of endogenous amino acids in the rumen and small intestine represents a route of N recycling, which has been estimated to account for 30–40% of N absorption as amino acids (Lapierre and Lobley, 2001). Based on their models, Ouellet et al. (2002) estimated that endogenous secretions account for as much as 30% of total PDV sequestration of amino acids. In our study (table 2), endogenous leucine flow in the duodenum and ileum accounted for 19–37% of total leucine sequestration by the PDV of dry and lactating dairy cows. While the reabsorption of endogenous N represents an opportunity for reuse of N, it also incurs a cost in terms of the rate of protein synthesis in gut tissues (Reeds et al., 1999; van der Schoor et al., 2002). In ruminants, the efficiency of utilization of absorbed essential amino acids for growth (largely muscle accretion) is typically lower (50–55%) than in nonruminants (70–75%), and this may well be a consequence of the disproportionately high rates of body protein turnover occurring in the gut compared to nonruminants (MacRae et al., 1997a). These high rates of protein turnover and endogenous secretion appear to be one cost of supporting the extensive development of metabolically active rumen tissues and the consumption of high-fiber diets that increase gut fill.

4. AMMONIA ABSORPTION AND UREA SYNTHESIS Initial studies of net amino acid absorption across the PDV in sheep fed alfalfa pellets (Wolff et al., 1972) also highlighted the extensive absorption of ammonia N compared to amino acid N. Across the PDV, net ammonia absorption accounted for a greater fraction of dietary N intake (48%) than did the net absorption of N as amino acids (26%). This in part reflects the diet fed, as the alfalfa pellets fed were relatively high in protein (20%), which is highly rumen-degradable. However, across a range of dietary treatments, net PDV absorption of ammonia N is highly correlated with N or digestible N intake, and typically exceeds the absorption of total amino acid N (Seal and Reynolds, 1993; Reynolds, 1995; Lapierre and Lobley, 2001; Lindsay and Reynolds, 2003). In a recent summary of published data from cattle (Lindsay and Reynolds, 2003), increased net PDV release of ammonia N accounted for 41% of incremental N intake, whilst simultaneous increases in α-amino N or total amino acid absorption accounted for 31% of the increments in N intake. The ammonia absorbed is derived from microbial degradation of nitrogenous compounds, including feed protein and endogenous urea and proteins, as well as any ammonia arising from metabolism within PDV tissues. As emphasized by Lapierre and Lobley (2001), a substantial portion of ammonia absorbed is derived from blood urea. They estimated that, on average, roughly two-thirds of urea synthesized is transferred to the lumen of the gut via saliva and direct blood transfer, which may involve a specific transporter

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(Waterlow, 1999). Of the urea N transferred to the gut, 40% was recycled as absorbed ammonia, whilst 50% was absorbed as amino acids synthesized in the rumen. The remainder was lost in the feces, presumably in the form of microbial protein synthesized in the hindgut. As microbial protein synthesis is a major route of urea and ammonia N utilization in the gut lumen, dietary N supply and digestion is not the only determinant of ammonia absorption, and the supply of fermentable energy and other factors affecting microbial growth also impacts net ammonia appearance in the portal vein. For example, starch infusion into either the rumen or abomasum of lactating dairy cows decreased net PDV release of ammonia (Reynolds et al., 1998), and abomasal starch infusion increased fecal excretion of N, presumably as a consequence of incomplete starch digestion in the small intestine and increased microbial protein synthesis in the hindgut (Reynolds et al., 2001b). 4.1. Costs of excess protein digestion and urea synthesis in the liver Ammonia absorbed into the portal vein is efficiently cleared by the liver and detoxified by incorporation into urea and to a lesser extent other nitrogenous compounds such as glutamine (Lobley et al., 2000). When considered in isolation, the urea cycle requires four phosphate bonds per mole of urea synthesized, and on this basis ureagenesis in the liver appears to account for a substantial portion of liver oxygen use (Reynolds et al., 1991b; Lobley et al., 1995; Reynolds, 1995; Milano et al., 2000). This accounting is supported by calorimetry studies in which increased heat production was measured in sheep infused into the abomasum with ammonia or urea, or intravenously with urea (Martin and Blaxter, 1965). These studies suggested a greater systemic cost of urea synthesis than the theoretical cost of converting ammonia to urea, which was attributed to increased cycling of urea and ammonia between the blood and gut lumen (Martin and Blaxter, 1965). In addition, calorimetry studies with dairy cattle suggested an energetic cost of consuming digestible protein in excess of requirements equivalent to 30 kJ ME per g N (Tyrrell et al., 1970). However, these increases in heat production (i.e. oxygen consumption) were measured on a whole-body basis. In multicatheterized cattle fed isonitrogenous diets differing in forage:concentrate ratio at equal ME intakes, digested N, ammonia absorption and liver removal, and liver urea synthesis were greater when the high-forage diet was fed, but liver oxygen consumption was not affected significantly (table 3; Reynolds et al., 1991a,b). Liver urea release and oxygen consumption increased with intake of both rations, but this was associated with an increase in ME, glucose synthesis, and presumably liver mass. In other work, adding urea to a high-protein diet fed at maintenance to beef cattle caused a large increase in ammonia absorption and liver urea release, but again had no significant effect on liver oxygen consumption (table 3; Maltby et al., 1993). Subsequent studies in sheep in which ammonia delivery to the liver and ureagenesis were increased by ammonia infusion (table 3) have also failed to show a statistically significant increase in liver oxygen consumption (Lobley et al., 1995, 1996), although numerical trends for increased oxygen uptake were observed in one study when ammonia absorption was increased by 165% (Milano et al., 2000). These observations suggest that increases in ammonia absorption and subsequently liver urea synthesis do not require increased oxidative metabolism in the liver. This may reflect shifts in other metabolic processes within the liver, to provide the ATP required for ureagenesis without increasing oxidative metabolism in total. An alternative explanation is that the net cost of urea synthesis is lower when the ATP gain of fumarate metabolism is included in the balance sheet, which reduces the energy cost of urea formation by 75% (Reynolds et al., 1991b). This accounting does not explain the observed increase in body oxygen consumption reported in sheep infused with ammonium bicarbonate

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(Martin and Blaxter, 1965), but differences in the relative amount of ammonia infused may be a factor (Milano et al., 2000). In contrast to the studies cited in the preceding discussion, increases in dietary protein level, via the addition of soybean meal to a corn-based diet fed to growing beef steers, significantly increased body heat production (Reynolds et al., 1992a), in line with the results of Tyrrell et al. (1970). In this case, increases in body heat production with higher dietary protein intake were associated with increases in ammonia absorption (table 3), as well as increased absorption of some amino acids (Reynolds et al., 1995a), and the increases in body oxygen consumption were attributable to relatively small, but significant, increases in both liver and PDV oxygen consumption. The significant increase in liver oxygen use in this later study may in part reflect the larger number of animals sampled, and low variation observed. However, in sheep fed low-protein concentrate-based diets supplemented with urea or two sources of protein, liver oxygen consumption was increased when the supplemental protein Table 3 Effects of diet intake and composition on net portal-drained visceral ammonia absorption (PDV NH3N) and net liver release of urea N and use of oxygen in beef cattle kg/d Animal and diet

DMI

mmol/h N intake

Growing heifersa Low intake 75% alfalfa 4.75 0.133 75% corn:SBMb 3.60 0.098 High intake 75% alfalfa 7.78 0.209 75% corn:SBM 6.51 0.174 Diet effect, P < 0.001 0.001 Mature steersc 75% alfalfa 5.67 0.153 75% alfalfa + urea 5.80 0.209 Diet effect, P < 0.001 0.001 Growing steersd Medium intake 75% corn 5.10 0.096 75% corn:SBM 4.98 0.131 High intake 75% corn 6.87 0.128 75% corn:SBM 6.96 0.179 Diet effect, P < 0.718 0.001 Mature sheep–mesenteric vein NH3N infusione Alfalfa pellets 0.71 0.018 + 12.6 mmol/h NH4Cl 0.71 0.018 NH3 effect, P < Mature sheep–mesenteric vein NH3N infusionf Grass pellets 1.09 0.035 + 7.5 mmol/h N4CO3 1.09 0.035 Grass pellets–barley 1.08 0.031 + 7.5 mmol/h N4CO3 1.08 0.031 NH3 effect, P <

PDV NH3N

Liver urea N

Liver O2 use

186 143

354 235

719 621

340 250 0.067

593 491 0.080

1205 1192 0.460

253 398 0.001

363 537 0.004

926 947 0.457

121 212

199 323

821 865

165 261 0.001

289 473 0.001

1091 1180 0.051

27.1 39.5 0.001

37.7 61.9 0.071

92.4 110.4 0.535

37.0 47.8 42.6 57.7 0.001

69.0 84.9 71.2 85.8 0.001

151.2 194.4 173.4 188.4 0.252 Continued

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Table 3—Cont’d Effects of diet intake and composition on net portal-drained visceral ammonia absorption (PDV NH3N) and net liver release of urea N and use of oxygen in beef cattle kg/d Animal and diet

DMI

mmol/h N intake

Mature sheep–mesenteric vein NH3N infusiong Grass pellets 0.78 0.017 + 9 mmol/h NH4CO3 0.78 0.017 + 24 mmol/h NH4CO3 0.78 0.017 NH3 effect, P < Sheep–95% concentrateh Control 1.10 0.012 + Urea 1.13 0.021 + SBM 1.19 0.021 + BFM 1.01 0.018 Protein effect,i P < 0.57 0.43

PDV NH3N

Liver urea N

Liver O2 use

20.6 35.3 54.5 0.001

42.6 54.7 81.4 0.002

96.6 96.0 120.2 0.130

6.5 12.8 11.7 11.8 0.40

37.4 52.3 55.2 68.3 0.18

178 185 229 239 0.01

a Reynolds

et al. (1991a,b). meal. c Maltby et al. (1993). d Reynolds et al. (1992a). e Lobley et al. (1995). Sheep received 5-day mesenteric vein infusions of low (1.5 mmol/h) or high (14.1 mmol/h) NH4Cl. f Lobley et al. (1996). Sheep received 4-day mesenteric vein infusions of low (1.5 mmol/h NH Cl) 4 or high (1.5 mmol/h NH4Cl plus 7.5 mmol/h NH4CO3) ammonium salt. g Milano et al. (2000). Sheep received 4-day mesenteric vein infusions of 0, 9, or 24 mmol/h NH CO . 4 3 h Ferrell et al. (2001). Sheep were fed a low-protein control concentrate (6.6% crude protein) supplemented (to 11.2% crude protein) with urea, SBM, or blood and feather meal (BFM). i Comparison of urea with SBM and BFM protein supplements. b Soybean

was fed, but not urea (Ferrell et al., 2001; table 3). This was also associated with a trend for greater net PDV release of α-amino N when the protein-based supplements were fed. Therefore, increases in oxygen consumption observed in animals fed protein in excess of requirement may reflect an increased oxidation of amino acids, rather than an energy cost of urea synthesis from ammonia per se. As emphasized by Bergman and Heitmann (1980), interorgan cycles involving a number of amino acids shuttle N and carbon between the liver and peripheral tissues, and provide spatial separation of the urea cycle. With the exception of branched-chain amino acids, oxidation of essential amino acids occurs to a large extent in the liver, but oxidation also occurs in peripheral tissues such as muscle, which is the primary site of branched-chain amino acid catabolism (Layman, 2003). It is apparent that PDV tissues participate in the oxidative metabolism of leucine and other amino acids (Lapierre et al., 1999, 2002; Lobley et al., 2001, 2003). Urea synthesis and excretion essentially represents the catabolism of amino acids available in excess of their requirement for anabolic and metabolic functions, and the fraction of ammonia N absorbed that is not recycled to the gut. In the hepatocyte, one N in urea arises directly from ammonia, whilst the other is derived from aspartate, which is derived from glutamate and thus indirectly from ammonia. Previous studies in multicatheterized cattle observed an increase in liver amino acid removal under conditions of increased ammonia absorption, suggesting that the synthesis of aspartate from ammonia via glutamate might be limiting urea synthesis, thus increasing liver catabolism of amino acids to support ureagenesis (Reynolds, 1992; Parker et al., 1995). However, variables other than ammonia supply to

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the liver were also altered in the studies forming the basis of this hypothesis (e.g. Reynolds et al., 1991b). In the intervening period a detailed, systematic series of studies has been conducted by G.E. Lobley and his colleagues at the Rowett Research Institute to explore the effects of increased ammonia supply on liver metabolism in sheep, under experimental conditions that sought to control variation in other metabolic variables affecting liver metabolism, and incorporating novel isotopic labelling experimentation and mathematical modeling (Lobley et al., 1995, 1996; Milano et al., 2000; Milano and Lobley, 2001). Initial indications suggested an effect of mesenteric vein NH4Cl infusion on leucine oxidation (Lobley et al., 1995). However, more recent studies in which shifts in acid–base status were avoided by infusing NH4HCO3 (Lobley and Milano, 1997; Milano et al., 2000; Milano and Lobley, 2001) found no indication that increased amino acid deamination is required for the synthesis of urea under conditions of increased ammonia load, at least in maintenance-fed sheep. 4.2. Regulation of urea synthesis Since its description seventy years ago, the regulation of the ornithine cycle and liver urea synthesis has been the subject of numerous reviews and much discussion (e.g. Waterlow, 1999; Lobley et al., 2000), and will not be considered in detail in the present chapter. Generally, liver removal of amino acids is determined by the functional requirements of the liver (e.g. constitutive and export protein synthesis) and the availability of amino acids in the blood pool relative to body requirements. The extent to which ureagenesis in the liver is directly regulated by factors other than plasma amino acid concentrations has been the subject of debate, but after decades of research, current thinking is that “it is very unlikely that the signal (integrating protein intake and urea cycle activity) is an alteration in the plasma concentration either of total amino-N or any single amino acid” (Waterlow, 1999). In growing steers treated with growth hormone-releasing factor, increases in body N retention, at equal N intake, were accompanied by decreases in liver removal of α-amino and ammonia N and release of urea, but with no change observed in blood α-amino N concentration (Lapierre et al., 1992; Reynolds et al., 1992b). Similar responses were observed in growing steers treated with growth hormone (Bruckenthal et al., 1997). In nonruminants, growth hormone treatment caused a decrease in the activity of urea cycle enzymes in the liver, but the effects may have been an indirect consequence of increased protein retention in vivo (McLean and Gurney, 1963; Palekar et al., 1981). Abomasal infusion of glucose decreased liver urea production and increased N retention in maintenance-fed sheep (Obitsu et al., 2000), presumably through effects of insulin, but the effects on urea production may also reflect indirect responses to increased amino acid retention. Similarly, abomasal infusion of starch for 2 weeks markedly increased tissue N retention in late-lactation, pregnant dairy cows (Reynolds et al., 2001b).

5. LIVER AMINO ACID METABOLISM In maintenance-fed sheep (Wolff et al., 1972; Bergman and Heitmann, 1980), liver removal of a number of amino acids accounted for substantial portions of their net PDV absorption and release, but as emphasized by Bergman (1986), the animals were not in positive tissue N balance. Regardless, these studies identified a number of interorgan cycles involving specific groups of amino acids. For the glucose precursors alanine, serine, and glycine, their net removal by the liver was greater than their net absorption, such that their net total splanchnic flux was negative. A negative total splanchnic flux for these amino acids represents the contributions of peripheral tissues to their liver metabolism, and the hind limbs released them

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on a net basis, reflecting the catabolism of glucose and other amino acids, such as leucine (Layman, 2003). The branched-chain amino acids were removed by the liver, but in the lowest amount relative to net PDV release, such that their net total splanchnic release was more positive than for other amino acids, and they were removed by the hindlimbs. Glutamate was released by the liver, whilst glutamine was removed, and the opposite occurred in the hindlimbs, representing a means of transporting ammonia N from peripheral tissues to the liver for urea synthesis. A similar interorgan “cycle” was apparent for arginine and ornithine plus citrulline, which shuttled N from the kidney and hindlimbs to the liver as arginine. These data suggest that some of this arginine was synthesized via the action of arginine synthetase in peripheral tissues, using ornithine and citrulline which were released by the liver and subsequently removed by the kidney and hind limbs (Bergman and Heitmann, 1980). Again, this spatial separation of the urea cycle provides opportunity for metabolic flexibility within and among tissues of the body. On average, the liver removed more amino acid N than appeared across the PDV (Wolff et al., 1972), but this reflects the level of feed intake and N status of the animals, and the fact that net absorption underestimates true rates of absorption to the extent that amino acids are utilized by the PDV. With greater intake, the relationship between liver removal of amino acids and their true or net absorption across the PDV will vary, depending in part on the energy status of the animal, which determines protein requirement in growing animals. Similarly, relationships between PDV and liver flux of amino acids in dairy cattle will be determined by stage of lactation and relative milk protein production. In attempting to ascertain the fractional clearance of amino acids during their absorption and passage through the liver, information needed for the development of mechanistic models of amino acid utilization, amino acids mixtures (or proteins) have been infused into the abomasum, to increase absorption, or mesenteric veins, to mimic increased absorption, over short (hours) or long (days) periods (e.g. Reynolds and Tyrrell, 1991; Reynolds et al., 1995b, 1999, 2000; Bruckenthal et al., 1997; Wray-Cahen et al., 1997; Lobley et al., 2001). The interpretation of these results must consider a variety of factors, such as the amounts infused relative to requirements, the time allowed for adaptation to the infused amino acids, the form in which the amino acids are provided (protein vs free amino acids), and the balance of amino acids provided (see Wolfe and Miller, 2002). Essential amino acids are not required in isolation, thus provision of a limiting amino acid often creates a deficiency of a second limiting amino acid. Therefore, the efficiency of utilization of one amino acid, and thus liver clearance, is dependent on a balanced supply of other amino acids relative to body requirements. The inverse of this concept of a balanced amino acid supply is illustrated by a model for the creation of a deficiency of a specific amino acid through the feeding of a low-protein diet to lactating goats, and the infusion of a mixture of essential amino acids into the abomasum which is balanced relative to milk protein composition but for the absence of a specific amino acid, such as histidine (Bequette et al., 2000). Under these circumstances, the metabolic adaptations of the mammary gland to maximize histidine supply for milk protein synthesis (increased blood flow and mammary histidine extraction) are nothing short of amazing. Similarly, the metabolic and production responses of the gut and liver to mesenteric vein infusion of mixtures of amino acids based on milk protein composition were affected by the presence or absence of nonessential amino acids (Reynolds et al., 1995b). In other studies, the response of net PDV amino acid release and liver removal of essential amino acids (Reynolds et al., 1999), and PDV sequestration of leucine and phenylalanine (Caton et al., 2001), differed when the same quantity of essential amino acids was provided as a component of casein, which also provided supplemental nonessential amino acids, or a mixture of free essential

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amino acids. Differences in whole-body and splanchnic leucine metabolism were also observed in rats fed supplemental free amino acids compared to the same mixture of amino acids provided as casein (Daenzer et al., 2001). Despite these factors, some general patterns of liver metabolism are apparent across studies in cattle and sheep (Lobley et al., 1996, 2001; Lobley and Milano, 1997; Wray-Cahen et al., 1997; Lapierre et al., 1999; Reynolds et al., 1999, 2000, 2001a; Blouin et al., 2002). As a proportion of net PDV absorption, liver removal of alanine, serine, glycine, phenylalanine, and histidine is typically high. This in part reflects the role of the first three as glucose precursors (Bergman and Heitmann, 1980), the role of glycine in detoxification processes, and the high concentration of phenylalanine and histidine in plasma protein synthesized by the liver (Lobley et al., 2001). In contrast, net liver removal of the branched-chain amino acids is typically low relative to their rate of absorption, as observed in maintenance-fed sheep (Bergman, 1986). In cattle, the liver typically releases leucine and often valine and isoleucine (Blouin et al., 2002). Their net removal was increased when their supply to the liver was increased by abomasal casein infusion, but not a mixture of free essential amino acids, which caused an increase in their net liver release (Reynolds et al., 1999). Similar responses were observed when mixtures of free amino acids, based on the composition of milk protein, were infused into the mesenteric vein of early-lactation dairy cows fed a low-protein diet for 3 days (Reynolds et al., 1995b). Infusion of the mixture of essential amino acids increased net liver release of leucine and tended to increase the release of valine, while the same amino acid mixture infused along with nonessential amino acids had no effect on their net flux across the liver. On a net basis, the liver releases leucine in dogs as well, but this could be attributable in part to liver removal and transamination of MOP (Abumrad et al., 1982). In dairy cows, net liver removal of MOP is insufficient to account for the leucine released (Lapierre et al., 2002), suggesting other sources such as peptides or blood-borne proteins arising in other tissues and degraded in the liver (Elwyn, 1970). i-Valerate is a product of leucine catabolism in the liver, and the synthesis of branched amino acids from branchedchain volatile fatty acids in the ruminant liver has been proposed (van der Walt, 1993). In lactating dairy cows, the absorption and liver removal of i-valerate cows (e.g. Reynolds et al., 2003) is typically much greater than net leucine release (e.g. Lapierre et al., 2002). Leucine delivery to peripheral tissues is important not only as an essential amino acid for protein synthesis, but also as a regulator of protein synthesis and other anabolic processes (Abumrad et al., 1982), in part through modification of insulin signalling (Layman, 2003). 5.1. Gluconeogenesis In sheep, Wolff and Bergman (1972b) estimated that amino acid carbon accounts for from 11% to 30% of liver glucose synthesis, based on measured transfers of alanine, glutamate, aspartate, glycine, and serine 14C to glucose (11% of glucose production), or the maximal potential net contribution of all the plasma amino acids removed by the liver (30% of glucose production). There is no question that the carbon from these glucogenic amino acids makes an important contribution to liver glucose synthesis in ruminants. Net liver removal of other precursors (propionate, lactate, glycerol, i-butyrate, and n-valerate) is seldom adequate to account for all of the glucose released by the liver, but the extent to which amino acid supply limits glucose production is not certain. Glucose production by the liver is regulated by requirement, thus the provision of additional precursor that is efficiently removed by the liver, such as propionate or alanine, typically decreases liver lactate removal, without affecting glucose production in fed animals

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(Reynolds, 1995). However, in early-lactation dairy cows, glucose production is often considered to be limited by precursor supply from the diet, as occurs in fasting, and increases in pyruvate carboxylase activity have suggested a greater contribution of alanine, and by conjecture other amino acids, to glucose production in early lactation (Drackley et al., 2001). However, in early-lactation cows fed low-protein diets, mesenteric vein infusion of a mixture of nonessential amino acids for 3 days had no significant effect on liver glucose production, and significantly reduced milk yield (Reynolds et al., 1995b). In contrast, abomasal infusions of casein, or an equivalent mixture of essential amino acids, increased liver glucose production (Reynolds et al., 1999). This suggests that the response to casein was not due to the provision of nonessential amino acids, but perhaps an effect of essential amino acid supply on liver metabolism or glucose requirement. Even in very early lactation, the net balance of liver glucose release and precursor removal does not support the concept of an obligatory increase in nonessential amino acid contributions to liver glucose synthesis, apart from an increased potential contribution of alanine (Reynolds et al., 2003). Between 9 days before calving and 11 days after calving, the potential contribution of alanine to liver glucose release doubled (from 2.5% to 5%), but increases in net liver removal of propionate, lactate, alanine, and glycerol were sufficient to account for all of the increase in liver glucose release. This suggests that the amino acid contributions to liver glucose synthesis in early lactation arise from the obligatory deamination of amino acids in the liver, rather than being a metabolic requirement of early lactation.

6. CONCLUSIONS The extensive development of the ruminant forestomach distinguishes their N economy from that of nonruminants. The microbial fermentation that occurs there markedly alters the profile and form of protein and amino acids presented to the small intestine for digestion and absorption, and provides opportunities for extensive recycling of N between the body and lumen of the gut. This recycling occurs via exchanges of ammonia and urea with blood and luminal pools, endogenous gut and secretory N entry to the gut lumen, microbial protein synthesis, and the subsequent digestion and absorption of microbial and endogenous amino acids. The costs of this exquisite microbial symbiosis include the costs of urea synthesis, which may be less then hypothesized at the level of the liver, and an extensive utilization of absorbed amino acids from arterial blood, which masks their net appearance from the lumen of the gut into the portal vein. Liver metabolism of amino acids includes substantial requirements for liver functions and the integration of the supply of nitrogenous compounds from the diet with body requirements. A more detailed understanding of these processes within the splanchnic tissues and their response to changes in diet composition and intake, relative to nutrient requirements for production, is needed to improve current empirical models of the efficiency of amino acid utilization for production. Such data are forthcoming; however, the prediction of productive responses to changes in amino acid supply from the small intestine can not be based simply on estimates of nutrient supply. The genetic and environmental propensity of the animal for the productive use of their nutrient supply will ultimately determine their N economy.

7. FUTURE PERSPECTIVES The ability of current feed-rationing and nutrient requirement systems to predict productive response of ruminants to changes in the supply of protein to the small intestine is currently

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limited by an oversimplification of postabsorptive metabolism. Future approaches will need to address the quantitative metabolism of individual amino acids, which is highly integrated, complex, flexible, and adaptive. Ultimately, the efficiency with which nitrogenous compounds are utilized will be determined by the propensity of the animal fed for productive nutrient use. However, the exact mechanisms by which nutrient absorption and tissue requirements are integrated are poorly understood. For example, the production of urea from amino acids is largely a consequence of the availability of amino acids in excess of requirement, yet the signal by which this oversupply is communicated to the urea cycle remains a mystery. Although the urea cycle was the first metabolic cycle delineated, and despite years of research on its regulation, it remains a potentially fruitful area for future research. Unraveling the costs of excess protein intake in terms of the integration of amino acid catabolism and urea genesis is particularly relevant. The processes of amino acid catabolism do not occur solely in the liver for all amino acids, yet the response of amino acid metabolism in the gut and other extrahepatic tissues to increased protein intake are still poorly described. The regulation of branched-chain amino acid metabolism and their role in regulating metabolic processes deserves particular attention in this regard. In addition, the metabolic source of the branchedchain amino acids released by the liver of ruminants should be determined. As restrictions on N losses from animal production facilities increase, so to will the need to more precisely formulate diets to meet the requirements for specific amino acids. This will only be achieved through a greater understanding of the metabolic integration of dietary supply with tissue demand.

REFERENCES Abumrad, N.N., Wise, K.L., Williams, P.E., Abumrad, N.A., Lacy, W.W., 1982. Disposal of α-ketoisocaproate: roles of liver, gut and kidneys. Amer. J. Physiol. 243, E123–E131. Benson, J.A., Reynolds, C.K., Aikman, P.C., Lupoli, B., Beever, D.E., 2002. Effects of abomasal long chain fatty acid infusion on splanchnic nutrient metabolism in lactating diary cows. J. Dairy Sci. 85, 1804–1814. Bequette, B.J., Hanigan, M.D., Calder, A.G., Reynolds, C.K., Lobley, G.E., MacRae, J.C., 2000. Kinetics of amino acid transport in the mammary gland of lactating goats when histidine supply is limiting for milk production. J. Dairy Sci. 83, 765–775. Bergman, E.N., 1975. Production and utilisation of metabolites by the alimentary tract as measured in portal and hepatic blood. In: McDonald, I.W., Warner, A.C.I. (Eds.), Digestion and Metabolism in the Ruminant. The University of New England, Armidale, Australia, p. 292. Bergman, E.N., 1986. Splanchnic and peripheral uptake of amino acids in relation to the gut. Fed. Proc. 45, 2277–2282. Bergman, E.N., Heitmann, R.N., 1980. Integration of whole-body amino acid metabolism. In: Buttery, P.J., Lindsay, D.B. (Eds.), Protein Deposition in Animals. Butterworth, London, pp. 69–84. Berthiaume, R., Dubreuil, P., Stevenson, M., McBride, B.W., Lapierre, H., 2001. Intestinal disappearance and mesenteric and portal appearance of amino acids in dairy cows fed ruminally protected methionine. J. Dairy Sci. 84, 194–203. Blouin, J.P., Bernier, J.F., Reynolds, C.K., Lobely, G.E., Dubreuil, P., Lapierre, H., 2002. Effect of diet quality on splanchnic fluxes of nutrients and hormones in lactating diary cows. J. Dairy Sci. 85, 2618–2630. Bruckenthal, I., Huntington, G.B., Baer, C.K., Erdman, R.A., 1997. The effect of abomasal infusion of casein and recombinant somatotropin hormone injection on nitrogen balance and amino acid fluxes in portal-drained viscera and net hepatic and total splanchnic blood in Holstein steers. J. Anim. Sci. 75, 1119–1129. Burrin, D.G., Ferrell, C.L., Britton, R.A., Bauer, M., 1990. Level of nutrition and visceral organ size and metabolic activity in sheep. Brit. J. Nutr. 64, 439–448. Caton, J.S., Reynolds, C.K., Bequette, B.J., Lupoli, B., Aikman, P.C., Humphries, D.J., 2001. Effects of abomasal casein or essential amino acid infusions on splanchnic leucine and phenylalanine metabolism in lactating dairy cows. J. Dairy Sci. 84, Suppl. 1, 363.

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Daenzer, M., Petzke, K.J., Bequette, B.J., Metges, C.C., 2001. Whole-body and splanchnic amino acid metabolism differ in rats fed mixed diets containing casein or its corresponding amino acid mixture. J. Nutr. 131, 1965–1972. Dijkstra, J., Mills, J.A.N., France, J., 2002. The role of dynamic modelling in understanding the microbial contribution to rumen function. Nutr. Res. Rev. 15, 67–90. Drackley, J.K., Overton, T.R., Douglas, G.N., 2001. Adaptations of glucose and long-chain fatty acid metabolism in liver of dairy cows during the periparturient period. J. Dairy Sci. 84, E. Suppl. E100–E112. Elwyn, D.H., 1970. The role of the liver in regulation of amino acid and protein metabolism. In: Munro, H.N. (Ed.), Mammalian Protein Metabolism. Academic Press, New York, p. 523. Ferrell, C.L., Freetly, H.C., Goetsch, A.L., Kreikemeier, K.K., 2001. The effect of dietary nitrogen and protein on feed intake, nutrient digestibility, and nitrogen flux across the portal-drained viscera and liver of sheep consuming high-concentrate diets ad libitum. J. Anim. Sci. 79, 1322–1328. Habel, R.E., 1992. Guide to the Dissection of Domestic Ruminants. Habel, R.E., Ithaca, NY. Huntington, G.B., Reynolds, C.K., Stroud, B., 1989. Techniques for measuring blood flow in the splanchnic tissues of cattle. J. Dairy Sci. 72, 1583–1595. Huntington, G.B., Eisemann, J.H., Whitt, J.M., 1990. Portal blood flow in beef steers: comparison of techniques and relation to hepatic blood flow, cardiac output and oxygen uptake. J. Anim. Sci. 68, 1666–1673. Katz, M.L., Bergman, E.N., 1969. Simultaneous measurements of hepatic and portal venous blood flow in the sheep and dog. Amer. J. Physiol. 216, 946–952. Lapierre, H., Lobley, G.E., 2001. Nitrogen recycling in the ruminant: a review. J. Dairy Sci. 84, E. Suppl. E223–E236. Lapierre, H., Reynolds, C.K., Elsasser, T.H., Gaudreau, P., Brazeau, P., Tyrrell, H.F., 1992. Effects of growth hormone-releasing factor and intake on energy metabolism in growing beef steers: net hormone metabolism by portal-drained viscera and liver. J. Anim. Sci. 70, 742–751. Lapierre, H., Bernier, J.F., Dubreuil, P., Reynolds, C.K., Farmer, C., Ouellet, D.R., Lobley, G.E., 1999. The effect of intake level on splanchnic protein metabolism in growing beef steers. Brit. J. Nutr. 81, 457–466. Lapierre, H., Blouin, J.P., Bernier, J.F., Reynolds, C.K., Dubreuil, P., Lobley, G.E., 2002. Effect of diet quality on leucine metabolism across splanchnic tissues in lactating dairy cows. J. Dairy Sci. 85, 2631–2641. Larsen, M., Madsen, T.G., Weisbjerg, M.R., Hvelplund, T., Madsen, J., 2000. Endogenous amino acid flow in the duodenum of dairy cows. Acta Agric. Scand. Sect. A. Anim. Sci. 50, 161–173. Layman, D.K., 2003. The role of leucine in weight loss diets and glucose homeostasis. J. Nutr. 133, 261S–267S. Lindsay, D.B., Reynolds, C.K., 2003. Metabolism of the portal-drained viscera. In: Dyjkstra, J., France, J., Forbes, J.M. (Eds.), Quantitative Aspects of Ruminant Digestion and Metabolism. CABI, Wallingford, UK. Submitted. Lobley, G.E., 1994. Amino acid and protein metabolism in the whole body and individual tissues of ruminants. In: Asplund, J.M. (Ed.), Principles of Protein Nutrition in Ruminants. CRC Press, London, p. 147. Lobley, G.E., Bremner, D.M., Brown, D.S., 2001. Response in hepatic removal of amino acids by the sheep to short-term infusions of varied amounts of an amino acid mixture into the mesenteric vein. Brit. J. Nutr. 85, 689–698. Lobley, G.E., Connell, A., Lomax, M.A., Brown, D.S., Milne, E., Calder, A.G., Faringham, D.A.H., 1995. Hepatic detoxification of ammonia in the ovine liver: possible consequences for amino acid catabolism. Brit. J. Nutr. 75, 217–235. Lobley, G.E., Milano, G.D., 1997. Regulation of hepatic nitrogen metabolism in ruminants. Proc. Nutr. Soc. 56, 547–563. Lobley, G.E., Milano, G.D., van der Walt, J.G., 2000. The liver: integrator of nitrogen metabolism. In: Cronje, P.B. (Ed.), Ruminant Physiology: Digestion, Metabolism, Growth and Reproduction. CAB International, Wallingford, UK, pp. 149−168. Lobley, G.E., Shen, X., Le, G., Bremmer, D.M., Milne, E., Calder, A.G., Anderson, S.E., Dennison, N., 2003. Oxidation of essential amino acids by the ovine gastro-intestinal tract. Brit. J. Nutr. 89, 617–629. Lobley, G.E., Weijs, P.J.M., Connell, A., Calder, A.G., Brown, D.S., Milne, E., 1996. The fate of absorbed and exogenous ammonia as influenced by forage or forage-concentrate diets in growing sheep. Brit. J. Nutr. 76, 231–248. MacRae, J.C., Bruce, L.A., Brown, D.S., Calder, A.G., 1997a. Amino acid use by the gastrointestinal tract of sheep given lucerne forage. Amer. J. Physiol. 273, G1200–G1207.

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MacRae, J.C., Bruce, L.A., Brown, D.S., Farningham, D.A.H., Franklin, M., 1997b. Absorption of amino acids from the intestine and their net flux across the mesenteric- and portal-drained viscera of lambs. J. Anim. Sci. 75, 3307–3314. Maltby, S.A., Reynolds, C.K., Lomax, M.A., Beever, D.E., 1993. The effect of increased absorption of ammonia and arginine on splanchnic metabolism of beef steers. Anim. Prod. 56, 462. Marsden, M., Bruce, C.I., Bartram, C.G., Buttery, P.J., 1988. Initial studies on leucine metabolism in the rumen of sheep. Brit. J. Nutr. 60, 161–171. Martin, A.K., Blaxter, K.L., 1965. The energy cost of urea synthesis in sheep. In: Blaxter, K.L. (Ed.), Energy Metabolism of Farm Animals. EAAP Publ. No. 11. Academic Press, London, pp. 83–91. McLean, P., Gurney, M.W., 1963. Effect of adrenalectomy and of growth hormone on enzymes concerned with urea synthesis in rat liver. Biochem. J. 87, 96–104. Milano, G.D., Hotston-Moore, A., Lobley, G.E., 2000. Influence of hepatic ammonia removal on ureagenesis, amino acid utilization and energy metabolism in the ovine liver. Brit. J. Nutr. 83, 307–315. Milano, G.D., Lobley, G.E., 2001. Liver nitrogen movements during short-term infusion of high levels of ammonia into the mesenteric vein of sheep. Brit. J. Nutr. 86, 507–513. NRC, 2001. Nutrient Requirements of Dairy Cattle, 7th Revised Edition. National Academy Press, Washington, DC. Obitsu, T., Bremmer, D., Milne, E., Lobley, G.E., 2000. Effect of abomasal glucose infusion on alanine metabolism and urea production in sheep. Brit. J. Nutr. 84, 157–163. Ouellet, D.R., Demers, M., Zuur, G., Lobley, G.E., Seoane, J.R., Nolan, J.V., Lapierre, H., 2002. Effect of dietary fiber on endogenous nitrogen flows in lactating dairy cows. J. Dairy Sci. 85, 3013–3025. Palekar, A.G., Collipp, P.J., Maddaiah, V.T., 1981. Growth hormone and rat liver mitochondria: Effects on urea cycle enzymes. Biochem. Biophys. Res. Commun. 100, 1604–1610. Parker, D.S., Lomax, M.A., Seal, C.J., Wilton, J.C., 1995. Metabolic implications of ammonia production in the ruminant. Proc. Nutr. Soc. 54, 549–563. Reeds, P.J., Burrin, D.G., Stoll, B., van Goudoever, J.B., 1999. Consequences and regulation of gut metabolism. Proceedings of the VIIIth International Symposium on Protein Metabolism and Nutrition. EAAP Publ. No. 96, Wageningen Press, Wageningen, The Netherlands, pp. 127–153. Reynolds, C.K., 1992. Nitrogen metabolism by ruminant liver. J. Nutr. 122, 850–854. Reynolds, C.K., 1995. Quantitative aspects of liver metabolism in ruminants. In: engelhardt, W., Leonhard-Marek, S., Breves, G., Giesecke, D. (Eds.), Ruminant Physiology: Digestion, Metabolism, Growth and Reproduction: Proceedings of the 8th International Symposium on Ruminant Physiology. Ferdinand Enke Verlag, Stuttgart, Germany, p. 351. Reynolds, C.K., 2002. Economics of visceral tissue metabolism in ruminants: toll keeping or internal revenue service? J. Anim. Sci. 80, E. Suppl., E74–E84. Reynolds, C.K., Aikman, P.C., Lupoli, B., Humphries, D.J., Beever, D.E., 2003. Splanchnic metabolism of dairy cows during the transition from late gestation through early lactation. J. Dairy Sci. 86, 1201–1217. Reynolds, C.K., Bequette, B.J., Caton, J.S., Humphries, D.J., Aikman, P.C., Lupoli, B., Sutton, J.D., 2001a. Effects of intake and lactation on absorption and metabolism of leucine and phenylalanine by splanchnic tissues of dairy cows. J. Dairy Sci. 84, Suppl. 1, 362. Reynolds, C.K., Cammell, S.B., Humphries, D.J., Beever, D.E., Sutton, J.D., Newbold, J.R., 2001b. Effects of post-rumen starch infusion on milk production and energy metabolism in dairy cows. J. Dairy Sci. 84, 2250–2259. Reynolds, C.K., Casper, D.P., Harmon, D.L., Milton, C.T., 1992a. Effect of CP and ME intake on visceral nutrient metabolism in beef steers. J. Anim. Sci. 70, Suppl. 1, 315. Reynolds, C.K., Crompton, L.A., Firth, K., Beever, D., Sutton, J., Lomax, M., Wray-Cahen, D., Metcalf, J., Chettle, E., Bequette, B., Backwell, C., Lobley G., MacRae, J., 1995b. Splanchnic and milk protein responses to mesenteric vein infusion of 3 mixtures of amino acids in lactating dairy cows. J. Anim. Sci. 73, Suppl. 1, 274. Reynolds, C.K., Harmon, D.L., Prior, R.L., Casper, D.P., Milton, C.T., 1995a. Splanchnic metabolism of amino acids in beef steers fed diets differing in CP content at two ME intakes. J. Anim. Sci. 73, Suppl. 1, 270. Reynolds, C.K., Humphries, D.J., Cammell, S.B., Benson, J., Sutton, J.D., Beever, D.E., 1998. Effects of abomasal wheat starch infusion on splanchnic metabolism and energy balance of lactating dairy cows. In: McCracken, K.J., Unsworth, E.F., Wylie, A.R.G. (Eds.), Energy Metabolism of Farm Animals: Proceedings of the 14th Symposium on Energy Metabolism, CAB International, Wallingford, UK, p. 39.

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PART III Lipid metabolism

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Hepatic fatty acid oxidation and ketogenesis in young pigs1 J. Odle, P. Lyvers-Peffer, and X. Lin Department of Animal Science, North Carolina State University, Raleigh, NC 27695, USA

A primary limitation to efficient pork production is morbidity and mortality during the perinatal period. Because pigs are born with low energy reserves, their survival hinges on timely consumption of milk. In contrast to carbohydrate-based fetal metabolism, the transition to a milk-based diet necessitates rapid biochemical adaptations to accommodate the oxidation of fatty acids that comprise more than 60% of milk energy. From research reported to date, the degree to which neonatal pigs make these adaptations is questionable. In stark contrast to other mammalian neonates, piglets do not demonstrate elevated ketogenesis despite high milk-fat intake. Ketone bodies play a pivotal role in the transition from carbohydrate-based metabolism to fat-based metabolism, providing an important alternative fuel for glucosedependent tissues. Impaired adaptation limits the piglets’ ability to oxidize fat which likely contributes to the etiology of mortality. Therefore, this review considers the developmental aspects of lipid oxidation in the young pig. The key regulatory enzymes previously elucidated in rodents are reviewed, with inclusion of the limited knowledge available in pigs. Further research in this area will hopefully assist in development of strategies (via nutritional and/or exogenous hormonal manipulation) to enhance development of fatty acid oxidation and ultimately improve piglet survival and growth.

1. INTRODUCTION Impaired growth and high mortality of neonatal pigs pose significant challenges to the swine industry. Postnatal mortality varies among production units, but has been recently estimated by the Agricultural Statistics Service of the United States Department of Agriculture to average approximately 12% of live births, and has shown only modest improvement over the past twenty years (USDA, 2002). In addition, it is estimated that prenatal (embryo and fetal) mortality in

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Supported in part by a grant from the USDA-NRI, No. 98-35206-6645.

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swine may be as high as 25% so that, collectively, these data imply that the number of piglets weaned per litter currently may be less than 65% of true potential. Associated problems of slow growth (morbidity) further add to the inefficiency. The combined economic impact of these losses is enormous. The cost is ultimately carried by the consumer in the price paid for pork. For this reason, there is great impetus for identifying and studying the stressors responsible for the high postnatal mortality. The etiology is complex, as a number of factors may contribute, including nutritional deficiency, low immunocompetence and disease resistance, hypothermia, and crushing by the dam. Many retrospective survey studies have attempted to determine the relative importance of these stressors, but epidemiological/survey approaches have contributed little useful information because the cause of death is difficult to determine precisely, and interactions among the factors complicate interpretation. Consequently, if progress is to be made, controlled experimentation is needed to better understand the developing piglet’s nutritional, immunological, and behavioral responses to its environment. This review addresses a metabolic component of this multifactorial etiology, examining the biochemical competency of piglets to oxidize fatty acids during early postnatal life. In particular, the ontogeny and regulation of hepatic fatty acid oxidation is highlighted owing to the dramatically low level of ketogenesis expressed in neonatal pigs compared to other species.

2. THE NEED FOR RAPID DEVELOPMENT OF FATTY ACID OXIDATION Prior to birth, the fetus oxidizes predominantly glucose, lactate, and amino acids (Battaglia and Meschia, 1978). At parturition, the newborn must elicit the behavioral responses necessary to acquire milk from the dam. This requires effective competition among littermates and occurs in a thermal environment that may be more than 10ºC below the animal’s critical temperature (Stanier et al., 1984). Rohde Parfet and Gonyou (1988) have shown that >30 min may lapse before the first milk is consumed and considerably longer time is required before positive energy balance is regained. Owing to limited body reserves at birth, negative energy balance quickly becomes life-threatening to the piglet. Survival therefore hinges on the timely consumption of the dam’s milk which provides 60% of its calories as fat (Ferre et al., 1986). These events necessitate rapid metabolic adaptations to shift from carbohydrate-based fetal metabolism to fat-based postnatal metabolism. Other mammalian species, faced with a similar challenge, demonstrate elevated ketogenesis during this transition (Girard et al., 1992). For example, ketogenesis measured in hepatocytes from newborn rats increases 6-fold between 0 and 16 h of age (Ferre et al., 1983), and blood ketone body concentrations may exceed 1.5 mM (Foster and Bailey, 1976) in the suckling rat. Thus, neonatal hyperketonemia plays a significant role in the energy economy of the neonate (Girard et al., 1992), sparing glucose and providing carbon for lipogenesis in neural tissue. In contrast, piglets do not display hyperketonemia (Bengtsson et al., 1969; Pegorier et al., 1981) (i.e. less than 0.2 mM) and this may further compromise their survival. Available data indicate that piglets apparently digest and absorb milk fat with high efficiency (digestion coefficients >95% at 2 days of age) and that a major portion of absorbed fat is deposited in adipose tissues prior to weaning (e.g. pigs are <2% fat at birth and ~15% at weaning). However, relatively little is known regarding the oxidative fate of lipid in the piglet and even less is known regarding the regulation of lipid oxidation in this species. Before reviewing the available literature addressing development of fatty acid oxidation in piglets, the major regulatory features of fatty acid oxidation (elucidated in other species) are described below.

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3. REGULATION OF FATTY ACID OXIDATION AND KETOGENESIS The major metabolic pathways involved in hepatic fatty acid metabolism are summarized schematically in fig. 1. In animal cells there are two fatty acid β-oxidation systems, one located in the mitochondria and the second in the peroxisome. The mitochondrion is considered the primary site for fatty acid β-oxidation, while the peroxisome is considered an alternative pathway. Under conditions that enhance peroxisome proliferation, the relative contribution of the peroxisome to total fatty acid oxidation in the liver may be as high as 30% in the rat (Kondrup and Lazarow, 1985) and 47% in the neonatal pig (Yu et al., 1997a).

Fig. 1. Pathways of hepatic lipid metabolism with emphasis on oxidative metabolism. Enzymes/pathways are numbered as follows: (1) long-chain acyl-CoA synthetase, (2) acetyl-CoA carboxylase, (3) various acyl-CoA transferases, (4) carnitine shuttle consisting of CPT I, translocase, and CPT II, (5) medium-chain acyl-CoA synthetase, (6) mitochondrial hydroxymethylglutaryl-CoA synthase, (7) acyl-CoA dehydrogenase, (8) long-chain acyl-CoA synthetase, (9) very long-chain acyl-CoA synthetase, (10) acyl-CoA oxidase, (11) carnitine octanoyltransferase.

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The mitochondria and peroxisome are often found in close proximity to lipid droplets and are believed to work in concert (Latruffe et al., 2001). The β-oxidation reactions of the two systems are similar beginning with an initial dehydrogenation followed by a hydration, a second dehydrogenation, and finally thiolytic cleavage to produce acetyl-CoA and an acylCoA shortened by two carbons (see Reddy and Mannaerts, 1994, for review). Although the reactions are similar, the actual proteins involved differ between the β-oxidation systems. 3.1. Biochemical dogma Long-chain fatty acids (LCFA) are activated to their CoA thioesters via synthetases (fig. 1, enzyme 1; EC 6.2.1.3) located in the ER and in the outer membrane of the mitochondria (Aas, 1971). To date, five genetic variants of the long-chain acyl-CoA synthetases have been cloned from rodent species (Oikawa et al., 1998) and their differential regulation may influence the metabolic fate of the activated fatty acids (Lewin et al., 2001). While preference is shown for LCFA substrates, those of medium-chain length (MCFA, i.e. C6–C10) may be activated as well. The fatty acyl-CoA (FA-CoA) may then be esterified via acyl-CoA transferases (fig. 1, enzyme 3; EC 2.3.1.15) located in the ER, forming various triglycerides, cholesterol esters, phospholipids, etc., which may be exported as lipoproteins (VLDL; Coleman et al., 2000). These transferases have low affinity for MCFA-CoAs such that MCFA are obligate fuels (Bach and Babayan, 1982). The FA-CoA may alternatively be transported into the mitochondria via the coordinated activities of three membrane proteins: carnitine acyltransferase I (CAT, fig. 1, enzyme 4; EC 2.3.1.21) catalyzing the formation of FA-carnitine from FA-CoA outside of the mitochondrial matrix, translocase catalyzing the exchange/diffusion (antiport) of FA-carnitine for free carnitine across the inner mitochondrial membrane, and carnitine acyltransferase II (CAT II), similar to CAT I except residing on the matrix side of the inner membrane. The CAT I and II activities are catalyzed by a family of acyltransferase enzymes. Proteins have been identified with optimum activity toward C2, C8, and C16 FA-CoAs in various tissues. The latter, referred to as carnitine palmitoyltransferase (CPT; discussed below) is likely of greatest importance in the young pig given the predominantly long-chain fatty acid composition of sow’s milk. Within the mitochondrial matrix, the FA-CoA are subjected to oxidation at the β-carbon-yielding acetyl-CoA, which may be further oxidized to CO2 in the TCA cycle. Alternatively, it may exit as acetylcarnitine (using the CAT system in reverse), may produce ketone bodies (acetoacetate, β-OH-butyrate, and acetone), or may be hydrolyzed to free acetate by acetyl-CoA hydrolase. Hydroxymethylglutaryl-CoA (HMG-CoA) synthase (fig. 1, enzyme 6; EC 4.1.3.5) is presumably the rate-limiting enzyme in the ketogenic pathway (Williamson et al., 1968). Cytosolic acetyl-CoA (derived from the mitochondria via CAT or citrate lyase or from the peroxisome) may be carboxylated to malonyl-CoA by acetyl-CoA carboxylase (ACC, fig. 1, enzyme 2; EC 6.4.1.2) within the lipogenic pathway wherein FA-CoA is synthesized de novo. Very long-chain and long-chain fatty acids are also activated to their CoA thioesters preceding catabolism in the peroxisome. The peroxisomal membrane contains two synthetases, a long-chain fatty acid synthetase positioned on the cytosolic side of the membrane (fig. 1, enzyme 8), and a very long-chain fatty acid (VLCFA) synthetase positioned toward the peroxisomal matrix (fig. 1, enzyme 9). The location of the VLCFA synthetase is responsible for the substrate specificity between the peroxisome and the mitochondrion. While the mitochondria can oxidize long, medium, and short-chain fatty acids, VLCFA are either poorly oxidized by this organelle or not at all (Lazo et al., 1990). Following CoA activation, the initial dehydrogenation step in the peroxisome is catalyzed by multiple acyl-CoA oxidases (fig. 1, enzyme 10); two have been identified in the human

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(Wanders et al., 2001) and three in the rat (Van Veldhoven et al., 2001). While acyl-CoA dehydrogenase, the first enzyme in mitochondrial β-oxidation, produces two ATP as electrons are donated directly to coenzyme Q of the electron transport chain (ETC), the first step in peroxisomal β-oxidation catalyzed by acyl-CoA oxidase results in the production of H2O2 as electrons are passed directly to molecular oxygen. As a result, the peroxisome is approximately half as efficient as the mitochondrion in producing energy from the β-oxidation of fatty acids. Acyl-CoA oxidase shows very little affinity for medium- and short-chain fatty acids (see Reddy and Mannaerts, 1994, for review); as a result, fatty acids are only chainshortened in the peroxisome. In addition, the peroxisome, lacking TCA cycle enzymes, cannot metabolize the acetyl-CoA to CO2, nor can it produce ketone bodies because the enzymes of ketogenesis are also absent. Therefore, the end-products of peroxisomal fatty acid oxidation include acetyl-CoA and chain-shortened acyl-CoA. The transport of fatty acids and the subsequent end-products of their oxidation across the peroxisomal membrane is a subject of much debate. It was originally believed that the peroxisomal membrane was highly permeable. However, isolation of the peroxisome results in a loss of the structural integrity of the membrane (Wanders et al., 2001); therefore, many earlier studies involving peroxisome permeability may have been misleading. Studies involving S. cerevisiae provide evidence for the involvement of transport proteins (see Hettema and Tabak, 2000, for review); in addition, peroxisomal half transporters have been identified in humans, although their definitive role in the transport of fatty acids has not been fully elucidated (Wanders and Tager, 1998). Although a CAT protein has been identified in the peroxisome, it is not membrane-bound and is therefore not implicated in the transport of FA across the membrane. The CAT identified in the peroxisome, or carnitine octanoyltransferase (COT, fig. 1, enzyme 11) as it is often referred to in literature, has optimum activity toward fatty acids of medium chain length. It is speculated that the peroxisomal COT catalyzes the conversion of the end-products of peroxisomal β-oxidation to their carnitine esters. Subsequently, these carnitine esters may exit the peroxisome and be directed toward the mitochondria where they may undergo complete oxidation to CO2. Studies have shown that 4,8-dimethylnonanoyl-CoA derived from the incomplete oxidation of pristanic acid in the peroxisome is indeed translocated to the mitochondria for complete oxidation (Verhoeven et al., 1998). Furthermore, the concerted action of the two β-oxidation systems is further supported by the observation that natural and synthetic ligands (i.e. ligands of the peroxisome proliferator-activated receptor or PPAR), which increase peroxisome proliferation and peroxisomal enzymes, also increase mitochondrial enzymes involved in fatty acid metabolism (i.e. CPT I and HMG-CoA synthase). 3.2. Regulation of carnitine palmitoyltransferase I (CPT I) Using fed, fasted, and alloxan-diabetic rats, McGarry and Foster (1980) have reported considerable evidence establishing the allosteric control of CPT I by malonyl-CoA. During physiological states in which lipogenesis is occurring, ACC is activated and the associated high level of malonyl-CoA serves to inhibit CPT I and thereby prevent the simultaneous and futile oxidation of fatty acids by preventing their entry into the mitochondria. As such, regulation at CPT I is thought to function in directing FA-CoA between esterification and oxidative fates. Beyond changes in malonyl-CoA concentration, changes in the sensitivity of CPT I to malonyl-CoA inhibition have also been reported in various physiological states including the perinatal period in rabbits (Prip-Buus et al., 1990). Furthermore, low levels of carnitine in tissues of colostrum-deprived neonates (Borum, 1983) could limit transport and

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thus oxidation of fatty acids. Milk, however, is high in carnitine (Kerner et al., 1984), and suckling results in elevated hepatic carnitine postnatally (Robles-Valdes et al., 1976). Medium-chain fatty acids also are a valuable probe in studying regulation at CPT I because they can diffuse across the mitochondrial membranes and be activated by an alternative acyl-CoA synthetase (fig. 1, enzyme 5; EC 6.2.1.2) located in the matrix (Groot et al., 1976). Thus, medium-chain fatty acids may bypass, in part, regulation via CPT I and be oxidized independently of carnitine. Medium-chain triglyceride utilization by neonatal pigs has been studied extensively in our laboratory and has been previously reviewed (Odle, 1997, 1998). Recent advances in regulation of CPT have accompanied cloning of the genes for CPT I and II (see McGarry and Brown, 1997, for review). Two isoforms of CPT I exist: the L-form (liver), which possesses relatively low sensitivity to malonyl-CoA inhibition, and the M-form (muscle), possessing very high sensitivity to malonyl-CoA. Both forms show high interspecies homology (>80%). In rats, hepatic concentrations of CPT I mRNA have been shown to increase markedly (up to 5-fold) within 24 h after birth, while CPT II expression was constitutive (Thumelin et al., 1994; Asins et al., 1995). Studying hepatocytes cultured from perinatal rats, Chatelain et al. (1996) have shown that mRNA levels for CPT I (L) respond markedly and rapidly to in vitro supplementation with clofibrate, linoleate, and dibutyrylcAMP, presumably through interaction with respective cis-acting elements and transcription factors (e.g. PPAR-RXR, FFAR, and CREB, respectively). 3.3. Regulation of acetyl-CoA carboxylase (ACC) Regulation of ACC plays a central role in controlling carbon flux through both anabolic and catabolic pathways of fatty acid metabolism. As the rate-limiting step in de novo fatty acid biosynthesis, and because of the allosteric influence of the product (malonyl-CoA) of this enzyme on CPT I (described above), its regulation takes many forms including rapid allosteric and phosphorylation/dephosporylation mechanisms as well as longer-term mechanisms at the level of gene expression (see Kim, 1997, for review). Hormonal stimulation (e.g. glucagon) results in increased intracellular cAMP and causes rapid inactivation of ACC via phosphorylation at multiple sites. Recent findings have suggested that different isozymes (designated α and β) of ACC, encoded for by different genes, may differentially regulate anabolic and catabolic carbon flux. Indeed, the recently cloned β form (Ha et al., 1996) has an additional 150 amino acids at the N-terminus that may direct it to insertion into the mitochondrial membrane where it may play a direct role in regulating CPT I. This may be of particular importance in tissues (e.g. piglet liver) in which fatty acid synthesis is negligible and yet CPT I is highly sensitive to malonyl-CoA inhibition. 3.4. Regulation of mitochondiral HMG-CoA synthase (mHMGCS) Since the establishment of the CPT I control theory, a growing body of evidence has accumulated suggesting other possible intramitochondrial regulatory sites, particularly in neonates (Pegorier et al., 1983; Escriva et al., 1986; Decaux et al., 1988). Much of the early evidence was indirect and speculative. However, researchers at Cambridge (Lowe and Tubbs, 1985a,b,c; Quant et al., 1989, 1990, 1991, 1993) reported compelling evidence that control of mHMGCS activity is likely. Specifically, they have shown that its activity is regulated by a succinylation–desuccinylation mechanism. In the first step of its normal catalytic cycle, mHMGCS becomes acetylated at the active site by reaction with acetyl-CoA (its first substrate). They have shown that succinyl-CoA (at physiological concentrations) may also react

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leading to competitive inhibition of the enzyme (Lowe and Tubbs, 1985c). Furthermore, various in vivo treatments which stimulate ketogenesis (fasting, glucagon or mannoheptulose injection, alloxan diabetes, high-fat feeding, etc.) all increased mHMGCS activity by decreasing its degree of succinylation (Quant et al., 1989). The enzyme was 40−50% succinylated (and inactive) in the livers of normally fed rats and could be rapidly (within minutes) activated in vitro. This led the authors to speculate that the succinylation-control mechanism could allow for rapid changes in ketogenic flux rate in vivo. More recently (Quant et al., 1991), the control of ketogenesis in the neonatal rat has been shown to be mediated, in part, by changes in the amount and activity of mHMGCS, presumably at the level of gene expression (Casals et al., 1992; see Hegardt, 1999, for review). Indeed, Thumelin et al. (1993) showed that hepatic mRNA concentrations for mHMGCS increased by about 3-fold within 24 h of birth in rats, remained constant throughout suckling, and then rapidly declined when animals were weaned onto a low-fat diet. Furthermore, mRNA in cultured fetal hepatocytes increased by 4-fold within 4 h after exposure to glucagon. Additional research (Ayte et al., 1993) has suggested that expression may be regulated in part by methylation/demethylation of the 5′ flanking region of the gene and has identified CRE and C-EBP as potential cis regulatory elements (Brady et al., 1993; Gomez et al., 1993) which could mediate the glucagon effects. Subsequent research also identified the PPAR-RXR diad as an important regulator of expression induced by clofibrate and fatty acids (Rodriguez et al., 1994). 3.5. Hormonal support of fatty acid oxidation and ketogenesis Regardless of the underlying biochemical regulatory mechanism(s), the major hormonal influence is most likely mediated by the insulin/glucagon ratio (McGarry and Foster, 1977). When the ratio is low, as observed during the neonatal period, fasting, or diabetes, ketogenesis is stimulated. The hormonal effect may be mediated through regulation of acetyl-CoA carboxylase (Borthwick et al., 1986), thus affecting malonyl-CoA levels and CPT I activity and/or by decreasing succinyl-CoA levels and thereby activating mHMGCS (Quant et al., 1989) and/or by affecting levels of TCA cycle intermediates. Similarly, hormonal alteration of intracellular cAMP concentrations (Pegorier et al., 1989) may directly impact gene transcription (as described previously) via interaction with cAMP response elements. Furthermore, in vivo and in vitro exposure of rodent tissues to the peroxisome-proliferating hypolipidemic drugs (e.g. clofibrate) and dehydroepiandrosterone (Brady et al., 1991) has been shown to upregulate fatty acid oxidation and/or ketogenesis by increasing transcription of CPT and/or mHMGCS genes. 3.6. Hepatic lipid metabolism in the piglet Clearly, a fundamental understanding of the developmental aspects of lipid metabolism is essential in order to optimize postnatal fat utilization by the piglet. Putative changes in metabolic capacity within the first week of life are of greatest concern because 75% of mortality occurs during this time period (USDA, 1991). Unfortunately, relatively little research has been focused on these animals. The following review examines pig-specific studies. 3.7. Development of fatty acid oxidation in piglets Postnatal increases in fatty acid oxidation have been reported (Wolfe et al., 1978), but must be interpreted in light of the general increase in metabolic rate that occurs after birth

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(Odle et al., 1991b). For example, the oxidation of [U-14C]palmitate to CO2 and acid-soluble products (considered to represent ketone bodies and/or TCA cycle intermediates) by liver homogenates was reported to increase 4-fold between 0 and 7 days of age (Mersmann and Phinney, 1973). However, marked increases in mitochondrial respiration from several TCA cycle intermediates has likewise been reported (Mersmann et al., 1972) wherein oxygen consumption increased by 5-fold between 6 and 12 h postpartum. Subsequent histological work suggested rapid mitochondrial proliferation during this early neonatal period. Odle et al. (1991b) have likewise observed developmental increases in hepatic fatty acid oxidation in small and normal-birth-weight pigs during the first 48 h of life which could be largely explained by increases in oxygen consumption. Thus, increases in fatty acid oxidation postnatally may be accounted for, in part, by increased oxidative metabolism in general, and may not necessarily infer an increased reliance upon fat as a fuel (Adams et al., 1997a). 3.8. Lack of ketogenesis in piglets While piglets appear to display a hormonal profile (low insulin/glucagon) that would support ketogenesis (Pegorier et al., 1981), and have ample substrate from the fat present in milk, they do not display hyperketonemia (Bengtsson et al., 1969; Pegorier et al., 1981), despite elevated plasma nonesterified fatty acids (Adams and Odle, 1993b). This starkly contrasts with other mammalian species (e.g. rats, rabbits, etc.) which show pronounced hyperketonemia during suckling (Foster and Bailey, 1976) as well as ruminant species which under extreme lactational stress can die from ketoacidosis. Because ketone bodies provide important glucose-sparing carbon, aiding otherwise glucose-dependent tissues (e.g. neural tissues), their absence may be detrimental to the survival of the piglet, which is keenly susceptible to hypoglycemia (Swiatek et al., 1968). Furthermore, insofar as fatty acid oxidation also is required to support active gluconeogenesis, impaired fat oxidation also could contribute indirectly to hypoglycemia (Lepine et al., 1993; Duee et al., 1996). In theory, low ketone concentrations could be due to a low production rate (i.e. ketogenesis) and/or a high rate of utilization. Using continuous-infusion isotope kinetics, we have observed limited β-OH-butyrate oxidation rates in vivo (Tetrick et al., 1995). Piglets were arterially infused with 14C-β-OHbutyrate at rates sufficient to supply 15%, 30%, 45%, and 60% of their estimated ATP turnover. Michaelis–Menten analysis of measured oxidation rates versus plasma concentrations showed that β-OH-butyrate could supply a maximum of 32% of the piglet’s total body ATP turnover, but at physiological concentrations would supply <5% of the animal’s energy need. More likely, an impaired rate of hepatic ketogenesis (Pegorier et al., 1983; Duee et al., 1994) is the cause. Comparative in vivo research (Adams and Odle, 1993b) showed that the relative ketogenic capacity (measured by regression of plasma β-OH-butyrate on plasma octanoate concentrations following intraperitoneal injection of octanoate) of neonatal pigs is greatly attenuated (by up to 1−2 orders of magnitude) compared to weanling or mature swine and to neonatal or mature rabbits. Furthermore, using radio-HPLC to characterize products of radiolabeled fatty acid oxidation, we have observed trivial accumulation of isotope in ketone bodies from piglet liver compared with neonatal rat liver preparations (Adams et al., 1997a). Concurrent with these findings, measurements of hepatic mHMGCS have shown 70% lower activities in neonatal pigs than in neonatal rabbits (Adams and Odle, 1993a) or adult rats (Duee et al., 1994). Following the cloning of the pig gene (Adams et al., 1997b), most recent findings confirmed the attenuated expression of this enzyme (compared with rats) during suckling, but showed that starvation led to increased mRNA concentrations. However, substantial increases

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in mRNA did not occur until 2−3 weeks of age, indicating a developmental lag in induction compared with the rapid postnatal rise observed in the suckling rat (Thumelin et al., 1993). Subsequent examination of the upstream regulatory region of the pig gene has failed to identify any idiosyncrasies that might impair expression in that it contains the anticipated PPAR response element (Ortiz et al., 1999). Despite increased mRNA concentrations, enzyme activity still remained low. Current research findings indicate that pig mRNA codes for a catalytically active protein, and low synthase activity is a result of attenuated translation. Furthermore, the decrease in mRNA translation was not a result of alteration of the polyadenylate tail which can influence both mRNA stability and subsequent translation into protein (Barrero et al., 2001). 3.9. Regulation of fatty acid oxidation and ketogenesis in piglets The degree to which the regulatory features of ketogenesis (reviewed previously) described in other species extrapolates to the neonatal pig is not known. A putative limitation of fatty acid oxidation at the level of CPT I was suggested by data from Honeyfield and Froseth (1991), who reported a >10-fold increase in oxygen consumption when palmitoylcarnitine was compared to palmitoyl-CoA in heart and liver homogenates from newborn pigs. Likewise, using hepatocytes isolated from piglets at birth and from piglets fed or fasted 24 h from birth, we (Odle et al., 1995) have obtained evidence consistent with the hypothesis that CPT I is a potential regulatory site: (1) The molar oxidation rate of octanoate was 4 times higher than that of palmitate. This is 2-fold higher than can be explained by the molar energy difference between these fatty acids, and implies a putative limitation in the oxidation of palmitate compared to octanoate, given the measured oxygen consumption rates were similar in cells incubated with each fatty acid. While differences in the relative activities of the medium- and long-chain acyl-CoA synthetases cannot be excluded, this observation could be explained by a limitation of palmitoyl-CoA transport into the mitochondrion via CPT. (2) Carnitine (a co-substrate for CPT) increased oxidation and decreased esterification of palmitate, but had no effect on octanoate metabolism. (3) TDGA (an irreversible inhibitor of CPT I) reduced oxidation and increased esterification of palmitate, but again had no effect on octanoate metabolism. Developmental changes in liver and muscle CPT activity during the neonatal period have been reported (Bieber et al., 1973; Lin and Odle, 1995; Schmidt and Herpin, 1998). The activity doubled in the liver of pigs between 0 and 1 day of age but then plateaued to equal the activity in mitochondria from 24-day-old animals. Interestingly, palmitoyl-CoA oxidation continued to increase, doubling between 1 and 2 days postpartum. This suggests that something other than CPT activity was limiting oxidation as the animals aged. Perhaps decreases in the sensitivity of CPT to malonyl-CoA (Duee et al., 1994; Lin and Odle, 1995; Schmidt and Herpin, 1998) are responsible, as has been reported in rabbits (Prip-Buus et al., 1990). Indeed, Pegorier et al. (1983) challenged the idea of control mediated via CPT I. In contrast to our findings (Odle et al., 1991a,b, 1995; Lin et al., 1996), they reported low oxidation rates for octanoate (vs oleate). In addition, they were unable to increase oleate oxidation by isolated piglet hepatocytes incubated with glucagon (which should have decreased malonyl-CoA levels). This may not be surprising because the liver is not a major site of lipogenesis in the pig (Mersmann et al., 1973). In general, malonyl-CoA concentrations are higher in lipogenic tissues as the synthesis of malonyl-CoA is the first committed step of lipogenesis. The role of malonyl-CoA in nonlipogenic tissues, such as skeletal muscle, is regulation of CPT I activity. Thus, in nonlipogenic tissues the concentration of malonyl-CoA is much less than what is

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observed in lipogenic tissues and CPT I exhibits greater sensitivity to malonyl-CoA. Recent cloning of the pig M- and L-CPT I isoforms has revealed that L-CPT I is a natural chimera of rat M- and L-CPT I isoforms (Nicot et al., 2001). Pig L-CPT I shows similar kinetics toward carnitine as rat L-CPT I; however, IC50 for malonyl-CoA is similar to rat M-CPT I. Thus, pig L-CPT I has a greater sensitivity to malonyl-CoA than rat L-CPT I. In addition to the potential control by CPT I, regulation may also shift to an intramitochondrial site (e.g. mHMGCS). Research reported by Duee et al. (1994) verified the low rate of ketogenesis in mitochondria from 2-day-old piglets, and implicated an intramitochondrial constraint; namely, when the mitochondria were treated with malonate (a blocker of succinate dehydrogenase) to decrease TCA cycle flux, the major end-product of palmitoylcarnitine oxidation was presumed to be acetoacetate. In this case, the rate of palmitoylcarnitine utilization was only 2.5 nmol/min/mg protein. However, when malonate was added, the end-product was citrate and the rate of palmitoylcarnitine utilization increased by 3-fold. 3.10. Peroxisomal β-oxidation in piglets We have characterized the postnatal development and tissue distribution of peroxisomal fatty acid oxidation in piglets (Yu et al., 1997a,b, 1998). In general, activity measured in liver, kidney, and heart as either antimycin/rotenone-insensitive palmitate oxidation or palmitoylCoA dependent KCN-insensitive reduction of NAD, was high (i.e. 40−50% of total fat β-oxidation) compared with published rat values (e.g. 25%) and developed rapidly after birth. Hepatic activity of peroxisomal oxidation was increased when suckling-aged piglets were fasted, presumably due to elevated plasma free fatty acids and/or glucagon. Most recent data from our laboratory (Yu et al., 2001) showed that hepatic activities of fatty acid oxidase (an enzyme unique to peroxisomes) as well as CPT were dramatically induced in piglets fed milk replacer containing clofibrate. Clofibrate is a hypolipidemic drug that is known to induce peroxisome (and possibly mitochondrial) biogenesis in rodents (Brady et al., 1991), through interaction with the peroxisome proliferator-activated receptor (PPAR). We hypothesize that the extra thermogenesis associated with peroxisomal β-oxidation may be important in the suckling piglet’s maintenance of homeothermy.

4. FUTURE PERSPECTIVES Research findings to date indicate impairment in ketone body synthesis for the neonatal pig when compared to other species. This reduction in ketone bodies during a period when fatty acids are the main source of energy implies that the young pig is inefficient in utilizing dietary fat, and thus may impact piglet survival and performance. In order to optimize nutrition for the neonatal pig, an understanding of the underlying mechanisms of lipid metabolism must be elucidated. Further insight into the regulation of key lipid enzymes, including rate of synthesis and degradation of mRNA and protein, and subsequent protein activity is required. Recent research on CPT I and HMG-CoA synthase has shown key differences between these enzymes in piglets when compared to the rat, a popular model in lipid research. These differences highlight the need for more research in the neonatal pig; only then will the mechanisms responsible for lipid oxidation be defined so that improvements in lipid metabolism can be made at a cellular level. Research in the arena of molecular mechanisms of lipid oxidation will not provide overnight insight into improving current practices for piglet survival and performance. However, as swine production becomes increasingly specialized, the demand for the formulation of milk

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replacers that optimize early growth will increase also. The use of such milk replacers will allow dietary manipulation that cannot be achieved through nutritional management of the dam. Control over the piglet’s diet would allow for manipulation of the relative percent of energy contributed by carbohydrates versus fat and could have a profound impact on piglet morbidity and mortality. The specific fatty acids added to the diet also could be altered to increase the amount of MCT (for example), which serve as obligatory fuels. Furthermore, current research in the use of peroxisome proliferators indicate that their supplementation into the diet may provide a useful tool in improving fatty acid utilization by the pig. Further research in these areas will hopefully assist in development of strategies to enhance fatty acid oxidation and ultimately improve piglet survival and growth.

REFERENCES Aas, M., 1971. Organ and subcellular distribution of fatty acid activating enzymes in the rat. Biochim. Biophys. Acta 23, 32–47. Adams, S.H., Odle, J., 1993a. Does HMG-CoA synthase play a role in limitation of ketogenesis in neonatal pigs? FASEB J. 7, A379. Adams, S.H., Odle, J., 1993b. Plasma β-hydroxybutyrate after octanoate challenge: attenuated ketogenic capacity in neonatal swine. Amer. J. Physiol. 265, R761−R765. Adams, S.H., Lin, X., Yu, X.X., Odle, J., Drackley, J.K., 1997a. Hepatic fatty acid metabolism in pigs and rats: major differences in end-products, O2 uptake and β-oxidation. Amer. J. Physiol. 272, R1641−R1646. Adams, S.H., Alho, S.C., Asins, G., Hegardt, F.G., Marrero, P.F., 1997b. Gene expression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in a poorly ketogenic mammal: effect of starvation during the neonatal period of the piglet. Biochem. J. 324, 65−73. Asins, G., Serra, D., Arias, G., Hegardt, F.G., 1995. Developmental changes in carnitine palmitoyltransferases I and II gene expression in intestine and liver of suckling rats. Biochem. J. 306, 379−384. Ayte, J., Gomez, G.G., Hegardt, F.G., 1993. Structural characterization of the 3′ noncoding region of the gene encoding rat mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase. Gene 123, 267−270. Bach, A.C., Babayan, V.K., 1982. Medium-chain triglycerides: an update. Amer. J. Clin. Nutr. 36, 950−962. Barrero, M.J., Alho, C.S., Oritz, J.A., Hegardt, F.G., Haro, D., Marrero, P.F., 2001. Low activity of mitochondrial HMG-CoA synthase in liver of starved piglets is due to low levels of protein despite high mRNA levels. Arch. Biochem. Biophys. 385, 364−371. Battaglia, F.C., Meschia, G., 1978. Principal substrates of fetal metabolism. Physiol. Rev. 58, 499. Bengtsson, N.J., Gentz, J., Hakkarainen, J., Hellstrom, R., Persson, B., 1969. Plasma levels of FFA, glycerol, β-hydroxybutyrate and blood glucose during the postnatal development of the pig. J. Nutr. 97, 311−315. Bieber, L.L., Markwell, M.A.K., Blair, M., Helmrath, T.A., 1973. Studies on the development of carnitine palmitoyltransferase and fatty acid oxidation in liver mitochondria of neonatal pigs. Biochim. Biophys. Acta 326, 145−154. Borthwick, A.C., Edgell, N.J., Denton, R.M., 1986. Mechanisms involved in the short-term regulation of acetyl-CoA carboxylase by insulin. Biochem. Soc. Trans. 14, 563−565. Borum, P.R., 1983. Carnitine. Annu. Rev. Nutr. 3, 233−259. Brady, L.J., Ramsay, R.R., Brady, P.S., 1991. Regulation of carnitine acyltransferase synthesis in lean and obese zucker rats by dehydroepiandrosterone and clofibrate. J. Nutr. 121, 525−531. Brady, P.S., Barke, R.A., Brady, L.J., 1993. Regulation of the 68-kDa hepatic carnitine palmitoyltransferase. In: Berdanier, C.D. (Ed.), Nutrition and Gene Expression. CRC Press, Boca Raton, FL, pp. 319−334. Casals, N., Roca, N., Guerrero, M., Gil-Gomez, G., Ayte, J., Ciudad, C.J., Hegardt, F.G., 1992. Regulation of the expression of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene: its role in the control of ketogenesis. Biochem. J. 283, 261−264. Chatelain, F., Kohl, C., Esser, V., McGarry, J.D., Girard, J., Pegorier, J.P., 1996. Cyclic AMP and fatty acids increase carnitine palmitoyltransferase I gene transcription in cultured fetal rat hepatocytes. Eur. J. Biochem.235, 789−798.

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Lowe, D.M., Tubbs, P.K., 1985c. Succinylation and inactivation of 3-hydroxy-3-methylglutaryl-CoA synthase by succinyl-CoA and its possible relevance to the control of ketogenesis. Biochem. J. 232, 37−42. McGarry, J.D., Brown, N.F., 1997. The mitochondrial carnitine palmitoyltransferase system: from concept to molecular analysis. Eur. J. Biochem. 244, 1−14. McGarry, J.D., Foster, D.W., 1977. Hormonal control of ketogenesis. Arch. Intern. Med. 137, 495−501. McGarry, J.D., Foster, D.W., 1980. Regulation of hepatic fatty acid oxidation and ketone body production. Annu. Rev. Biochem. 49, 395−420. Mersmann, H.J., Phinney, G., 1973. In vitro fatty acid oxidation in liver and heart from neonatal swine (Sus domesticus). Comp. Biochem. Physiol. 44B, 219−223. Mersmann, H.J., Goodman, J., Houk, J.M., Anderson, S., 1972. Studies on the biochemistry of mitochondrial and cell morphology in the neonatal swine hepatocyte. J. Cell. Biol. 53D, 335−347. Mersmann, H.J., Phinney, G., Sanguinetti, M.C., Houk, J.M., 1973. Lipogenic capacity of liver from perinatal swine (Sus domesticus). Comp. Biochem. Physiol. 46B, 493−497. Nicot, C., Hegardt, F.G., Woldegiorgis, G., Haro, D., Marrero, P.F., 2001. Pig liver palmitoyltransferase I, with low Km for carnitine and high sensitivity to malonyl-CoA inhibition, is a natural chimera of rat liver and muscle enzymes. Biochemistry 40, 2260−2266. Odle, J., 1997. New insights into medium-chain triglyceride utilization by the neonate: observations from a piglet model. J. Nutr. 127, 1061−1067. Odle, J., 1998. Medium-chain triglycerides: a unique energy source for neonatal pigs. Pig News 20, 25−32. Odle, J., Benevenga, N.J., Crenshaw, T.D., 1991a. Utilization of medium-chain triglycerides by neonatal piglets: chain length of even- and odd-carbon fatty acids and apparent digestion/absorption and hepatic metabolism. J. Nutr. 121, 605−614. Odle, J., Benevenga, N.J., Crenshaw, T.D., 1991b. Postnatal age and the metabolism of medium- and long-chain fatty acids by isolated hepatocytes from small-for-gestational-age and appropriate-forgestational-age piglets. J. Nutr. 121, 615−621. Odle, J., Lin, X., van Kempen, T.A.T.G., Drackley, J.K., Adams, S.H., 1995. Carnitine palmitoyltransferase modulation of hepatic fatty acid metabolism and radio-HPLC evidence for low ketogenesis in neonatal pigs. J. Nutr. 125, 2541−2549. Oikawa, E., Iijima, H., Suzuki, T., Sasano, H., Sato, H., Kamataki, A., Nagura, H., Kang, M., Fujino, T., Suzuki, H., Yamamoto, T.T., 1998. A novel acyl-CoA synthetase, ACS5, expressed in intestinal epithelial cells and proliferating preadipocytes. J. Biochem. 124, 679−685. Ortiz, J.A., Mallolas, J., Nicot, C., Bofarull, J., Rodriguez, J.C., Hegardt, F.G., Haro, D., Marrero, P.F., 1999. Isolation of pig mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene promoter: characterization of a peroxisome proliferator-responsive element. Biochem. J. 337, 329−335. Pegorier, J.P., Duee, P.H., Peret, J., Girard, J., 1981. Changes in circulating fuels, pancreatic hormones and liver glycogen concentration in fasting or suckling newborn pigs. J. Dev. Physiol. 3, 203−217. Pegorier, J.P., Duee, P.H., Girard, J., Peret, J., 1983. Metabolic fate of non-esterified fatty acids in isolated hepatocytes from newborn and young pigs: evidence for a limited capacity for oxidation and increased capacity for esterification. Biochem. J. 212, 93−97. Pegorier, J.P., Garcia-Garcia, M.-V., Prip-Buus, C., Duee, P.-H., Kohl, C., Girard., J., 1989. Induction of ketogenesis and fatty acid oxidation by glucagon and cyclic AMP in cultured hepatocytes from rabbit fetuses. Biochem. J. 264, 93−100. Prip-Buus, C., Pegorier, J., Duee, P., Kohl, C., Girard, J., 1990. Evidence that the sensitivity of carnitine palmitoyltransferase I to inhibition by malonyl-CoA is an important site of regulation of hepatic fatty acid oxidation in the fetal and newborn rabbit. Biochem. J. 269, 409−415. Quant, P.A., Tubbs, P.K., Brand, M.D., 1989. Treatment of rats with glucagon or mannoheptulose increases mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity and decreases succinylCoA content in liver. Biochem. J. 262, 159−164. Quant, P.A., Tubbs, P.K., Brand, M.D., 1990. Glucagon activates mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in vivo by decreasing the extent of succinylation of the enzyme. Eur. J. Biochem. 187, 169−174. Quant, P.A., Robin, D., Robin, P., Ferre, P., Brand, M.D., Girard, J., 1991. Control of hepatic mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase during the foetal/neonatal transition, suckling and weaning in the rat. Eur. J. Biochem. 195, 449−454. Quant, P.A., Robin, D., Robin, P., Girard J., Brand, M.D., 1993. A top-down control analysis in isolated rat liver mitochondria: can the 3-hydroxy-3-methylglutaryl-CoA pathway be rate-controlling for ketogenesis? Biochim. Biophys. Acta 1156, 135−143. Reddy, J.K., Mannaerts, G.P., 1994. Peroxisomal lipid metabolism. Annu. Rev. Nutr. 14, 343−370.

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10

Essential fatty acid metabolism during early development S. M. Innis Department of Paediatrics, University of British Columbia, Vancouver, British Columbia, Canada V5Z 4H4

The n-6 and n-3 polyunsaturated fatty acids are essential nutrients that are required for growth and normal cell function. These fatty acids are present in cells as the acyl moieties of phospholipids which make up the structural matrix of cell and subcellular membranes, and function directly, or as precursors to other molecules that modulate cell growth, metabolism, inter- and intracellular communication and gene expression. The n-3 fatty acid docosahexaenoic acid (22:6n-3) is accumulated in the retina and brain grey matter during development, but is continually turned over, recycled and replenished by uptake from plasma during the dynamic processes of signal transduction in the retina and neuronal membranes. Depletion of 22:6n-3 from retinal and neural membranes results in reduced visual function, behavioural abnormalities, and alterations in the metabolism of neurotransmitters, and in membrane proteins, receptors and ion channel activities. Large gaps still exist in understanding of dietary requiremements for n-3 fatty acids at different stages of the life cycle, species differences in essential fatty acid metabolism, and the process that controls the partitioning of n-3 fatty acids for generation of energy and further metabolism for incorporation into membrane lipids.

1. OVERVIEW OF ESSENTIALITY OF n-6 AND n-3 FATTY ACIDS Studies in animals over 70 years ago provided the first demonstration that dietary fat contains components that are essential to normal growth and development. The signs of deficiency, which included scaly skin, growth retardation, reproductive failure and histological abnormalities (table 1), were ascribed to the absence of n-6 fatty acids, and were reversed or prevented by feeding the n-6 fatty acid linoleic acid (18:2n-6) (Innis, 1991). The importance of a second class of polyunsaturated fatty acids, the n-3 fatty acids, did not emerge until the 1970s when altered electroretinograph (ERG) recordings and behaviour were found in rats fed diets deficient in n-3 fatty acids (Benolken et al., 1973; Wheeler et al., 1975; Lamptey and Walker, 1976). Despite the knowledge that the n-3 fatty acid docosahexaenoic acid (22:6n-3)

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Table 1 Signs of n-6 and n-3 fatty acid deficiency Essential fatty acid/n-6 fatty acid deficiency

n-3 fatty acid deficiency

Decreased growth Scaly dermatoses Inflamed skin Decreased skin pigmentation Increased transepidermal water loss Impaired wound healing Alopecia Tail necrosis Fatty liver Kidney degeneration Reproductive failure

Reduced electroretinogram A and/or B wave recording Reduced visual acuity Reduced discrimination learning Reduced exploratory behaviour Increased stereotyped behaviour

represents a major proportion of the polyunsaturated fatty acids in brain and retina (Fliesler and Anderson, 1983; Sastry, 1985; Giusto et al., 2000), the acceptance of the essential role of n-3 fatty acids, particularly in human nutrition, grew only slowly, perhaps because of the absence of overt effects of n-3 fatty acid deficiency on growth or health. The formation and extension of neural and retinal membranes, however, requires a large amount of 22:6n-3, all of which must be derived from n-3 fatty acids in the diet. It is well appreciated that deficiency of one or more key nutrients during brain development can, depending on the timing, severity and duration, decrease cell division, dendritic arborization and myelination with resultant long-term effects on cognitive and behavioural functions (Dobbing et al., 1971; Dobbing, 1972; Wiggins, 1986; Morgane et al., 1992; Levitsky and Strupp, 1995). Because of this, much of the recent interest on essential fatty acids in human growth and development has focused on the requirements for n-3 fatty acids for visual and neural function. This chapter reviews current research on polyunsaturated fatty acid metabolism in development, the supply of n-6 and n-3 fatty acids before and after birth, and the role of n-3 fatty acids in the developing brain and retina.

2. ESSENTIAL FATTY ACID STRUCTURE AND METABOLISM 2.1. Structure and nomenclature Fatty acids are identified using a systematic nomenclature which identifies the fatty acid by the number of carbon atoms, and the number and position of any unsaturated double bonds relative to the carboxyl group (table 2, fig. 1). More commonly, fatty acids are referred to using a structural designation, or trivial name. The structural designation describes a fatty acid by the number of carbons, the number of double bonds, and the position of the first double bond from the methyl terminal carbon which is designated by the Greek letter omega (ω) or “n”. The delta (Δ) notation is used to designate the position of a carbon from the carboxyl terminus and is used to denote the site of action of the fatty acid desaturase enzymes. Polyunsaturated fatty acids, which are often referred to as PUFA, are grouped into families based on the position of the first methylene interrupted double bond from the methyl (ω or n) end.

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Table 2 Systematic names and abbreviations for essential n-6 and n-3 fatty acids Systematic name n-6 family 9,12-octadecadienoic 6,9,12-octadecatroenic 8,11,14-eicosatrienoic 5,8,11,14-eicosatetraenoic 7,10,13,16-docosatetraenoic 4,7,10,13,16-docosapentaenoic n-3 family 9,12,15-octadecatrienoic 6,9,12,15-octadecatetraenoic 5,8,11,14,17-eicosapentaenoic 4,7,10,13,16,19-docosahexaenoic 6,9,12,15,18,21-tetracosahexanenoic

Abbreviation

Trivial name

18:2n-6 18:3n-6 20:3n-6 20:4n-6 22:4n-6 22:5n-6

Linoleic γ-Linolenic Dihomo-γ-linolenic Arachidonic Adrenic

18:3n-3 18:4n-3 20:5n-3 22:6n-3 24:6n-3

α-Linolenic Stearidonic

Nisinic

2.2. Metabolism The Δ12- and Δ15-desaturase enzymes necessary to form n-6 and n-3 fatty acids, respectively, are present in plant but not in animal cells. Consequently, animals require a dietary source of n-6 and n-3 fatty acids, all of which are ultimately derived from plants either directly, or after transfer up the food chain. The parent 18-carbon n-6 and n-3 fatty acids are linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3). These fatty acids are considered essential dietary nutrients for most animals. Linoleic acid (18:2n-6) and 18:3n-3 can be further metabolized by desaturation and elongation, as well as chain shortening, but cannot be interconverted (Innis, 1991) (fig. 2). The major site of desaturation and elongation is the liver, although desaturase activity is also present in some other cells. Desaturation and elongation proceed alternately, by Δ6-desaturation, elongation and Δ5-desaturation, to yield arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3) from 18:2n-6 and 18:3n-3, respectively (fig. 2). These metabolites of 18:2n-6 and 18:3n-3 with 20 or more carbon atoms are often referred to as long-chain polyunsaturated fatty acids, and abbreviated as LC-PUFA. For many years it was believed that docosapentaenoic acid (22:5n-3) was desaturated by a microsomal Δ4-desaturase to form docosahexaenoic acid (22:6n-3). Such a Δ4-desaturase, however, has not been found in animal cells (Moore et al., 1991; Voss et al., 1991; Mohammed et al., 1995, 1997; Luthria et al., 1996). The pathway for

Fig. 1.

Schematic representation of a fatty acid.

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Fig. 2.

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Schematic representation of the major pathways of essential fatty acid desaturation and elongation.

synthesis of 22:6n-3 is now known to involve synthesis of a 24-carbon n-3 fatty acid by two successive elongations of 20:5n-3 to form 24:5n-3, which is then desaturated at position 6 to form tetracosahexaenoic acid (24:6n-3), and translocated to the peroxisomes where one cycle of β-oxidation leads to formation of 22:6n-3 (Sprecher et al., 1995, 1999; Ferdinandusse et al., 2001). The 22:6n-3 thus formed is then shuttled back to the endoplasmic reticulum, which is the major site of phospholipid biosynthesis (Vance, 1990; Choy, 1997). Synthesis of the n-6 docosapentaenoic acid (22:5n-6) from 20:4n-6 occurs in an analogous pathway (Sprecher et al., 1995, 1999). The regulation and steps involved in the intracellular trafficking of fatty acids between the endoplasmic reticulum and peroxisomes is still incompletely understood. The desaturase enzymes necessary for synthesis of 20:4n-6 and 22:6n-3 are present in animal but not in plant cells. Thus, omnivores and carnivores obtain 20:4n-6 and 22:6n-3 from the flesh of other animals, while herbivores consume only the precursors 18:2n-6 and 18:3n-3. The activity of the essential fatty acid desaturation and elongation pathway appears to vary markedly among species, being higher in rodents and low in pigs and humans (Pooviah et al., 1976; Yamazaki et al., 1992; Emken et al., 1994; Li et al., 2000; Pawlosky et al., 2001). Rats, for example, have very high amounts of 22:6n-3 in liver phosphatidylethanolamine (PE) when compared to humans and other primates (table 3). The felinae, which are obligate carnivores, have very low activity of Δ6-desaturase, and thus require a dietary source of

0.3 1.6 1.5 1.3 0.9 0.5 0.9 1.0 0.1 1.1 1.5 0.8 1.2 2.3 1.7 1.4 0.9 1.2 0.7 0.8 0.1 0.3 0.7

Adapted from Crawford et al. (1976).

Rat Guinea pig Hamster Lemming Bat Vole Warthog Horse Zebra Elephant Hartebeest Red deer Ox Buffalo Eland Giraffe Cat Civet Leopard Marmoset Vervet Man Dolphin

18:2n-6

11 17 9.6 10 15 11 12 12 10 11 11 10 11 12 14 14 13 14 14 11 6.8 14 6.9

20:4n-6 5.1 7.9 4.2 4.3 5.2 1.1 6.3 7.1 4.9 7.2 7.2 6.9 6.3 6.5 7.0 6.2 6.7 5.9 7.1 4.7 5.3 6.0 3.6

22:4n-6

Brain

2.1 0.0 1.2 0.8 1.1 1.2 0.9 1.1 1.3 1.0 1.6 1.2 2.2 0.2

0.6 2.4 0.2 1.5 2.5 0.5 1.2 1.3

22:5n-6 21 19 22 29 21 20 23 19 18 25 17 25 22 16 21 24 27 26 22 29 21 23 27

22:6n-3 6.0 23 11 16 8 7.2 22 17 47 10 11 5.1 4.0 9.7 8.5 12 5.5 3.4 3.8 10 9.2 6.3 1.7

18:2n-6

Essential n-6 and n-3 fatty acids in brain grey matter and liver phosphatidylethanolamine of different species

Table 3

21 11 16 7.6 11 5.5 12 6.2 4.2 10 14 11 16 13 14 12 18 15 15 23 17 16 30

20:4n-6 0.6 1.3 0.3 0.3 0.6 0.2 0.6 0.6 0.1 0.4 0.7 0.1 2.3 0.3 0.6 1.1 1.3 3.6 2.4 0.7 0.3 3.5 0.9

22:4n-6

Liver

0.4 0.9 0.5 0.9 0.2 0.1 0.2 0.4 0.1 0.1 0.3 0.1 0.6 0.1 0.2 0.8 2.0 1.2 0.7 1.3 0.4 2.3 0.9

22:5n-6

22 21 10 23 3.9 26 1.6 3.6 0.2 0.6 0.9 0.3 0.3 2.3 1.8 0.8 22 20 18 8.2 5.9 7.7 11

22:6n-3

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20:4n-6 and 22:6n-3 (Rivers et al., 1975, 1976; MacDonald et al., 1983; Pawlosky et al., 1997), which is consumed as part of their natural meat diet. Many of the essential roles of n-6 and n-3 fatty acids are fulfilled by 20:4n-6 and 20:5n-3 and 22:6n-3, rather than their precursors 18:2n-6 and 18:3n-3, respectively. Linoleic acid (18:2n-6) and metabolites in the pathway to 20:4n-6, however, have important roles in cholesterol metabolism and in the skin, and 20:3n-6 is the precursor for synthesis of series 1 eicosanoids (Wertz et al., 1987; Ziboh and Chapkin, 1988; Innis, 1996). α-Linolenic acid, on the other hand, is not known to have any essential functions other than as a precursor for synthesis of 20:5n-3 and 22:6n-3. Acetyl-CoA derived from β-oxidation of 18:3n-3 is extensively recycled for synthesis of saturated and monounsaturated fatty acids and cholesterol synthesis in developing tissues (Cunnane et al., 1994, 1999; Sheaff-Greiner et al., 1996; Menard et al., 1998). Desaturation of 18:2n-6 and 18:3n-3 is believed to involve the same enzymes. In vitro, the Δ6-desaturase enzyme shows clear preference in order 18:3n-3 > 18:2n-6 > 18:1n-9 (Brenner and Peluffo, 1966, 1969; Brenner et al., 1969). Owing to the abundance of 18:2n-6 in commonly used vegetable oils, such as safflower, corn and soybean oil (Chow, 2000), human diets in many Western countries have much higher 18:2n-6 than 18:3n-3 (Simopoulos, 1999). The high proportion of 18:2n-6 to 18:3n-3 in the diet has implications for reducing synthesis of 22:6n-3, which is important for brain and visual development, and for increasing the risk of health problems associated with increased production of 20:4n-6-derived eicosanoids. Synthesis of 22:6n-3 and 20:4n-6 is also reduced by products of the same and opposing series of fatty acids. For example, high intakes of 20:5n-3 or 22:6n-3 from fish or fish oil reduces tissue 20:4n-6, decreases the synthesis of n-6 fatty acid-derived eicosanoids, and increases the synthesis of eicosanoids derived from 20:5n-3 (Fischer et al., 1989; Broughton and Morgan, 1994; Ferretti et al., 1998; Broughton and Wade, 2002). Changes in the balance of n-6 and n-3 fatty acid-derived eicosanoids can have important effects on inflammation and immunity, hemostatic and endothelial function, and reproductive functions including ovulation rate, progesterone production by the corpus luteum, timing of luteolysis and gestational length (von Schacky and Weber, 1985; Rogers et al., 1987; Kristensen et al., 1989; Tremoli et al., 1995; Calder, 1998, 2001; Abayasekara and Wathes, 1999). β-Oxidation of the 18-carbon essential fatty acids in the mitochondria depends on carnitinedependent translocation, and leads to generation of acetyl-CoA which then enters the tricarboxylic acid cycle. The first and rate-limiting step of β-oxidation of the longer-chain n-6 and n-3 fatty acids in the peroxisomes is catalysed by straight-chain fatty acyl-CoA oxidase and generates hydrogen peroxide (Wanders et al., 2001). In a similar process, 22:6n-3 and 22:4n-6 can be retroconverted to 20:5n-3 and 20:4n-6, respectively, thereby maintaining tissue pools of these fatty acids. In the absence of a dietary supply of n-6 and n-3 fatty acids, oleic acid (18:1n-9) derived from the diet or synthesized de novo from acetyl-CoA, undergoes Δ6- and Δ5-desaturation and elongation to form eicosatrienoic acid (20:3n-9), and concentrations of 20:4n-6 decrease due to the absence of 18:2n-6 (Innis, 1991, 1996). The usual biochemical method for establishing essential fatty acid deficiency is to calculate the ratio of 20:3n-9 to 20:4n-6. This is commonly referred to as the triene to tetraene ratio. An increase in the ratio of plasma 20:3n 9 to 20:4n-6 to >0.2 is considered to indicate essential fatty acid deficiency in humans (Holman et al., 1991; Jeppensen et al., 1998). Dietary deficiency of n-3 fatty acids results in decreased 22:6n-3 and increased desaturation of n-6 series fatty acids, leading to increased 22:4n-6 and 22:5n-3 in brain and retinal membranes (Bourre et al., 1984; Innis, 1991). Refeeding deficient developing and adult animals with 18:3n-3 results in recovery of neural cell membrane 22:6n-3, although the rate of recovery may be slower in the central nervous system than in

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other organs (Youyou et al., 1986; Bourre et al., 1989a; Connor et al., 1990; Weisinger et al., 1999; Moriguchi et al., 2001). Desaturation and elongation of n-9 fatty acids and the triene/ tetraene ratio is normal unless there is a concomitant deficiency of n-6 fatty acids. The compensatory increased desaturation and elongation of n-6 fatty acids in n-3 fatty acid-deficient animals results in maintenance of the normal total n-6 + n-3 polyunsaturated fatty acids in the brain (Galli et al., 1971; Neuringer et al., 1986; Hrboticky et al., 1990), although this may not be so for all neural membrane phospholipids (Murthy et al., 2002).

3. FUNCTIONAL ROLES OF ESSENTIAL FATTY ACIDS Long-chain polyunsaturated fatty acids (those with 20 or more carbon chains and 3 or more double bonds) are found predominately in phospholipids in which they form the hydrophobic core of all cell and subcellular membranes. Linoleic acid (18:2n-6) is also present in membrane phospholipids, adipose tissue triglycerides, plasma cholesterol esters, and in the specialized lipids of the skin. Reviews of the role of n-6 fatty acids in maintaining the normal epithelial cell–water barrier are available (Ziboh and Chapkin, 1988). The n-6 and n-3 fatty acids, 20:3n-6, 20:4n-6 and 20:5n-3 are precursors for prostaglandins, hydroxy fatty acids, leukotrienes and lipoxins, often collectively referred to as eicosanoids. These oxygenated metabolites, which are formed via cyclo-oxygenase and lipoxygenase, are synthesized following a stimulus and act locally as autocoids, often initiating a cascade of events. In general, metabolites formed from 20:5n-3 have weaker or opposing effects than the metabolites formed from 20:4n-6. Several reviews on eicosanoid metabolism have been published (Fischer, 1989; Kinsella and Lokesh, 1990; Funk, 2001). The importance of n-6 and n-3 fatty acids in metabolic and physiological processes can be summarized into three general mechanisms: the fatty acid moieties of membrane phospholipids contribute to the physical properties of the membrane bilayer, with secondary effects on the activity of membrane-associated proteins, receptors and ion channels; n-6 and n-3 fatty acids are precursors for generation of membrane-derived signal molecules, as well as eicosanoids; and n-6 and n-3 fatty acids have direct effects on gene expression. Docosahexaenoic acid, (22:6n-3), unlike the n-6 fatty acids, has a highly specific distribution in tissues and phospholipids. Concentrations of 22:6n-3 are particularly high in the amino phospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE) of neural grey matter, and in the outer segments of rod and cone photoreceptors in the retina (Fliesler and Anderson, 1983; Sastry, 1985; Giusto et al., 2000). Large amounts of 22:6n-3 are also present in specific phospholipids in the heart and sarcoplasmic reticulum of skeletal muscle sarcolemma, and in sperm (Fiehn and Pewter, 1971; Poulos et al., 1973; Gudbjarnason et al., 1978; Charnock et al., 1983; Ollero et al., 2000). Severe restriction of n-3 fatty acids throughout gestation, lactation and postnatal development, when there is a need for new tissue synthesis, results in reduced tissue 22:6n-3, decreased visual function, decreased performance on discrimination learning tasks, and increased stereotyped behaviour in rodents and non-human primates (Benolken et al., 1973; Wheeler et al., 1975; Neuringer et al., 1984, 1986; Yamamoto et al., 1988; Bourre et al., 1989b; Reisbick et al., 1990, 1994; Frances et al., 1996a,b; Okada et al., 1996; Gamoh et al., 1999; Moriguchi et al., 2000; Greiner et al., 2001) (table 1). In adult animals, 22:6n-3 is aggressively retained, even during longstanding and severe dietary n-3 fatty acid restriction (Tinoco et al., 1979; Tinoco, 1982). Some species, including many fish, insects and pulmonates, require a dietary source of 18:3n-3 for normal growth and feed efficiency (Tinoco et al., 1979; Tinoco, 1982). n-3 fatty acids do not appear to be essential for growth and feed efficiency in mammals, although 22:6n-3 is involved in energy metabolism and calcium ion channel activity in the

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heart (Kang and Leaf, 1996; Leaf et al., 1999; Leifert et al., 1999; McLennan, 2001; Ferrier et al., 2002), which may explain the relation of high dietary intakes of n-3 fatty acids with reduced heart rate variability and sudden death (Christensen et al., 1996, 1997; Nair et al., 1997; Albert et al., 1998). When released by phospholipases, membrane phospholipid n-6 and n-3 fatty acids become available as unesterified fatty acids and function as important signal molecules, in addition to serving as substrates for eicosanoid synthesis. Their functions include regulation of the activity of protein kinases, G-proteins, adenylate and guanylate cyclases, phospholipases, ion channels and multiple other proteins and receptors (Bernsohn and Spitz, 1974; Bourre et al., 1989b; Park and Ahmed, 1992; Gerbi et al., 1994; Poling et al., 1995; Kang and Leaf, 1996; Litman and Mitchell, 1996; Koenig et al., 1997; Huster et al., 1998; Leifert et al., 1999; Bonin and Khan, 2000; Litman et al., 2001). In addition, polyunsaturated fatty acids regulate the expression of genes for regulatory proteins of lipid and carbohydrate metabolism through peroxisome proliferator-activated receptor (PPAR)-dependent and independent mechanisms (Duplus et al., 2000; Clarke, 2001; Jump, 2002), and influence leptin gene expression (Reseland et al., 2001). New information also suggests that n-6 fatty acids are involved in adipogenesis during development by pathways involving PPAR γ2, although polyunsaturated fatty acids also suppress genes related to lipogenesis (Ntambi et al., 1988; Clarke et al., 1990; Reginato et al., 1998). Recently, it has become clear that n-3 fatty acids alter the expression of genes related to endocytosis, signal transduction, synaptic vesicle recycling and formation, lipid metabolism, nuclear ligand-activated transcription factor receptors in brain, retinoic acid receptor (R × R) (Khair-El-Din et al., 1996; Mata de Urquiza et al., 2000; Kitajka et al., 2002), and intestinal nutrient absorption (Lampen et al., 2001). 3.1. n-3 essential fatty acids and visual function The retina is an integral part of the central nervous system, which is composed of six cell types – the photoreceptor cells, horizontal, bipolar, amacrine, interplexiform and ganglion cells – and communicates directly with the brain via ganglion cells passing through the optic nerve. The two photoreceptor cell types are the rods and cones. Rods are elongate and cylindrical and function as dim light receptors, while cones are shorter and usually cylindrical, mediate colour vision and function at relatively higher light intensities (Giusto et al., 2000). Rods and cones are highly specialized differentiated neurons that contain a stack of photosensitve membranes at the distal end (known as the outer segments), a central region containing mitochondria, golgi and nucleus, and a synaptic terminal. The outer segments are made up of densely stacked disks, each of which is a double layer of infolded plasma membrane which is highly enriched in 22 :6n-3. Vertebrate retina photoreceptor cells contain 50% protein and 50% lipid, with 90–95% of the lipid present as phospholipid and 4–6% as cholesterol (Giusto et al., 2000). The major phospholipid species are PE, PC and PS. Within the outer segment disks, as much as 80% of the polyunsaturated fatty acids are 22:6n-3, with species of PE, PS and PC in which both fatty acids are present as 22:6n-3 (Fliesler and Anderson, 1983; Aveldano, 1987; Giusto et al., 2000). In bovine rod outer segment membranes, about 30% of the PC, 20% of the PE and 50% of the PS have a long-chain polyunsaturated fatty acid esterified at both the sn-1 and the sn-2 position (Aveldano et al., 1983; Aveldano, 1987; Aveldano and Sprecher, 1987). It is notable that this unusual and characteristic membrane enrichment of 22:6n-3 is present even in the bovine retina, a herbivore species that obtains no dietary 22:6n-3. Specific pathways allow efficient recycling of 22:6n-3 from photoreceptor cells during shedding (turnover) from the disk tips. This involves phagocytosis by retinal pigment

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epithelium cells, then recycling of 22:6n-3 for reuse in synthesis of new disk membranes (Rodriguez de Turco et al., 1999). About 80–90% of the retina rod and cone outer segment protein is the visual pigment rhodopsin (opsin plus the carotenoid 11-cis-retinal), which functions as a photon receptor coupled to regulatory G-proteins (Giusto et al., 2000). A light-induced change in the conformation of rhodopsin triggers a cascade of reactions that result in increased phosphodiesterase activity and decreased cyclic GMP, which leads to closure of the photoreceptor membrane sodium channels and hyperpolarization, followed by depolarization. These events correspond to the positive A and negative B waves, respectively, of the electroretinograph (ERG). Recent studies have provided evidence that 22:6n-3 may influence photoreceptor signal transduction by influencing the ability of photons to transform rhodopsin from metarhodopsin I to the activated metarhodopsin II state (Mitchell et al., 1992; Mitchell and Litman, 1998; Litman et al., 2001). Retina concentrations of 22:6n-3 increase during gestation and reach adult concentrations by the time of term birth in humans (Martinez, 1992). Dietary deficiency of n-3 fatty acids during development results in reduced 22:6n-3 in the retina of animals (Hrboticky et al., 1991). In addition to 22:6n-3, n-3 fatty acids with up to 36 carbons are present in small amounts in the retina (Aveldano et al., 1983; Aveldano, 1987; Aveldano and Sprecher, 1987), and these are reduced in rats fed a diet deficient in n-3 fatty acids throughout development (Suh et al., 1996, 2000). The role of these fatty acids in retinal function has not yet been elucidated, although they may be related to rhodopsin kinetics. Several studies have addressed the effect of decreased retina 22:6n-3 on retinal and visual function in animals and human infants. Early studies in this field reported increased A and B wave amplitudes in ERG responses of rats fed a fat-free diet (Benolken et al., 1973; Wheeler et al., 1975; Anderson et al., 1976). Later, Neuringer et al. (1984, 1986) reported prolonged recovery times of dark-adapted A and B wave responses, and reduced rod and cone A wave responses in full-field ERG of monkeys fed a severely n-3 fatty acid-restricted diet through fetal and neonatal development. The role of n-3 fatty acids in retinal and visual development in human infants is discussed in section 8. 3.2. n-3 essential fatty acids and brain function The brain contains the second highest concentration of lipid in the body, after adipose tissue, with 50% lipid on a dry weight basis, 10% lipid on a wet weight basis (Sastry, 1985). Unlike adipose, however, the brain contains minimal amounts of triglyceride, the lipids of brain being almost entirely composed of the membrane structural components. About half of the lipid is phospholipid, with about 20% cholesterol, 15–20% cerebrosides and smaller amounts of sulphatides and gangliosides. The phospholipids of brain grey matter contain large amounts of 20 : 4n-6 and 22: 4n-6, particularly in PI and PE, and high amounts of 22:6n-3 in grey matter PE and PS (Sastry, 1985). Myelin, on the other hand, contains mainly saturated and monounsaturated fatty acids (O’Brien and Sampson, 1965). The brain of herbivores, like the visual photoreceptor cells, is enriched in 22:6n-3 despite the absence of a dietary intake of preformed 22 : 6n-3, and only low amounts of 22:6n-3 in liver phospholipids (table 3). Only small amounts of 18 : 2n-6 are present in neural phospholipids, usually less than 1% of all the fatty acids, and concentrations of 18:3n-3 are even lower. This unusual characteristic feature of brain suggests the presence of specific pathways for selective uptake of 20:4n-6 and 22:6n-3 from plasma against a considerable concentration gradient, and that 20:4n-6 and 22:6n-3 are important to normal neural metabolism. The enrichment of 22:6n-3 in mammalian brain grey matter, together with the inability of animals to form n-3 fatty acids

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de novo, has focused attention on the need to elucidate the role of 22:6n-3 in neural function and requirements for n-3 fatty acids during brain growth and development. The behaviour changes in developing animals fed diets deficient in n-3 fatty acids include reduced performance in maze tasks, habituation, exploratory activity in novel environments and brightness and olfactory-based discrimination learning (Yamamoto et al., 1988; Bourre et al., 1989b; Enslen et al., 1991; Innis, 1991; Yonekubo et al., 1993; Frances et al., 1996a,b; Greiner et al., 1997, 2001; Wainwright et al., 1998). Polydypsia, increased stereotypic (locomotor) activity and altered performance on a task of recognition looking memory have also been reported for monkeys fed a diet very low in n-3 fatty acids (<0.1% dietary energy 18:3n-3) through gestation and postnatal development (Reisbick et al., 1990, 1994). In addition to the decrease in 22:6n-3, n-3 fatty acid deprivation results in increased n-6 fatty acids, including 20:4n-6, 22:4n-6 and 22:5n-6, in the brain of animals (Galli et al., 1971; Bourre et al., 1984; Hrboticky et al., 1989, 1990; Favrelière et al., 1998) and human infants (Farquharson et al., 1995). It needs to be considered whether or not functional and biochemical changes associated with n-3 fatty acid deficiency are due to a specific requirement for 22:6n-3, the increase in n-6 fatty acids, or a combination of effects. Although considerable information has been published to describe the changes in brain and other tissues of animals fed diets varying in essential fatty acids, less is known on how changes in n-3 fatty acids alter behaviour. Recent research has established that 22:6n-3 alters metabolism of several neurotransmitters, including dopamine, serotonin (5-HT), epinephrine, acetylcholine, GABA and N-methyl-D-aspartate (NMDA) channel activity (Delion et al., 1994, 1996, 1997; Nishikawa et al., 1994; Hamano et al., 1996; Jones et al., 1997; Minami et al., 1997; Young et al., 1998; Zimmer et al., 1998, 1999, 2000a,b, 2002; de la Presa Owens and Innis, 1999a,b; McGahon et al., 1999; Itokazu et al., 2000). The cortical dopaminergic system is important in modulation of learning, attention and motivation, and in the visual pathways (Gava and McKean, 1977; Brozoski et al., 1979; Le Moal and Simon, 1991; Antal et al., 1997; Basmak et al., 1999) and has been the focus of several studies with n-3 fatty acid-deficient animals. Usually, these studies involve feeding a diet severely restricted in n-3 fatty acids through several generations in order to deplete adipose tissue n-3 fatty acids that can be transported from mother to offspring during gestation and suckling. Newer research has shown that the effects of n-3 fatty acid deficiency on the brain are complex and region specific. The effects described include changes in dopamine concentration, vesicular monoamine transporter 2, dopamine D2 receptor, tyrosine hydroxylase (the ratelimiting enzyme in dopamine synthesis), and dopamine storage pools (Delion et al., 1994, 1996, 1997; Yoshida et al., 1997; Zimmer et al., 1998, 1999, 2000a,b, 2002). Newer techniques of dual-probe microdialysis have shown that although dopamine is decreased in frontal cortex of n-3 fatty acid-deficient animals, dopamine may be increased in the nucleus accumbens (Zimmer et al., 2002). This could suggest that the mesocorticolimbic area dopaminergic system functions more, but the mesocortical pathway is less active in n-3 fatty acid-deprived animals. Newer studies also suggest that the effects of n-3 fatty acid deficiency may be more apparent after a learning task, or administration of drugs that deplete endogenous dopamine storage pools (Yoshida et al., 1997; Zimmer et al., 1998, 2000a,b). Dietary deficiency of n-3 fatty acids leading to decreased 22:6n-3 in the developing brain is also associated with reduced serotonin and serotonin receptor binding in frontal cortex (Delion et al., 1996; de la Presa Owens and Innis, 1999a,b). This is of interest because 5-HT2 receptors are believed to play an important role in behaviour (Leysen and Pauwels, 1990). Cross-sectional studies in humans have described a negative relation between 5-hydroxy indolaceteic acid (5-HIAA, which is the metabolite of serotonin) in cerebrospinal fluid and

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plasma 22:6n-3 among adults with violent behaviours (Hibbeln et al., 1998). Other aggressive and depressive affective disorders have also been postulated to be linked to altered n-3 fatty acid metabolism (Adams et al., 1995; Hamazaki et al., 1996; Edwards et al., 1998; Hibbeln, 1998; Maes et al., 1999; Stoll et al., 1999). In vascular smooth muscle cells, enrichment with 22:6n-3 results in failure to respond to serotonin (Pakala et al., 2000). In other tissues, 20:5n-3 and 22:6n-3 both block the effect of growth factors that act through signalling pathways involving receptor tyrosine kinase (such as platelet-derived growth factor, fibroblast growth factor, epidermal growth factor and insulin-like growth factor) and G-protein coupled receptors (such as bomberin, bradykinin, vasopressin, thrombin, serotonin and thromboxane A2) (Kaminski et al., 1993). These findings have potential implications for linking the effects of 22:6n-3 to altered behaviour, and for a wide spectrum of physiological processes in developing animals. The abundance of 22:6n-3 in synaptic terminals and cortical growth cone membranes suggests n-3 fatty acids could be important for brain structural growth. Morphological analysis of brain from rats fed a n-3 fatty acid-deficient diet through three generations have described a lower cell body size in CA1 pyrimidal neurons at the septal location, although no differences were found in other hippocampal regions, or in neuron volume, density or number (Ahmad et al., 2002a). Consistent with this, others have shown reduced nerve growth factor concentrations in hippocampus of n-3 fatty acid-deficient rats (Ikemoto et al., 2000). Lower neuron size in hypothalamus and parietal cortex of weanling rats, and in periform cortex of mature rats with a 90% decrease in brain 22:6n-3, has also been reported (Ahmad et al., 2002b). Non-randomized studies in children with disorders of peroxisomal biogenesis have provided evidence that supplemental 22:6n-3 can lead to improvements in the marked deficits in cognitive and visual function, and impaired myelination in these disorders (Martinez and Vazquez, 1998; Martinez et al., 2000). In these disorders, the absence of peroxisomes results in the inability to form 22:6n-3. A mechanism through which n-3 fatty acids could stimulate oligodendrocyte metabolism and the synthesis of myelin has not been described. Information to suggest that n-3 fatty acids influence synthesis and turnover of brain phospholipids has also been published. Elucidation of this possibility is important because decreased phospholipid synthesis or turnover could have profound effects on membrane phospholipid-dependent signalling pathways. Studies with F2 generation 8-week-old rats found decreased PS in olfactory bulb, brain cortex and brain mitochondria, while PS in liver and adrenal were not affected (Hamilton et al., 2000). In other studies, PS was reduced by 28% and PC was increased in hippocampus phospholipids of rats fed a n-3 fatty acid-deficient diet (Murthy et al., 2002). Further evidence for a role of 22:6n-3 in PS metabolism has come from studies showing that intra-amniotic injection of ethyl-22:6n-3 increased both 22:6n-3 and PS in brain of fetal rats (Green and Yavin, 1995). PS synthesis was also increased in C6 glioma cells cultured with 22:6n-3 (Garcia et al., 1998). The significance of these findings relates to the role of PS in signal transduction through a regulatory role in protein kinase C (PKC) activation (Bell, 1986; Bell and Burns, 1991; Casamenti et al., 1991; Borghese et al., 1993; Mosier and Newton, 1998). Some evidence to suggest that dietary essential fatty acids affect the activity of enzymes involved in PC biosynthesis in brain synaptic membranes has also been published (Hargreaves and Clandinin, 1987). Studies using radiolabelled tracers have also provided evidence of decreased docosahexaenoyl-CoA, and phospholipid synthesis and turnover in vivo in n-3 fatty acid-deficient animals (Gazzah et al., 1995; Contreras et al., 2001). Chronic n-3 fatty acid deficiency, however, does not affect 20:4n-6 recycling in the brain, which suggests that phospholipases involved in release of 20:4n-6 and 22:6n-3 may be regulated independently (Contreras et al., 2001). Decreased turnover of membrane phospholipids

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enriched in 22:6n-3, reflecting adaptation by the brain to conserve important membrane polyunsaturated fatty acids during limited dietary availability, has important implications for optimal signal transduction.

4. ESSENTIAL FATTY ACID TRANSFER IN GESTATION All of the n-6 and n-3 fatty acids accumulated in the fetus must ultimately be derived from the mother by placental transfer. Placental tissue contains Δ6- and Δ5-desaturases. The activity of Δ6-desaturase in ovine placenta increases towards term (Shand and Noble, 1981; Choy et al., 1997). Synthesis of 22:6n-3 by the placenta, however, has not been demonstrated. Whether placental desaturase activity contributes 20:4n-6 to the foetus does not appear to be known. Concentrations of 20:4n-6 and 22:6n-3 are much higher, whereas 18:2n-6 is lower in foetal than maternal plasma lipids (Crawford et al., 1981; Elias and Innis, 2001) (fig. 3). Current information suggests that placental transfer of 20:4n-6 and 22:6n-3 involves a multi-step process of cell uptake and intracellular translocation that is facilitated by several membraneassociated and cytosolic fatty acid binding proteins. Specific membrane binding of n-6 and n-3 fatty acids, favouring 20:4n-6 and 22:6n-3 over 18:2n-6 or 18:3n-3, and preferential transfer of n-6 and n-3 fatty acid compared to non-essential fatty acids by human placenta have been reported (Campbell et al., 1996, 1998a,b; Haggarty et al., 1997; Dutta-Roy, 2000). Despite the presence of pathways to facilitate the transfer of n-6 and n-3 fatty acids across the placenta, the maternal intake of essential fatty acids during pregnancy can have a marked effect on n-6 and n-3 fatty acid accretion in developing foetal tissues. Dietary deficiency of n-3 fatty acids in gestation results in decreased 22:6n-3 and increased 22:4n-6 and 22:5n-6 in growth cones, the amoeboid leading edge of the growing neurite, in the fetal rat brain while high intakes of 20:5n-3 and 22:6n-3 result in increased 22:6n-3 and decreased n-6 fatty acids (Innis and de la Presa Owens, 2001) (fig. 4). In the latter studies, differences in maternal

Fig. 3. The relative enrichment of 18:2n-6, 18:3n-3, 20:4n-6 and 22:6n-3 in fetal compared to maternal plasma was calculated for each mother–fetal cord plasma pair as the difference in the given fatty acid in the maternal compared to fetal plasma/maternal plasma × 100%. Values shown are means ± SEM, n=55. Adapted from Elias and Innis (2001).

Fig. 4. Effect of maternal essential fatty acid intake on n-6 and n-3 fatty acids in fetal rat brain neuronal growth cone phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidyliniositol (PI). Rats were fed safflower, soybean or fish oil throughout pregnancy and fetal brain was analysed at birth. The bars represent means + SEM, values with a different superscript are different (P< 0.05). Adpated from Innis and de la Presa Owens (2001).

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dietary fat intake also resulted in altered dopamine in the fetal rat brain. Levels of 20:4n-6 and 22:6n-3 in maternal plasma are also significantly correlated with the concentration of the same fatty acid in newborn human infant plasma (Elias and Innis, 2001), and infants born with higher blood levels of 22:6n-3 and 20:4n-6 maintain this advantage for several weeks after birth (Foreman-van Drangelen et al., 1995; Guesnet et al., 1999). This adipose tissue 22:6n-3 and 20:4n-6 accumulated during foetal development contributes to the postnatal blood pool of 22:6n-3 and 20:4n-6. Several studies have shown that supplementation of pregnant women with 22:6n-3 increases 22:6n-3 in plasma and red blood cell lipids of infants at birth (van Houwelingen et al., 1995; Connor et al., 1996; Helland et al., 2001), providing convincing evidence that the maternal intake of 22:6n-3 is an important factor in the placental transfer of this fatty acid. Research is now starting to address the functional significance of the supply of 20:4n-6 and 22:6n-3 to the developing foetus. A positive relation between 20:4n-6 and birth weight, despite high 18:2n-6, has been found in several studies (Koletzko and Braun, 1991; Elias and Innis, 2001). Although this is consistent with the essentiality of 20:4n-6 in growth (Mohrhauer and Holman, 1963), the biological explanation for this is not known. Electroencephalography (EEG) at 2 days, as a measure of infant CNS maturity, indicated infants with a more mature EEG pattern have significantly higher 22:6n-3 in cord plasma phospholipids than infants with a less mature EEG pattern (Helland et al., 2001). Other recent studies have found an inverse relationship between maternal plasma 22:6n-3 and active sleep and sleep–wake transitions, and a positive association with wakefulness in 2-day-old infants (Ceruku et al., 2002), also suggesting that lower 22:6n-3 during gestation may be associated with delayed CNS maturation. Evidence of long-term sequelae, should these be present, due to early differences in 22:6n-3 status at birth is still needed, as are intervention studies to rule out the possibility that these associations are explained by differences in other dietary and lifestyle variables that accompany differences in 22:6n-3 intake. Long-chain polyunsaturated n-3 fatty acids, and the balance of n-6 to n-3 fatty acids in the diet, also influences gestation length in animals and humans (Olsen et al., 1992, 1995; Abayasekara and Wathes, 1999). In humans, high habitual intakes of 20:5n-3 and 22:6n-3, and supplementation with 2.7 g/day fish oil have been associated with longer gestation, possibly due to suppression of n-6 fatty acid-derived eicosanoids that are involved in initiation of parturition (Olsen et al., 1991, 1992). However, no association was found between length of gestation and the intake of n-3 fatty acids among women for whom the 95% range of intake was 0 to 0.75 g/day (Olsen et al., 1995). Typical intakes of long-chain n-3 fatty acids in North America are in the range of 100–200 mg/person/day (Innis and Elias, 2003). Whether the small increase in length of gestation in humans (of about 3–4 days) found at very high intakes of 20:5n-3 + 22:6n-3 is of functional benefit to the infant is not known.

5. ESSENTIAL FATTY ACID TRANSFER IN MILK The n-6 and n-3 fatty acid composition of milk varies considerably among different species, probably reflecting differences in diet and lipid metabolism. The milks of ruminants, such as cattle and goats, have low concentrations of all the n-6 and n-3 fatty acids, while concentration of 20:5n-3 and 22:6n-3 are particularly high in the milk of marine mammals (table 4). In addition to the effects of the diet, species differences in the desaturation and elongation, or β-oxidation, of 18:2n-6 and 18:3n-3 also influence the amount of 20:4n-6 and 22:6n-3 in milk. For example, milk from rats fed a diet containing 18:2n-6 and 18:3n-3, but no 20:4n-6 or 22:6n-3, had 0.8% 20:4n-6 and 0.5% 22:6n-3. Milk from pigs fed the same fat source, on the

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Table 4 Fatty acid composition of selected animal and human milk lipids

<10:0 12:0 14:0 16:0 18:0 16:1 18:1 18:2n-6 20:4n-6 18:3n-3 20:5n-3 22:6n-3

Cowa

Goatb

Pigc

Catd

Dogd

Rate

10.8 3.0 10.6 33.7 12.6 1.8 21.4 2.9 0.2 0.3

22.4 6.4 12.4 33.7 3.8

1.0 4.1 3.1 28.0 5.1 9.4 13.2

0.4 0.9 5.4 26.8 10.1 5.1 40.3 5.8

0.2 0.7 4.9 26.5 3.7 7.8 10.9 9.2

0.6

1.3

2.1

14.8 10.7 11.6 23.5 4.8 1.3 17.8 10.4 0.8 0.8 0.4 0.5

11.3 2.7 1.1 0.3 0.2 0.1

Whalee Dolphine Sea lione

0.1 6.1 12.8 5.0 7.3 25.6 0.3 0.8 0.8 6.5 9.6

0.2 5.6 12.9 4.8 8.1 38.2 1.1 0.5 1.1 4.2 6.0

0.1 3.7 18.0 2.1 7.6 40.1 1.5 1.1 1.5 5.5 7.1

Humanf 0.6 4.1 6.1 19.4 7.2 2.5 35.7 12.1 0.4 1.4 0.1 0.2

Adapted from: a Jensen (2002); b Le Doux et al. (2002); c Foote et al. (1990); d Parodi (1982); e Unpublished data; f Innis and King (1999).

other hand, had 0.6% 20:4n-6 and only 0.1% 22:6n-3. Human milk also typically has about 0.5–0.7% 20:4n-6 and 0.2–0.4% 22:6n-3, with the amount of 22:6n-3 decreasing to 0.1% fatty acids among women following vegan diets with no preformed 22:6n-3 (Sanders et al., 1978; Innis, 1992; Innis and King, 1999). Studies in multiple species have shown that the amounts of 18:2n-6, 18:3n-3, 20:5n-3 and 22:6n-3 secreted in milk depends on the amount of the same fatty acid in the maternal diet. As for other dietary situations, this can be expected to influence the amount of essential fatty acids deposited in the tissues of the growing milk-fed neonate. Studies in animals, including cattle, have shown that increasing the dietary intake of 18:2n-6 results in increased secretion of 18:2n-6 in milk (Bayourthe et al., 2000; Morales et al., 2000). Similarly, supplementation of diets with fish oil results in increased secretion of 20:5n-3 and 22:6n-3 in milk (Arbuckle and Innis, 1993). The increased 20:5n-3 and 22:6n-3 in the milk of lactating sows fed 1% weight diet as fish oil results in marked increases in 20:5n-3 and 22:6n-3 in plasma, red blood cells and liver, and a smaller increase in 22:6n-3 in brain of the suckling piglets (table 5). The implications of the polyunsaturated fatty acid supply in milk to the growth, behaviour and health of livestock is worthy of consideration. Autposy analysis has confirmed that, as in animals, the essential fatty acid composition of the diet does influence the accretion of n-6 and n-3 fatty acids in developing human infant tissues (Makrides et al., 1994; Farquharson et al., 1995; Jamieson et al., 1999). Because of this, it is important to understand the effects of diet on the essential fatty acid composition of human milk. The essential fatty acid composition of human milk shows considerable variability both among and within populations (Jensen, 1989, 1999; Innis, 1992). This variability appears to be explained by differences in the maternal essential fatty acid intake. Human milk in North America and Europe currently has 12–16% total fat as 18:2n-6, representing about 6–8% of the infant’s energy intake (Innis, 1992; Innis and King, 1999). Reports from the 1950s found 7% 18:2n-6 in human milk lipids, and clearly showed that a diet high in 18:2n-6 results in a marked increase in 18:2n-6 in the milk of lactating women (Insull and Ahrens, 1959; Insull et al., 1959). Whether the 2-fold increase in 18:2n-6 in human milk is explained by changes in dietary fat over the last half century is unclear.

28.6 4.5 28.6 20.7 0.6

2.6 0.4 0.4 1.5

27.6 4.4 37.5 11.0 0.6

1.1 0.1 0.2 0.1

+Fish oil

23.7 20.5 14.9 20.8 11.1 0.1 0.1 0.4 1.8 2.6

Veg. oil 23.8 22.6* 9.7* 19.7 9.2* 0.2* 0.5* 1.1 1.2 7.9*

+ Fish oil

Piglet plasma

10.4 31.8 9.8 8.7 24.0 0.5 0.1 0.4 2.8 7.3

Veg. oil

Liver PE

10.2 26.5* 5.8* 10.0 19.8* 0.3 0.1 1.4* 1.9* 21.1*

+ Fish oil 10.4 28.7 14.7 0.9 15.4 2.4 0.1 0.2 0.7 13.1

Veg. oil 8.5 23.9 18.4 0.7 15.7 2.0 0.1 0.1 0.5 16.6*

+ Fish oil

Cerebrum PE

8.1 25.6 9.1 1.3 14.8 1.6 0.1 0.1 1.6 31.4

Veg. oil

Retina

9.4 27.9 9.8 0.9 12.8 0.7 0.1 0.1 1.0 32.3

+ Fish oill

Adapted from Arbuckle et al. (1993) Sows were fed diets with 4% by weight fat as vegetable oil with 35% 18:2n-6 and 5.3% 18:3n-3, or vegetable plus fish oil with 47.7% 18:2n-6, 7.2% 18:3n-3, 0.7% 20:5n-3, 0.7% 20:5n-3 and 0.2% 22:6n-3 from 5 days before parturition through lactation. The piglet tissue fatty acids were determined at 15 days of age. PE, phosphatidylethanolamine. *P < 0.05 between groups.

16:0 18:0 18:1 18:2n-6 20:4n-6 22:5n-6 18:3n-3 20:5n-3 22:5n-3 22:6n-3

Veg. oil

Sow milk

Polyunsaturated fatty acids in milk lipids and suckling piglets from sows fed vegetable and fish oils

Table 5

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Human milk concentrations of 22:6n-3 also show considerable variability, by over an order of magnitude among populations and individuals. The average amount of 22:6n-3 in human milk has been reported to be 2.8% in Zhangzi, China, 1.4% fatty acids in Canadian Inuit, 1.0% in Japan, 0.8–0.9% on the Malay Peninsula, 0.5% in Norway, 0.2–0.4% in Canada, Europe and the USA (Kneebone et al., 1985; Innis and Kuhnlein, 1988; Innis, 1992; Chulei et al., 1995; Helland et al., 2001), and 0.1% among women in the UK following vegan diets (Sanders et al., 1978). While 18:2n-6 has increased, the amount of 22:6n-3 in human milk in Western countries has decreased. Analyses conducted over the last 15 years in Australia and Canada show human milk 22:6n-3 has declined by 25–50% (Makrides et al., 1995b; Innis, 2002). Supplementation of lactating women with fish or fish oil increases the secretion of 20:5n-3 and 22:6n-3 into human milk (Harris et al., 1984; Henderson et al., 1992; Helland et al., 2001). Supplementation of lactating women with 22:6n-3 from single-cell oils with >40% 22:6n-3 and <0.1% 20:5n-3, on the other hand, increases 22:6n-3 in human milk without increasing 20:5n-3 (Makrides et al., 1996; Gibson et al., 1997; Fidler et al., 2000). Higher amounts of 22:6n-3 in milk result in higher intakes and higher blood lipid levels of 22:6n-3 in breast-fed infants (Henderson et al., 1992; Gibson et al., 1997; Innis and King, 1999; Jensen et al., 2000). The amount of 20:4n-6 in milk appears to be much more tightly regulated, and does not seem to be reduced even with high intakes of 20:5n-3 and 22:6n-3 (Innis and Kuhnlein, 1988; Makrides et al., 1996; Fidler et al., 2000; Jensen et al., 2000). Recent studies using stable isotope tracer methodologies have estimated that 20–25% of 22:6n-3, 33% of 18:2n-6 and 12% of 20:4n-6 secreted in human milk are derived from the dietary intake of the previous 48 hours (del Prado et al., 2000; Fidler et al., 2000). This may suggest that adipose tissue provides a significant portion of the fatty acids secreted in milk. Consequently, the essential fatty acid content of the diet fed during gestation may also be important to the essential fatty acid quality of the milk in later lactation. The importance of the supply of essential fatty acids in milk or milk substitutes to infant growth and development is discussed in section 8.

6.

ESSENTIAL FATTY ACID ACCRETION IN THE BRAIN AND RETINA

Arachidonic acid (20:4n-6) and 22:6n-3 are accumulated in large amounts in the brain and retina during brain growth, particularly the brain growth spurt when the relative increase in brain weight is at its highest (Dobbing and Sands, 1979). Animals that are born with a relatively mature brain, such as the monkey and guinea pig, known as precocial animals, have the highest requirement for 20:4n-6 and 22:6n-3 for brain growth in utero. Altricial animals, such as rats, which are born immature, have the largest period of brain growth after birth. The increase in rat brain 20:4n-6 and 22:6n-3 is most rapid between days 10 and 15 after birth. At birth, rat brain contains about 0.6 mg 20:4n-6 and 0.6 mg 22:6n-3. This increases to 2.46 mg 20:4n-6 and 2.48 mg 22:6n-3 by 10 days of age, then almost doubles over the next 5 days to 4.21 mg 20:4n-6 and 4.8 mg 22:6n-3 at 15 days of age, while the adult brain has 6.3 mg 20:4n-6 and 10.2 mg 22:6n-3 (Sinclair and Crawford, 1972). In the human and pig, brain growth is intermediate about the time of birth. The human brain weighs about 100 g at the beginning of the third trimester of gestation, 370 g at term birth, and 2000 g at 2 years of age. The pig brain weighs about 5.8 g at 70 days of gestation, 10.7 g at 80 days, then increases another 3-fold to 31 g at 110 days of gestation, and weighs about 46 g by 4 weeks after birth (Pond et al., 2000). However, the rate of brain growth does not reflect the rate of maturation within individual regions of the brain, or the sensitivity of particular developing anatomical, biochemical or functional systems to the supply of essential fatty acids. For example, in the human, the growth of dendritic arbors and peak formation of synapses extends from about

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34 weeks of gestation through 24 months after birth, during which time new connections form at a rate of up to almost 40,000 synapses/sec (Huttenlocher et al., 1982; Borgeois, 1997; Huttenlocher and Dabholkar, 1997; Levitt, 2003). Autopsy analyses of fetal and infant tissue from 22 weeks gestation to 17 weeks after birth has been used to estimate the amounts of n-6 and n-3 fatty acids accumulated in developing human tissues (Clandinin et al., 1980a,b, 1981). Estimates of intrauterine accretion of essential fatty acids indicate deposition of 552 mg/day n-6 fatty acids and 67 mg/day n-3 fatty acids during the last trimester of gestation. Most of this is accumulated in white adipose tissue (368 mg n-6 and 52 mg n-3 fatty acids/day). Accretion in the human fetal brain amounts to 5.8 mg n-6 and 3.1 mg n-3 fatty acids/day, representing about 1.1% and 4.65% of total body accretion, respectively. It is not known if the brain is protected, for example at the expense of adipose tissue during limited dietary availability of essential fatty acids. The extent to which the developing brain and retina depend on plasma 22:6n-3, or are able to convert 18:3n-3 to 22:6n-3, is important to understand the dietary requirements for n-3 fatty acids and the interpretation of differences in plasma 22:6n-3. Several studies have shown that brain is able to take up and convert 14C-labelled 18:2n-6 and 18:3n-3 into longer-chain metabolites (Sinclair and Crawford, 1972; Dhopeshwarkar and Subramanian, 1975a,b, 1976; Cohen and Bernsohn, 1978; Anderson and Connor, 1988; Washizaki et al., 1994; Chang et al., 1997). Conversion of 18:3n-3 and 22:5n-3 to 22:6n-3 in the retina and retinal pigment epithelium has also been demonstrated (Wang and Anderson, 1993; Alvarez et al., 1994). In vitro, brain cerebroendothelial cells take up 18:2n-6 and 18:3n-3 and can form 20:4n-6 and 22:4n-6, and 20:5n-3 and 22:5n-3 (Moore et al., 1991; Moore, 1994) as well as 22:6n-3 in a pathway involving carbon chain 24 intermediates (Delton-Vandenbroucke et al., 1997). However, cerebral and cerebellar neuronal cells do not form 22:6n-3 (Moore, 2001). Astrocytes from suckling rat brain, however, can form 22:6n-3, which after release can be taken up by neurons (Moore et al., 1991; Moore, 2001). Although the pathway for 22:6n-3 formation is present, the major product of 18:3n-3 metabolism in neonatal brain astrocytes is 22:5n-3, not 22:6n-3 (Innis and Dyer, 2002, Williard et al., 2002); in vivo, brain has only minor amounts of 22:5n-3 (Sastry, 1985). In the absence of n-3 fatty acids, neonatal brain astrocytes cultured with 18:2n-6 accumulate 20:4n-6 and 22:4n-6, rather than 22:5n-6 as occurs in n-3 fatty acid- deficient animals (Innis and Dyer, 2002). These findings indicate that while astrocytes have the ability to form 22:6n-3 uptake of 22:6n-3 from plasma derived from synthesis in the liver, or from placental transfer before birth or provided preformed in the diet after birth is likely to be quantitatively more important for brain 22:6n-3 accretion. The pathways of transfer of 22:6n-3 from plasma to the developing brain are still incompletely understood. Several studies have shown that albumin-bound (unesterified) 20:4n-6 and 22:6n-3 is taken up by the brain (Washizaki et al., 1994; Jones et al., 1997). Other studies have shown that 18:2n-6 and 20:4n-6, but not 16:0, from physiological amounts of radiolabelled 2-arachidonyl-lysophosphatidylcholine, 2-palmitoyl-lysophosphatidylcholine, and 2-linoleoyl-lysophosphatidylcholine, rapidly appear in the brain, and that uptake of 18:2n-6, 20:4n-6 and 22:6n-3 occurs more readily from lysophospholipids than from unesterified fatty acids (Thies et al., 1992, 1994; Bernoud et al., 1999; Lagarde et al., 2001). Lysophospholipids represent a significant (5–20%) proportion of total phospholipids in mammalian plasma, and thus could play an important role in the transfer of 20:4n-6 and 22:6n-3 to extrahepatic tissues. Other recent studies have shown that HDL PE may deliver long-chain polyunsaturated fatty acids to the brain via the sequential methylation of PE to PC at the blood–brain barrier (Magret et al., 1996). Further elucidation of the role of different plasma lipids in transporting essential fatty acids to the developing brain is important in order

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to better identify the dietary and metabolic conditions that place developing infants at risk of delayed neural and retinal development.

7. ESSENTIAL FATTY ACID METABOLISM IN DEVELOPMENT Prior to birth and during suckling, all of the n-6 and n-3 fatty acids accumulated by the developing foetus and neonate must be derived by transfer from the mother, either by placental transfer or from milk. The extent to which the foetus and young animal are able to use 18:2n-6 and 18:3n-3 or depend on a supply of preformed 20:4n-6 and 22:6n-3 is important in understanding dietary essential fatty acid requirements during development. Both Δ6- and Δ5-desaturase activities are present in liver and brain from early in development. Desaturase activities are relatively low in fetal rat liver and higher in fetal brain (Sanders and Rana, 1987). In mouse, brain Δ6-desaturase activity decreased about 4-fold from 3 days before birth to weaning, and activity in liver increased about 7-fold until 11 days after birth (Bourre and Piciotti, 1992). Linolenic acid (18:3n-3) deficiency increased, and feeding with 20:5n-3 + 22:6n-3 decreased, activity of the Δ6-desaturase (Dinh et al., 1993). Similarly, pig liver and brain at 63 days of gestation, 3 days postpartum (term birth, about 115 days) and at 12 weeks of age are able to convert [1-14C]18:2n-6 and 20:3n-6 to tetraenes (20:4n-6, 22:4n-6) and pentaenes (22:5n-6), but the rate of conversion in the fetal piglet liver and brain is 3–5-fold lower than in the animals after birth, and also much lower in brain than liver (Clandinin et al., 1985a,b). Li et al. (2000) found no synthesis of 22:6n-3 from [U-13C]18:3n-3 in liver of foetal piglets at 70–72 or 110–112 days gestation. Instead, desaturation of 18:3n-3 was limited at 20:5n-3, suggesting that the final steps of elongation and peroxisomal chain shortening may be limiting. Biosynthesis of 22:6n-3, however, increased rapidly over the first 14 days after birth (Li et al., 2000). This is compatible with placental supply of 22:6n-3 before birth, and the low amount of 22:6n-3 in sow milk. Stable isotope tracer methodology has been used to show that foetal baboons can form 18:3n-3 from a dose of intravenous [U-13C]18:3n-3 (Su et al., 2001). However, only about 0.6% of the 18:3n-3 administered was recovered in brain 22:6n-3. Again, this is consistent with low desaturation of n-3 fatty acids during prenatal development when placental transfer facilitates high concentrations of 22:6n-3 in foetal plasma. Activity of Δ6- and Δ5-desaturase has also been shown in human foetal liver microsomes from as early as 17 weeks of gestation (Chambaz et al., 1985; Poisson et al., 1993). The activity of the pathway to 22:6n-3 in human foetal liver prior to birth, however, is not known. Studies over a decade ago established that blood lipid 20:4n-6 and 22:6n-3 are lower in infants fed formula with 18:2n-6 and 18:3n-3 as the only n-6 and n-3 fatty acids than in breast-fed infants (Putnam et al., 1982; Ponder et al., 1992). Higher amounts of 18:3n-3 in the diet do not result in higher plasma or red blood cell 22:6n-3 in infants (Ponder et al., 1992). Studies in this field interpreted these findings as evidence of “immature” activity of a putative Δ4-desaturase believed to be responsible for synthesis of 22:6n-3 from 22:5n-3 (Putnam et al., 1982; Carlson et al., 1993). However, 20:4n-6, which is formed by Δ6- and Δ5-desaturation of 18:2n-6, is also lower in infants fed formula than in breast-fed infants. Advances in stable isotope tracer technologies have now allowed the demonstration that conversion of isotopically labelled 18:3n-3 to 22:6n-3 and of 18:2n-6 to 20:4n-6 is as high or higher in preterm as in term infants, and higher in infants not receiving preformed 20:4n-6 and 22:6n-3 from human milk (Carnielli et al., 1996; Salem et al., 1996; Sauerwald et al., 1997; Uauy et al., 2000; Pawlosky et al., 2001). The conversion of 18:3n-3 to 22:6n-3, however, appears to be highly variable among individuals and could be as low as <1 to 4% 18:3n-3 converted to 22:6n-3 in humans (Emken et al., 1994; Pawlosky et al., 2001). These tracer methodologies

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Table 6 Cerebral cortex long-chain polyunsaturated fatty acids in human infants fed human milk or infant formula PE

PS

Formula 18:3n-3 Fatty acid 20:4n-6 22:4n-6 22:5n-6 22:6n-3 22 :5n-6/22:6n-3

Formula 18:3n-3

Human milk

1.5%

0.4%

Human milk

1.5%

0.4%

17.6 12.0 3.2 17.7 0.18

20.1* 12.6 4.8* 13.4* 0.36

19.6∗∗ 14.3* 7.0* 11.6* 0.60

7.9 7.9 5.3 23.5 0.22

9.0 8.5 7.7* 19.3* 0.40

9.0 9.3 10.4** 14.4* 0.72

*P <0.01, **P < 0.5

compared to human milk. The formula contained 1.5% 18:3n-3 and 16.0% 18:2n-6 or 0.4% 18:3n-3 and 14.5% 18:2n-6. Adapted from Farquharson et al. (1995). PE, phosphatidylethanolamine; PS, phosphatidylserine.

have not yet been able to provide quantitative estimates of the contribution of 22:6n-3 synthesis to accumulation at the level of the tissues. Data from autopsy tissue also provides information on essential fatty acid metabolism in human infants. Infants fed formula containing 0.4% or 1.5% 18:3n-3 but no long-chain polyunsaturated fatty acids had lower brain 22:6n-3 and higher 22:4n-6 and 22:5n-6 in cerebral cortex PE and PS than infants who were breast-fed (Farquharson et al., 1995). Brain 22:5n-6 was also higher and the 22:6n-3 to 22:5n-3 ratio lower in infants fed the formula with 0.4% 18:3n-3 rather than 1.5% 18:3n-3 (table 6). The increase in 22:5n-6 is consistent with deficiency of 18:3n-3, and also provides evidence that the final steps of elongation, Δ6-desaturation and peroxisomal partial chain shortening of essential fatty acids are active, even in young infants. Stable isotope studies have also shown that 22:6n-3 synthesis is higher in infants with higher intakes of 18:3n-3 (Sauerwald et al., 1996). The findings, however, do not address whether a higher intake of 18:3n-3 can support sufficient endogenous synthesis of 22:6n-3 to meet the needs of the developing brain.

8. LONG-CHAIN FATTY ACIDS IN HUMAN INFANT NUTRITION Several studies have reported that groups of infants who are breast-fed perform better on tests of neurodevelopment than bottle-fed infants (Anderson et al., 1999). This advantage appears to remain even when many factors, such as socio-demographic variables, maternal eduction and birth order, are controlled, although further work still needs to be done with respect to clearly identifying and controlling extent and duration of breast-feeding in these comparative studies (Drane and Logemann, 2000). Although milk contains numerous biologically active components not present in infant formulas, the presence of 22:6n-3 in human milk and the critical role of this fatty acid in normal retina and brain development has led to intense investigation of the essential fatty acid needs of the human infant for growth and development. The central question is whether the rate of conversion of 18:3n-3 to 22:6n-3 in human infants is sufficient to provide enough 22:6n-3 for optimal brain and retinal function. These studies are complicated because the relationship between plasma or red blood cell and brain 22:6n-3 is curvilinear, rather than linear (Arbuckle et al., 1991; Rioux et al., 1997; Ward et al., 1998). At intakes above requirement, progressive increases in plasma and red blood cell 22:6n-3 with increasng dietary intake are not accompanied by similar increases in 22:6n-3 in the brain

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and retina. As might be expected, increasing the dietary intake of 20:4n-6 and 22:6n-3 in young infants results in an increase in these fatty acids in blood lipids, with the increase dependent on the amount of 20:4n-6 and 22:6n-3 fed, and also the concurrent intake of 18:2n-6 (Carlson et al., 1993b; Makrides et al., 1995a; Innis et al., 1996). Because of this, the assessment of dietary n-3 fatty acid requirements requires an approach combining measurement of growth, functions dependent on neural or other tissue 22:6n-3, and blood lipid 22:6n-3. Premature infants are considered particularly vulnerable to nutritional deficiency because of their limited adipose tissue mass and immaturity in many metabolic and physiological pathways at birth. Following birth, there is a rapid decline in the high 20:4n-6 and 22:6n-3 characterisitic of foetal plasma, and a large increase in 18:2n-6 (Innis, 1991). The increase in brain weight relative to body weight is also more rapid during the last trimester of gestation than at later ages. Numerous studies have documented an increased incidence of developmental problems in prematurely born children at school age (Bhutta et al., 2002), which is likely to reflect in part failure to provide optimal essential nutrients to support third trimester brain growth and development ex utero. Several options are available for including 20:4n-6 and 22:6n-3 in infant formulas. For 20:4n-6, these include egg total lipids, egg-derived triglycerides and egg phospholipids, or triglycerides from the single-cell fungus Mortierella alpina (which are highly enriched in 20:4n-6). Sources of 22:6n-3 include fish oils, either with high 20:5n-3 and 22:6n-3, fish oils such as tuna oils specifically selected to be low in 20:5n-3 and high in 22:6n-3, egg lipids, or oils from the single-cell micro-algae Crypthecodinium cohnii which contains >40% 22:6n-3 and virtually no 20:5n-3. Early studies by Birch et al. reported a higher rod threshold and lower maximal amplitude values in the B wave in ERG recordings of infants fed formula containing corn oil with only 0.5% 18:3n-3 (about 0.25% dietary energy) (Birch et al., 1992; Uauy et al., 1992). The higher threshold and lower maximum amplitude suggest that greater luminescence was needed to elicit a response, and that signal transduction was reduced, respectively. Subsequent studies found that supplementation of preterm infant formula containing 1.2% energy as 18:3n-3 (about 2.4% fatty acids) with 0.12% or more energy as 22:6n-3 and 0.23% or more energy as 20:4n-6 resulted in higher visual acuity when measured either by VEP techniques or with behavioural measures based on the ability of the infant to demonstrate a looking response to black and white gratings (stripes) of varying size (Carlson et al., 1993b, 1996a; Faldella et al., 1996; San Giovanni et al., 2000; O’Connor et al., 2001). Recently, a large multicenter trial found an advantage in Bayley mental developmental inventories and the MacArthur communicative inventories in preterm infants of birth weight less than 1250 g fed formula supplemented with 20:4n-6 and 22:6n-3 (O’Connor et al., 2001). The long-term significance of these early changes in visual and neural development is not yet known. The findings, however, show that 1.2% dietary energy as 18:3n-3 does not meet the n-3 fatty acid requirements of preterm infants, and that visual and some aspects of neural development are increased by small dietary intakes of 22:6n-3. Several studies, however, have found evidence of reduced growth in preterm infants fed formulas containing 22:6n-3 from fish oils (Carlson et al., 1992, 1996a; Ryan et al., 1999), and a positive relation between 20:4n-6 and growth has been described (Carlson et al., 1993a). Recent clinical studies have also provided evidence of higher growth in preterm infants fed formulas supplemented with 0.27% energy 20:4n-6 and 0.14% 22:6n-3 from single-cell triglycerides (Innis et al., 2002). While it is not clear if this is related to the role of 20:4n-6 in regulating early aspects of adipogenesis (Reginato et al., 1998), bone growth (Weiler and Fitzpatrick-Wong, 2002) or other mechanisms, it is evident that both the n-6 and the n-3 long-chain polyunsaturated fatty acids play an important role in the growth and development of preterm infants. Following these

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studies, preterm infant formulas containing both 20:4n-6 and 22:6n-3 are now available in many countries. Some evidence also suggests that inclusion of 20:4n-6 and 22:6n-3 in formula fed to preterm infants may increase immune system development, as assessed by ex vivo mitogen stimulation of peripheral blood lymphocytes (Field et al., 2001). Much more work on the role of essential fatty acids and their role in the developing immune and other systems is needed. Although many studies have addressed the role of dietary 22:6n-3 and 20:4n-6 in the growth and development of term infants, current findings are not consistent. Several early studies reported evidence of higher looking acuity and higher VEP acuity in term gestation infants fed formula supplemented with 22:6n-3 in amounts similar to that in human milk (Makrides et al., 1995c; Carlson et al., 1996b). Recent large multicenter trials, however, have not found a benefit of adding 0.12% to up to 0.36% 22:6n-3 (about 0.06–0.18% energy) of the formula fat on looking acuity, VEP acuity, or scores on tests of mental and motor skill development (Auestad et al., 1997, 2001; Lucas et al., 1999; Makrides et al., 2000). Birch et al. (1998, 2000), however, found higher VEP acuity during the the first year of life, and higher scores on the Bayley scales of infant development at 18 months of age among term infants fed formula containing a fat blend with 0.36% 22:6n-3 compared with no 22:6n-3 and 1.49% 18:3n-3. VEP acuity was also higher in 12-month-old infants who were weaned from breastfeeding to formula with 22:6n-3 rather than unsupplemented formula at about 5 months of age (Birch et al., 2002). Higher scores on a three-step problem-solving task have also been reported for term infants fed a formula containing fat with 0.15–0.25% 22:6n-3 and 0.7% 18:3n-3 (Willatts et al., 1998). No evidence of altered growth was found in any of the latter studies with term infants fed formulas containing 22:6n-3 and 20:4n-6. The discrepancy among studies with term infants could involve the low 18:3n-3 content and thus dependence on 22:6n-3 in some of the formulas tested, addition of inadequate amounts of 22:6n-3 to see a positive effect, differences in the formula balance of 20:4n-6 to 22:6n-3, variability in the 22:6n-3 status of the infants at birth, age of the infants at testing, and lack of test sensititivity to detect biologically important differences in development. Recent studies have also addressed whether the variability in 22:6n-3 in human milk, and as a result in the diet and blood lipids of the breast-fed infant, is of physiological significance to infant development. Infants in the lowest tertile of RBC PE 22:6n-3 who received milk with 0.17% fat as 22:6n-3 had significantly lower visual acuity at 2 and 12 months of age than infants in the highest tertile of RBC PE 22:6n-3 who received milk with 0.31% 22:6n-3 (Innis et al., 2001; fig. 5). No relation was found between the infants’ 20:4n-6 or 22:6n-3 status and scores on the Bayley II mental or motor developmental indices, novelty preference assessed using the Fagan test, or on a standardized object search task (Piaget’s A not B). However, the infants’ 22:6n-3 status at 2 months of age was significantly related to the ability to discriminate a non-native (Hindi) retroflex and dental phonetic contrast at 9 months of age, and to language production and comprehension assessed with the CDI at 14 and 17 months of age, after adjusting for confounding variables (Innis et al., 2001; Innis, 2002). A significant association between sweep VEP acuity and human milk 22:6n-3 was also recently reported in a cross-sectional study of breast-fed infants (Jorgensen et al., 2001). These associations between 22:6n-3 and visual and neural development in breast-fed infants, while consistent with the essential role of 22:6n-3 in retina and brain function, cannot be interpreted as a demonstration of causality. This requires dietary intervention that modifies the maternal intake of 22:6n-3 but not other nutrients. However, newer research to show dependence of the fetal and infant 22:6n-3 on the maternal intake of 22:6n-3 raises important questions about the n-3 fatty acid requirements of pregnant and lactating women with respect to supporting optimal visual and neural development in the infant.

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Fig. 5. Relation of milk and infant red blood cell phosphatidylethanolamine (PE) 22:6n-3 at 2 months of age to visual acuity at 2 and 12 months of age in healthy term gestation infants. The bars represent mean visual acuity ± SD; * significantly different from infants in the lowest tertile of 22:6n-3. Adapted from Innis et al. (2001).

9. DIETARY ESSENTIAL FATTY ACID REQUIREMENTS The physiological effects of essential fatty acid deficiency have been well described, particularly in rats fed a fat-free diet and replenished with varying amounts of n-6 fatty acids (Mohrhauer and Holman, 1963; Holman, 1991; Innis, 1992; table 1). Studies in rodents have shown that a dietary intake of as low as 1–2% energy 18:2n-6 is sufficient to avoid any adverse effects on reproduction, gestation, perinatal mortality or growth. Careful doseresponse studies have shown that the minimum intake of 18:2n-6 required to sustain a constant level of 20:4n-6 in all organs (including brain, liver, lung, heart, kidney, muscle and adipose) is 2.4% of dietary energy (Bourre et al., 1990). A similar effect, with maintenance of normal growth, is achieved with a much smaller amount of 20:4n-6, of about 0.2% dietary energy (Mohrhauer and Holman, 1963). Similar studies on the requirement for n-3 fatty acids have shown that a dietary intake of 0.26% energy 18:3n-3 in adults and 0.4% energy 18:3n-3 in developing animals maintains 22:6n-3 in brain, liver, heart and other organs (Bourre et al., 1989a, 1993). There is no evidence that rodents require a dietary source of 22:6n-3 if fed a diet containing sufficient 18:3n-3. The requirement for n-3 fatty acids, however, can be met by small amounts of 22:6n-3 rather than conversion from 18:3n-3. Studies in young piglets have shown that an intake of <0.7% energy from 18:3n-3 is inadequate to support accretion of 22:6n-3 in neural tissues (Hrboticky et al., 1989, 1990, 1991). The accretion of 22:6n-3 was reduced when the diet of the neonatal piglet contained a ratio of 18:2n-6 to 18:3n-3 of 16:1 or higher, rather than 8:1 or lower (Arbuckle et al., 1992, 1994). Further understanding of the importance of the concurrent intake of 18:2n-6 to tissue accretion of 20:4n-6 and 22:6n-3 is needed. Higher intakes of 2% energy 18:3n-3 resulted in higher 22:6n-3 in the developing piglet brain (Arbuckle et al., 1992, 1994), compatible with studies showing postnatal desaturation and elongation of 18:3n-3 to 22:6n-3 in this species (Li et al., 2000). However, studies in young piglets also provide ample evidence that 22:6n-3, either in sows’ milk or in a milk replacer diet, is much more efficacious than 18:3n-3 in increasing 22:6n-3 in blood, liver, and brain and retina 22:6n-3 (Arbuckle et al., 1991; Arbuckle and Innis 1992, 1993; de la Presa Owens and Innis, 1999a,b). A dietary intake of as little as 0.15% energy as 22:6n-3 supports similar or higher brain and retina 22:6n-3 than a diet with 2% energy as 18:3n-3.

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Stable isotope tracer studies in pregnant and fetal baboons have also provided convincing evidence that conversion of 18:3n-3 to 22:6n-3 is much less efficient in supplying 22:6n-3 for incorporation into developing brain and other organs than preformed 22:6n-3 (Greiner et al., 1997; Su et al., 1999, 2001). The available data suggest that 90% and perhaps more of dietary 18:3n-3 undergoes β-oxidation, with the acetyl-CoA either further oxidized to generate ATP, or recycled for de novo synthesis of other lipids. Although the increase in brain and retina 22:6n-3 with increasing intakes of 22:6n-3 is much lower than the increase in 22:6n-3 in liver or plasma, high intakes of 20:5n-3 and 22:6n-3 have been shown to decrease visual function in guinea pigs, and to decrease auditory brainstem evoked potentials in neonatal rats (Weisinger et al., 1995; Stockard et al., 2000). Although the mechanism of these effects is not yet known, these findings suggest the need for dose-response studies to determine the safe upper limit of 22:6n-3 intake in developing animals. Studies in the 1960s identified skin lesions and growth failure among human infants fed skimmed cows’ milk, which provides minimal amounts of n-6 fatty acids (Hansen et al., 1958). Clinical and biochemical signs of n-6 fatty acid deficiency, many of which are related to the role of n-6 fatty acids in the skin, are not seen in human infants fed diets providing 18:2n-6 in amounts above recommended intakes of 3–4% energy as 18:2n-6 (FAO/WHO, 1993). Expert groups have suggested an acceptable range of intake of 18:2n-6 for preterm and term human infants of 3.2–12.8% energy (LSRO, 1998, 2001). The absence of skin signs, normal growth, and a triene/tetraene ratio of <0.2 in plasma lipids, however, ensures neither optimal 20:4n-6 nor balance between n-6 and n-3 fatty acids in developing tissues. Definitive information on n-6 fatty acid requirements that are based on functional endpoint indicators related to 20:4n-6 metabolism are largely lacking. Similarly, the n-3 fatty acid requirements of human infants are still unclear. The suggested acceptable range of 18:3n-3 intake for preterm and term infants is 0.7–2.1% of dietary energy (LSRO, 1998, 2001). The requirement for 18:3n-3, however, depends on the amount of 22:6n-3 provided to the infant. Recent clinical studies in premature infants suggest that in the absence of a dietary intake of 22:6n-3, an intake of 1.2% energy as 18:3n-3 does not meet the needs of the developing brain and retina (O’Connor et al., 2001). Another approach is to estimate a dietary intake likely to meet the needs for accretion, based on knowledge gained from autopsy tissue analyses. These analyses suggest that about 67 mg n-3 fatty acids (mostly 22:6n-3) and 552 mg n-6 fatty acids are accumulated per day in fetal tissue during the last trimester of gestation (Clandinin et al., 1981). Thus, an infant fed human milk or a milk substitute that provides the only dietary source of polyunsaturated fatty acids would need to recieve about 0.23% fat as 22:6n-3 and 2% fat as n-6 fatty acids, assuming an intake of 780 ml/day. Definitive data on the amount of dietary 18:3n-3 converted to 22:6n-3 during development are not available. Assuming this to be 10%, then the infant diet would need to contain at least 2.3% 18:3n-3 in order to meet the needs for n-3 fatty acids. Some expert groups have suggested a dietary intake of essential polyunsaturated fatty acids based on the amounts present in human milk. This is problematic because the essential fatty acid content of milk is greatly influenced by the amounts of n-6 and n-3 fatty acids in the maternal diet. The competition between 18:2n-6 and 18:3n-3 for desaturation has also led to recommendations for the n-6 to n-3 fatty acid in infant diets that are based on the composition of human milk. The total n-6 to total n-3 fatty acid ratio of human milk is generally in the range of 4:1 to 10:1 (Neuringer and Connor, 1986), but this ratio does not consider the differences in tissue handling and biological activity of 18:2n-6 and 20:4n-6, and 18:3n-3, 20:5n-3 and 22:6n-3. Dietary 22:6n-3 results in increased blood lipid 22:6n-3 in human infants, while the increasing dietary intake of 18:3n-3 has little effect. For example, the plasma phospholipids of infants fed formula with about 0.12% or 0% energy 22:6n-3 had 5.2 ± 0.2% and 2.0 ± 0.1% 22:2n-3,

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respectively, but infants fed formula with 0.4% or 2.4% 18:3n-3 and 0% 22:6n-3 had 2.3 ± 0.2% and 2.2 ± 0.3% 22:6n-3, respectively (Ponder et al., 1992; Innis et al., 1996). The increase in red blood cell and plasma phospholipid 22:6n-3 was lower in infants fed formula with 32% rather than 20% 18:2n-6 (Innis et al., 1996). The Food and Agriculture/World Health Organization recommend that term infants receive, per kg body weight, 600 mg 18:2n-6, 50 mg 18:3n-3, 40 mg 20:4n-6 and 20 mg 22:6n-3 per day, based on the amounts in human milk (FAO/WHO, 1993). Similarly, the Institute of Medicine of the National Academies of Medicine provides an adequate intake (AI) for infants of 0–12 months of 4.4–4.6 g/day of n-6 fatty acids and 0.5 g/day n-3 fatty acids, based on the intake of breast-fed infants (IOM, 2002). However, there is no evidence from clinical studies to indicate that these amounts exceed or meet the needs to maximize potential neural development in young infants (FAO/WHO, 1993).

10. CONCLUSIONS AND FUTURE PERSPECTIVES It is clear that essential fatty acid deficiency can lead to profound problems in growth, and functional disturbances in many organs including the developing central nervous system. Large gaps still exist in understanding the requirements, metabolism and functions of essential fatty acids, particularly the long-chain polyunsaturated fatty acids 20:4n-6 and 22:6n-3, during development. In particular, pathways and regulatory mechanisms involved in the transfer of 20:4n-6 and 22:6n-3 across the placenta and in milk, and the effect of the concurrent intake of 18:2n-6 in modulating fatty acid accretion in developing tissues, is incompletely understood. Likewise, much is yet to be learned regarding the role of n-6 and n-3 fatty acids in the molecular, biochemical and histological development of the central nervous system, and their relation to cognitive and behavioural impairments in developing animals and infants. Little information is available on the role of polyunsaturated fatty acids in the molecular and functional development of the immune system, intestine, bone, adipose tissue and many other organs; these areas afford considerable oportunities for new research. Future expert groups seeking to establish polyunsaturated fatty acid requirements in development will benefit from a greater understanding of species differences in essential fatty acid metabolism, and dose-response studies to elucidate the safe and adequate range and tolerable upper limit of intake of individual n-6 and n-3 fatty acids, and their appropriate balance in the diet. REFERENCES Abayasekara, D.R., Wathes, D.C., 1999. Effects of altering dietary fatty acid composition on prostaglandin synthesis and fertility. Prostagland. Leuk. Essent. Fatty Acids 61, 275–287. Adams, P.B., Lawson, S., Sanigorski, A., Sinclair, A.J., 1995. Arachidonic to eicosapentaenoic acid ratio in blood correlates positively with clinical symptoms of depression. Lipids 31, S157–S161. Ahmad, A., Moriguchi, T., Salem, N., 2002a. Decrease in neuron size in docosahexaenoic acid-deficient brain. Pediatr. Neurol. 26, 210–218. Ahmad, A., Murthy, M., Greiner, R.S., Moriguchi, T., Salem, N. Jr., 2002b. A decrease in cell size accompanies loss of docosahexaenoate in rat hippocampus. Nutr. Neurosci. 5, 103–113. Albert, C.M., Hennekens, C.H., O’Donnell, C.J., Ajani, U.A., Carey, V.J., Willett, W.C., Ruskin, J.N., Manson, J.E., 1998. Fish consumption and risk of sudden cardiac death. J. Amer. Med. Assoc. 279, 23–28. Alvarez, R.A., Aguire, G.D., Acland, G.M., Anderson, R.E., 1994. Docosapentaenoic acid is converted to docosahexaenoic acid in the retinas of normal and prcd-affected miniature poodle dogs. Invest. Ophthalmol. Vis. Sci. 35, 402–408. Anderson, G.J., Connor, W.E., 1988. Uptake of fatty acids by the developing rat brain. Lipids 23, 286–290.

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Sinclair, A.J., Crawford, M.A., 1972. The accumulation of arachidonate and docosahexaenoate in the developing rat brain. J. Neurochem. 99, 1753–1758. Sprecher, H., Chen, Q., Yin, F.Q., 1999. Regulation of the biosynthesis of 22:5n-6 and 22:6n-3, a complex intracellular process. Lipids 34, S153–S156. Sprecher, H., Luthria, D.L., Mohammed, B.S., Baykousheva, S.P., 1995. Reevaluation of the pathways for biosynthesis of polyunsaturated fatty acids. J. Lipid Res. 36, 2471–2477. Stockard, J.E., Saste, M.D., Benford, V.J., Barness, L., Auestad, J.D., 2000. Effect of docosahexaenoic acid content of maternal diet on auditory brainstem conduction times in rat pups. Dev. Neurosci. 22, 494–499. Stoll, A.L., Severus, W.E., Freeman, M.P., Rueter, S., Zboyan, H.A., Diamond, E.A., 1999. Omega-3 fatty acids in bipolar disorder, a preliminary double-blind, placebo-controlled trial. Arch. Gen. Psychiatry 56, 407–412. Su, H.M., Bernardo, M., Mirmiran, X.H., Ma, T.N., Corso, P.W., Nathanielsz, J.T., Brenna, J.T., 1999. Bioequivalence of dietary α-linolenate and docosahexaenoate acids as possible sources of docosahexaenoate accretion in brain and associated organs of neonatal baboons. Pediatr. Res. 45, 87–93. Su, H.-M., Huan, M.-C., Saad, N.M.R., Nathanielsz, P.W., Brenna, J.T., 2001. Fetal baboons convert 18:3n-3 to 22:6n-3 in vivo, a stable isotope tracer study. J. Lipid. Res. 42, 581–586. Suh, M., Wierzbicki, A.A., Lien, E., Clandinin, M.T., 1996. Relationship between dietary supply of long-chain fatty acids and membrane composition of long- and very long chain essential fatty acids in developing rat photoreceptors. Lipids 313, 61–64. Suh, M., Wierzbicki, A.A., Lien, E., Clandinin, M.T., 2000. Dietary 20:4n-6 and 22:6n-3 modulates the profile of long- and very-long-chain essential fatty acids, rhodopsin content, and kinetics in developing photoreceptor cells. Pediatr. Res. 48, 524–530. Thies, F., Delachambre, M.C., Bentejac, M., Lagarde, M., Lecerf, J., 1992. Unsaturated fatty acids esterified in 2-acyl-1-lysophosphatidylcholine bound to albumin are more efficiently taken up by the young rat brain than the unesterified from. J. Neurochem. 59, 1110–1116. Thies, F., Pillon, C., Moliere, P., Lagarde, M., Lecerf, J., 1994. Preferential incorporation of sn-2 lysoPC DHA over unesterified DHA in young rat brain. Amer. J. Physiol. 267, R1273–1279. Tinoco, J., 1982. Dietary requirements and functions of α-linolenic acid in animals. Prog. Lipid Res. 21, 1–45. Tinoco, J., Babcock, R., Hincenbergs, I., Medwadowski, B., Jiljanich, P., Williams, M.A., 1979. Linolenic acid deficiency. Lipids 14, 166–173. Tremoli, E., Maderna, P., Marangoni, F., Colli, S., Eligini, S., Catalano, I., Angeli, M.T., Pazzucconi, F., Gianfranceschi, G., Davi, G., 1995. Prolonged inhibition of platelet aggregation after n-3 fatty acid ethyl ester ingestion by healthy volunteers. Amer. J. Clin. Nutr. 61, 607–613. Uauy, R., Birch, D.G., Birch, E.E., 1992. Retinal development in very low birthweight infants fed diets differing in omega-3 fatty acids. Pediatr. Res. 28, 485–492. Uauy, R., Mena, P., Wegher, B., Nieto, S., Salem, N. Jr., 2000. Long chain polyunsaturated fatty acid formation in neonates, effect of gestational age and intrauterine growth. Pediatr. Res. 47, 127–135. Vance, D.E., 1990. Phosphatidylcholine metabolism, masochistic enzymology, metabolic regulation, and lipoprotein assembly. Biochem. Cell. Biol. 68, 1151–1165. van Houwelingen, A.C., Sorensen, J.D., Hornstra, G., Simonis, M.M., Boris, J., Olsen, S.F., Secher, N.J., 1995. Essential fatty acid status in neonates after fish-oil supplementation during late pregnancy. Brit. J. Nutr. 74, 723–731. von Schacky, C., Weber, P.C., 1985. Metabolism and effects on platelet function of the purified eicosapentaenoic and docosahexaenoic acids in humans. J. Clin. Invest. 76, 2446–2450. Voss, A., Reinhart, M., Sankarappa, S., Sprecher, H., 1991. The metabolism of 7,10,13,16, 19-docosapentaenoic acid to 4,7,10,13,16,19-docosahexaenoic acid in rat liver is independent of a 4-desaturase. J. Biol. Chem. 226, 19995–20000. Wainwright, P.E., Xing, H.C., Girard, T., Parker, L., Ward, G.R., 1998. Effects of dietary n-3 fatty acid deficiency on Morris water-maze performance and amphetamine conditioned place preference in rats. Nutr. Neurosci. 1, 281–293. Wanders, R.J., Vreken, P., Ferdinandusse, S., Jansen, G.A., Waterham, C.W., Van Grunsven, E.G., 2001. Peroxisomal fatty acid α- and β-oxidation in human enzymology, peroxisomal metabolite transporters and peroxisomal diseases. Biochem. Soc. Trans. 29, 250–267.

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Wang, N., Anderson, R.E., 1993. Synthesis of docosahexaenoic acid by retina and retina pigment epithelium. Biochemistry 32, 13703–13709. Ward, G.R., Huang, Y.S., Bobik, E., Xing, H.C, Mutsaers, L., Auestad, N., Montalto, M., Wainwright, P., 1998. Long-chain polyunsaturated fatty acid levels in formulae influence deposition of docosahexaenoic acid and arachidonic acid in brain and red blood cells of artificially reared neonatal rats. J. Nutr. 128, 2473–2487. Washizaki, K., Smith, Q.R., Rapoport, S.I., Purdon, A.D., 1994. Brain arachidonic acid incorporation and precursor pool specific activity during intravenous infusion of unesterified [3H]arachidonate in the anesthetized rat. J. Neurochem. 63, 727–736. Weiler, H.A., Fitzpatrick-Wong, S., 2002. Dietary long-chain polyunsaturated fatty acids minimize dexamethasone-induced reductions in arachidonic acid status but not bone mineral content in piglets. Pediatr. Res. 51, 282–289. Weisinger, H.S., Vinigrys, A.J., Bui, B.V., Sinclair, A.J., 1999. Effects of dietary n-3 fatty acid deficiency and repletion in the guinea pig retina. Invest. Ophthalmol. Vis. Sci. 40, 327–328. Weisinger, H.S., Vingrys, A.J., Sinclair, J.J., 1995. The effect of docosahexaenoic acid on the electroretinogram of the guinea pig. Lipids 31, 65–70. Wertz, P.W., Swartzendruber, D.C., Abraham, W., Madison, K.C., Downing, D.T., 1987 Essential fatty acids and epidermal integrity. Arch. Dermatol. 123, 1381–1384. Wheeler, T.G., Benolken, R.M., Anderson, R.E., 1975. Visual membranes, specificity of fatty acid precursors for the electrical response to illumination. Science 188, 1312–1314. Wiggins, R.C., 1986. Myelination, a critical stage in development. Neurotoxicology 7, 103–120. Willatts, P., Forsyth, P., Forsyth, J.S., DiModugno, M.K., Varna, S., Colvin, M., 1998. Effect of longchain polyunsaturated fatty acids in formula on infant problem solving at 10 months of age. Lancet 352, 688–691. Williard, D., Harmon, S., Kaduce, T., Spector, A., 2002. Comparison of 20-, 22-, and 24-carbon n-3 and n-6 polyunsaturated fatty acid utilization in differentiated rat brain astrocytes. Prostagland. Leuk. Essent. Fatty Acids 67, 99–104. Yamamoto, N., Hashimoto, A., Takemoto, Y., Okuyama, H., Nomura, M., Kitajima, R., Togashi, T., Tamai, Y., 1988 Effects of the dietary α-linoleate/linoleate balance on lipid compositions and learning ability of rats. II. Discrimination process, extinction process, and glycolipid compositions. J. Lipid Res. 29, 1013–1021. Yamazaki, K., Fujikawa, W., Hamazaki, T., Yano, S., Shono, T., 1992. Comparison of the conversion rates of α-linolenic acid (18,3(n - 3)) and stearidonic acid (18,4(n - 3)) to longer polyunsaturated fatty acids in rats. Biochim. Biophys. Acta 1123, 18–26. Yonekubo, A., Honda, S., Okano, M., Yamamoto, Y., 1993. Effects of dietary safflower oil or soybean oil on the milk composition of the maternal rat, and tissue fatty acid composition and learning ability of postnatal rats. Biosci. Biotech. Biochem. 57, 253–259. Yoshida, S., Yasuda, A., Kawazato, K., Sakai, K., Shimada, T., Takeshita, Y., Yuasa, S., Kobayashi, T., Watanabe, S., Okuyama, H., 1997. Synaptic vesicle ultrastructural changes in the rat hippocampus induced by a combination of α-linolenate deficiency and a learning task. J. Neurochem. 68, 1261–1268. Young, C., Gean, P., Wu, S., Lin, C., Shen, Y., 1998. Cancellation of low-frequency stimulationinduced long-term depression by docosahexaenoic acid in the rat hippocampus. Neurosci. Lett. 247, 198–200. Youyou, A., Durand, G., Pascal, G., Piciotti, M., Dumont, O., Bourre, J.M., 1986. Recovery of altered fatty acid composition induced by a diet devoid of n-3 fatty acids in myelin, synaptosomes, mitochondria and microsomes of developing rat brain. J. Neurochem. 46, 224–228. Ziboh, V.A., Chapkin, R.S., 1988. Metabolism and function of skin lipids. Prog. Lipid Res. 27, 81–105. Zimmer, L., Breton, P., Durand, G., Guilloteau, D., Besnard, J.C., Chalon, S., 1999. Prominent role of n-3 polyunsaturated fatty acids in cortical dopamine metabolism. Nutr. Neurosci. 2, 257–265. Zimmer, L., Delion-Vancassel, S., Durand, G., Guilloteau, D., Bodard, S., Besnard, J.C., Chalon, S., 2000a. Modification of dopamine neurotransmission in the nucleus accumbens of rats deficient in n-3 polyunsaturated fatty acids. J. Lipid Res. 41, 32–40. Zimmer, L., Delpal, S., Guilloteau, D., Aioun, J., Durand, G., Chalon, S., 2000b. Chronic n-3 polyunsaturated fatty acid deficiency alters dopamine vesicle density in the rat frontal cortex. Neurosci. Lett. 284, 25–28.

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11

Development of white adipose tissue lipid metabolism1 H. J. Mersmanna and S. B. Smithb a USDA/ARS

Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA b Department of Animal Science, Texas A & M University, College Station, TX 77843-2471, USA

Most mammals are born with little white adipose tissue; however, the guinea pig and human are exceptions. Limited adipose tissue places the newborn at risk when confronted with environmental challenges such as cold temperatures or limited milk supply. White adipose tissue develops rapidly after birth in those species with limited depots. Adipose tissue growth is a combination of cell proliferation coupled with differentiation and cell hypertrophy. Proliferation is a property of the undifferentiated preadipocyte and is predominant in neonatal development. It continues at a lower rate to accommodate growth and replace cells, but can be activated, even in adults, when caloric intake is excessive and continuous. Preadipocytes differentiate into adipocytes with subsequent growth of the adipocyte, increase in mass of the tissue being the result of accumulation of triacylglycerol in a large central intracellular lipid droplet. Glucose is the carbon precursor of fatty acid synthesis in adipocytes from nonruminant mammals, whereas acetate is the carbon precursor in ruminant species. The human has little or no capacity for adipocyte de novo fatty acid synthesis. Because milk is a high-fat food, there is little fatty acid synthesis during the suckling period. De novo fatty acid synthesis is primarily a process of importance in the postweaning mammal. Triacylglycerol is carried in lipoproteins and these are cleaved by lipoprotein lipase to yield fatty acids that are readily absorbed by adipocytes. Fatty acids are esterified in the adipocyte to triacylglycerol. The lipoprotein lipase and triacylglycerol biosynthetic activities increase rapidly after birth to allow uptake and esterification of fatty acids. The newborn mammal also needs fatty acids

1 This work is a publication of the USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas. This project has been funded in part with federal funds from the USDA/ARS under Cooperative Agreement No. 58-6250-6001. The contents of this publication do not necessarily reflect the views or policies of the USDA, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

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for an oxidative fuel. The source of these fatty acids is from the diet and immediately from circulating lipoproteins coupled with fatty acids released from the adipocyte by lipolysis, the breakdown of adipocyte triacylglycerol.

1. INTRODUCTION There are two types of adipose tissue in mammals, white and brown. The brown adipocyte is a specialized cell for generation of heat. It has multiple lipid droplets, i.e. stores of triacylglycerol (TG), but also a large number of mitochondria to oxidize long-chain fatty acids (FAs) mobilized from TG. Brown adipose tissue is present in most mammalian newborns. It is strategically located in the body, i.e. in the thorax and near the kidneys, to provide heat to essential organs, by FA mobilization and inefficient oxidation. Brown adipose tissue properties, developmental patterns, and functions are discussed by Smith and Carsten (Chapter 12). The newborn pig is different from most newborn mammals in that it has no brown adipose tissue. The white adipocyte is characterized by a large central lipid droplet that is the repository for storage of energy in the form of TG containing esterified FAs. Most mammalian species are born with little white adipose tissue (<5% of the body weight at birth). Most mammalian fetuses have a limited capacity to synthesize fatty acids de novo and the passage of FAs across the placenta is highly restricted (Noble, 1981; Martin et al., 1985; Hamosh, 1998; Kimura, 1998; Herrera, 2002). However, the guinea pig and human are born with 10% and 15% body fat (Bonnet, 1981; Widdowson and Lister, 1991); in these species, de novo FA synthesis increases markedly during the last trimester of pregnancy. White adipose tissue has multiple functions, but in the neonate, the two most important are for insulation, primarily provided by the subcutaneous fat depots, and as a repository for energy storage to be mobilized when the nutrient intake does not provide the required energy. The uterine environment provides shelter in the way of temperature control and provision of nutrients, both of which are lost at parturition. Mammalian newborns are generally in a precarious position if they are challenged by the environment, e.g. cold exposure, or if they cannot obtain sufficient nutrients by way of the diet, i.e. by suckling. The newborn pig is at a particular disadvantage because it has <2% body fat limiting the insulation and energy supplies, the FA mobilization and oxidation capacity is reduced, there is very little hair (for insulation), and there is no brown adipose tissue (Mersmann, 1974). The neonatal period is characterized by a multitude of metabolic changes as the organism adapts to the many challenges of the environment. The exact timing of a particular adaptation varies among species, but these changes occur during the window of time between the last days of gestation and the first days or weeks postpartum. More extensive discussions of metabolism in the neonatal pig are found in Chapter 14 by Herpin et al. and Chapter 9 by Odle et al. The distribution of adipose tissue is different in different species, as are the growth rates for the individual depots (Berg and Walters, 1983; Trenkle and Marple, 1983). For example, the predominant adipose tissue depot in the pig is the subcutaneous depot, with lesser fat deposition at the perirenal, mesenteric/omental, and intermuscular sites (Walstra, 1980; Kauffman et al., 1986; Kouba et al., 1999; Mitchell et al., 2001). In sheep, the subcutaneous adipose tissue depot is large, but the intermuscular depot is almost as large and the omental depot is about 50% of the subcutaneous depot (Moloney et al., 2002). In cattle, the subcutaneous depot is large, particularly in breeds that are selected for muscle production and that fatten readily, whereas in breeds with accentuated lactation rates, the internal fat depots are more extensively developed (Truscott et al., 1983). The metabolic activity and the differentiation of individual adipose tissue depots may be markedly different (Adams et al., 1997; Wajchenberg, 2000).

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We describe the development of white adipose tissue, and the key metabolic pathways that provide the primary functions of adipose tissue, the storage and mobilization of energy. There are several major reviews of the literature regarding these subjects in cattle, pigs, and sheep (Allen et al., 1976; Vernon, 1980, 1981; Mersmann, 1986; Smith and Smith, 1995).

2. DEVELOPMENT OF WHITE ADIPOSE TISSUE A single large central lipid droplet occupying much of the volume of the cell characterizes white adipocytes. The functional cytoplasm and nucleoplasm are pushed to the periphery; thus, the relative space occupied by the cytoplasm and nucleus shrinks as the cell stores more lipids. Adipocytes can become extremely large at full development, reaching sizes greater than almost any cell in the body. The diameter of completely expanded adipocytes is in excess of 100 μm in most mammalian species and can be 200–500 μm in pigs and cattle. 2.1. Hyperplasia The expansion of adipose tissue depots involves an increase in cell number coupled with an increase in the mass of individual adipocytes. The increase in cell number is a major factor in the increase of mammalian adipose tissue in young mammals (Allen, 1976). Ultimately, the cell number sets limits on the absolute mass of adipose tissue because differentiated adipocytes do not divide. The hyperplastic process is characteristic of the preadipocyte, the precursor cell that has not differentiated and begun to fill with TG; it is controlled by numerous factors including age, depot site, sex, and endocrine and growth factors (Hausman et al., 2001). In most adult mammals, the direct contribution of hyperplasia to an increase in the mass of adipose tissue is limited. A low rate of hyperplasia and differentiation is required for cell replacement during the entire life of the organism. However, after the major increase in cell number early in the life of a mammal, substantial hyperplastic rates are only measured, in most species, when the organism is exposed to excessive caloric intake. The increase in the size of an individual adipocyte by accumulation of lipid is limited. When some percentage of the adipocytes reach the size limits for that species, hyperplasia is increased to provide additional preadipocytes that can differentiate and fill with lipid to expand the depot. This has clearly been demonstrated in rodents where several adipose tissue depots (perirenal, perigonadal, inguinal) can be dissected in toto to enable determination of the entire number of cells per depot. Also, DNA synthesis can be determined readily in these small animals in individual depots. If the animals are fed excess energy for an extended period, hyperplasia is increased and is readily demonstrated (DiGirolamo and Mendlinger, 1971; Greenwood and Hirsch, 1974; Miller et al., 1984; Shillabeer and Lau, 1994). Hyperplasia and/or DNA synthesis are not readily quantified in larger mammals where the expense of measuring DNA synthesis is considerable and the individual depots are extremely difficult to remove quantitatively, limiting the capability for determination of cell number in the entire depot (Gurr and Kirtland, 1978). Attempts to measure total adipocyte number have been reported for depots in cattle and sheep (e.g. Hood and Allen, 1973b; Robelin, 1981; Vernon, 1986) and pigs (e.g. Anderson et al., 1972; Enser et al., 1976; Hood and Allen, 1977; Desnoyers et al., 1980; Hauser et al., 1997), as well as subcutaneous adipose tissue from specific portions of thoracic rib sections in pigs (Demaree et al., 2002) and cattle (Schiavetta et al., 1990). Also, DNA synthesis was demonstrated in young pigs (Gurr et al., 1977; Hausman and Kauffman, 1986a), and in subcutaneous adipose tissue explants from mature cattle (May et al., 1994). Measurement of cell number in

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a portion of an adipose tissue depot may not reflect changes in cell number in the entire depot because cell number in a small sample from a large depot, with expression of number per g tissue, may reflect changes in cell size and not hyperplasia. Likewise, changes in DNA synthesis in explants may not reflect hyperplasia in the entire depot. 2.2. Differentiation Adipocytes develop from precursor cells, preadipocytes. Preadipocytes can be prepared from digested adipose tissue and are obtainable from both young and older mammals. However, the concentration of preadipocytes is greater in neonatal mammals because, at this time, there is rapid cell division to provide preadipocytes to differentiate and fill with lipids to provide expansion of the adipose tissue depots. The stromal–vascular cell fraction, isolated from digested adipose tissue, contains numerous cell types, e.g. fibroblasts, reticulocytes, endothelial cells, blood cells, and preadipocytes. When plated with the appropriate medium, most of the attached cells appear as elongated, fibroblastic-like cells. Cell replication is rapid in the proper culture medium with serum being an important component. There are no morphological indications that any of these cells are preadipocytes. However, there are proteins characteristic of the preadipocyte, e.g. Pref 1 (Gregoire et al., 1998; Gregoire, 2001), and antigenic materials detectable in the preadipocyte that are specific to the adipocyte (Lee et al., 1986; Wright and Hausman, 1990; Cryer et al., 1992; Yu et al., 1997). When the appropriate growth factors and hormones are provided, many of the cells in the stromal–vascular fraction differentiate into adipocytes, i.e. they begin to deposit large amounts of lipid. The amount of differentiation seems to depend on the exact culture conditions. For example, if porcine stromal–vascular cells are differentiated in serum, the extent of differentiation (the total number of differentiated cells) is limited and differentiation occurs in clusters of cells. If the medium does not contain serum, differentiation is more uniform across the culture plate, i.e. there are no clusters, and the extent of differentiation is greater than in the presence of serum. In clonal lines of preadipocytes, derived from rodents, the extent of differentiation is extensive (80% to >90%), even when serum is present. Probably, there are species differences, as well as differences between the clonal cells and the primary preadipocytes directly derived from the adipose tissue stromal–vascular fraction. Most of the concepts about adipocyte development come from study of clonal cells coupled with a few studies of primary preadipocytes isolated from rodents. Primary preadipocytes that differentiate in culture under the appropriate conditions have been prepared from many species (Novakofski and Hu, 1987; Suryawan and Hu, 1995), including cattle (Plaas and Cryer, 1980; Cryer et al., 1984; Aso et al., 1995; Ohyama et al., 1998; Torii et al., 1998; Peixing et al., 2000; Wu et al., 2000), pigs (Hausman et al., 1984; Suryawan and Hu, 1993; Boone et al., 2000), rats (Bjorntorp et al., 1980), and sheep (Broad and Ham, 1983; Vierck et al., 1996; Soret et al., 1999; Arana et al., 2002), as well as humans (Hauner et al., 1989). The most extensive studies using preadipocytes isolated from a domestic species are with porcine preadipocytes with an overwhelming contribution by Hausman and coworkers. The distinguishing morphological feature of a cell beginning to differentiate into an adipocyte is the deposition of small lipid droplets. Seldom do normal cells deposit more than a very few small lipid droplets. In areas where adipose tissue is developing, elongated, fibroblastic-like cells can be observed with multiple small lipid droplets. As the cells accumulate more lipids, the number of droplets increases and droplets fuse to form larger droplets. The adipocytes with multiple lipid droplets are termed multilocular. Eventually the cell will contain a few large lipid droplets and as lipid deposition continues, fusion of lipid droplets will lead to a cell with

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a very large central lipid droplet, a unilocular adipocyte. Unilocular adipocytes also contain many small lipid droplets confined to the peripheral cytoplasmic space. These stages of adipocyte development are readily observed in the neonatal period, either before or after birth, depending on the species (Napolitano, 1963; Slavin, 1985; Cinti, 2001); the pig has been extensively studied (Mersmann et al., 1975; Hausman and Richardson, 1982; Hausman and Kauffman, 1986b). Coupling of cell culture systems and molecular biology techniques has led to a model for differentiation of preadipocytes to adipocytes (fig. 1). The events that characterize the differentiation from the totipotent stem cell to the multipotent mesenchymal precursor cell to the committed preadipocyte remain largely unknown. However, given a cell that is committed to become an adipocyte, i.e. a preadipocyte, a relatively clear series of sequential events seems to occur. The exact timing of the events may vary with the species from which the preadipocyte was derived and with the exact conditions used for cell culture. Preadipocytes grown in serum without the appropriate adipogenic factors continue to multiply with essentially no differentiation. When preadipocytes are presented with the proper stimuli, i.e. an appropriate combination of growth factors and hormones, the cells begin to differentiate. The factors needed to initiate differentiation vary with the cell type and/or the species, e.g. the clonal 3T3-L1 cells require insulin, a glucocorticoid, and a cAMP-phosphodiesterase inhibitor, whereas the related clonal cell, 3T3-F442A, requires only insulin. All cells require insulin, most require a glucocorticoid, many require a phosphodiesterase inhibitor, some require thyroid hormone, etc. (Hausman et al, 1989, 1993; Ramsay et al., 1989; Cryer et al., 1992; Jump and MacDougald, 1993). Porcine primary preadipocytes do not require thyroid hormone or a phosphodiesterase inhibitor; serum impedes the extent of differentiation but does not stop it (Hentges and Hausman, 1989; Suryawan and Hu, 1993). Porcine preadipocyte differentiation is suppressed by serum, but if oleic acid is added to the serum-containing medium, almost every cell in the culture differentiates (McNeel and Mersmann, unpublished data). It is important to recognize that the exact culture conditions dictate the extent of differentiation observed. An initial differentiation event is the decreased expression of several genes characteristic of the preadipocyte. This is followed by an increase in transcription factors that guide the development of the adipocyte phenotype (fig. 1). There are multiple reviews of adipocyte

Fig. 1.

Simplified model for adipocyte differentiation.

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differentiation (e.g. Smas and Sul, 1995; Brun et al., 1996; Loftus and Lane, 1997; Fajas et al., 1998; Morrison and Farmer, 2000; Ntambi and Young-Cheul, 2000; Rangwala and Lazar, 2000; Gregoire, 2001). Early in differentiation, there is an increase in the transcription factors CCAAT-enhancer binding proteins beta and delta (C/EBPβ and C/EBPδ). This is followed by an increase in another transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), and finally by an increase in C/EBPα. The active form of the PPARγ transcription factor is as a heterodimer with retinoid x receptor alpha (RXRα). This heterodimer must be activated by the binding of an appropriate ligand. Fatty acids are potential ligands that stimulate differentiation (Schoonjans et al., 1996; Kliewer et al., 1997). The transcription factor, adipocyte determination and differentiation-dependent factor 1 (ADD1), plays a key role in inducing both PPARγ and fatty acid synthase. The provision of FAs by synthase provides potential ligand for activation of PPARγ-RXRα (Kim and Spiegelman, 1996). The process of transformation to the adipocyte phenotype, a cell that is primarily geared to synthesize and mobilize lipid, is guided primarily by PPARγ and C/EBPα (Castillo et al., 1999; Lane et al., 1999; Lazar, 1999). A number of genes that characterize the adipocyte phenotype have response elements in their promoter region that bind either PPARγ or C/EBPα. Binding of the appropriate transcription factor activates the transcription of that gene (fig. 1). In most cases, an increase in the transcript for a gene, i.e. the mRNA, results in activation of the translation process with a resultant increase in the protein. The proteins that characterize the adipocyte each have a unique chronological pattern during differentiation, e.g. lipoprotein lipase (LPL) appears early in differentiation, whereas adipocyte fatty acid binding protein (aP2) or glucose transporter 4 (Glut 4) appear later (fig. 2). Clonal cells, which have been used to develop this model, appear to differentiate more synchronously than primary cells so that the chronological patterns may be more clearly observed with clonal cells. Also, there is evidence that primary preadipocytes are somewhat further along in development at isolation than the clonal preadipocytes. For example, porcine primary adipocytes have substantial concentrations of both the mRNA (Ding et al., 1999) and the protein (Kim et al., 2000) for PPARγ, whereas this

Fig. 2. Development of adipocyte transcripts in dorsal subcutaneous adipose tissue obtained from the neck region of milk-fed neonatal pigs. Data adapted from Ding et al. (1999) and McNeel et al. (2000).

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transcription factor is undetectable or present at very low concentration in clonal preadipocytes (Chawla et al., 1994; Tontonoz et al., 1994). The pattern of development of the mRNAs for numerous adipocyte transcription factors and adipocyte-characteristic genes has been documented in porcine primary preadipocytes differentiating in vitro (Ding et al., 1999; McNeel et al., 2000). Also, the pattern for development of the proteins for the key transcription factors, PPARγ (Kim et al., 2000) and the C/EBPs (Lee et al., 1998; Yu and Hausman, 1998; Chen et al., 1999), have been described for the differentiating porcine preadipocyte. It is important to emphasize that the culture conditions may modify the chronological pattern observed, even with clonal cells. It is difficult to study adipocyte differentiation in vivo because a tissue sample contains cells in many stages of differentiation. The pig offers a modestly reasonable model in vivo because at birth, there are many preadipocytes present, essentially all adipocytes are multilocular, and there is rapid transformation of many cells to the unilocular stage over a very few days. The tissue is undoubtedly more heterogeneous than clonal preadipocytes, or even primary preadipocytes differentiating in culture. However, development of several transcripts that characterize adipocyte differentiation or the adipocyte phenotype (Ding et al., 1999; McNeel et al., 2000), follow a pattern that is approximately the same as differentiation of porcine preadipocytes in culture (fig. 2). 2.3. Hypertrophy After differentiation of the adipocyte, most of the growth of the cell and, consequently, the tissue is by hypertrophy, which is the lipid filling process. Thus, the average adipocyte size in a given depot increases as the mammal grows, as indicated for ruminants (Hood and Allen, 1973b; Allen, 1976; Hood and Thornton, 1979; Hood, 1982; Robelin, 1986; Vernon, 1986) and pigs (Anderson and Kauffman, 1973; Mersmann et al., 1973c; Hood and Allen, 1977; Desnoyers et al., 1980; Hausman, 1985; Hauser et al., 1997). Adipocytes have flattened sides in vivo, but the shape approximates a sphere. Thus, the diameter is a reasonable expression of the cell size. It must be recognized that as the diameter doubles (e.g. from 20 to 40 μm), the volume increases 8 times (from 4200 μm3 to 33,500 μm3). Measurement of average cell size with extrapolation to calculate the number of cells packaged in a defined depot, e.g. in a rodent depot, is not unreasonable. However, extrapolation of the average cell size from a small sample obtained from a large mammal is probably not very valid because it is difficult to quantitatively dissect the depot. This is particularly applicable to the generally contiguous subcutaneous depot and the mesenteric depot, scattered within the entire gut mesentery. Furthermore, a single size determination on a small sample from a large depot is probably invalid because the cell size is not expected to be uniform over the entire depot. The composition of adipose tissue changes with growth (fig. 3). Most of the changes are attributable to the extensive accumulation of intracellular TG resulting in an increase in adipocyte size. Consequently, the TG expressed per g tissue increases with adipose tissue growth. In species of mammals with a marked increase in adipocyte size, the TG concentration easily reaches 700 mg per g tissue and may approach 900 mg per g tissue. Because the individual cells are increasing in size, the number of cells per g tissue is concomitantly decreasing. Consequently, the DNA and protein expressed per g tissue also decreases. The exact chronological pattern for these changes will depend on the species, the capacity of the adipocyte to fill with lipid, the nutritional status of the animals, and the particular tissue components measured. To a large extent, adipose tissue growth is governed by the caloric intake of the animal. Excess energy is deposited as fat. Thus, limits on energy intake limit the growth of adipose tissue.

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Fig. 3. Composition of porcine dorsal subcutaneous adipose tissue obtained from the neck region of suckling pigs until day 20, and from pigs weaned at day 21 and fed a low-fat grain-based diet. The 100% values are: triacylglycerol (TG) = 900 mg/g tissue; DNA = 1.9 ng DNA phosphorus/g tissue; protein = 71.6 mg/g tissue; collagen = 35.8 mg/g tissue. Data adapted from Mersmann et al. (1973c).

This is primarily the result of limits on adipocyte hypertrophy, but as indicated earlier, when caloric intake is elevated chronically, there is usually an increase in cell hyperplasia. Recent study of adipocyte biology indicates that the adipocyte is an endocrine cell exporting numerous peptides that have the potential to modify the biological function of the adipocyte and other tissues (Hwang et al., 1997; Kim and Moustaid-Moussa, 2000; Fruhbeck et al., 2001; Trayhurn and Beattie, 2001). The first factor discovered was leptin, a cytokinelike peptide that is made and secreted by the adipocyte. There are leptin receptors in the hypothalamus that regulate feeding behavior. Thus, as adipocytes grow and the amount of adipose tissue increases, the amount of leptin secreted to the blood increases. The increased leptin signals the brain to decrease feed intake. There are leptin receptors in other tissues, including adipocytes themselves. Leptin decreases adipocyte lipid anabolism and increases lipid catabolism in porcine adipocytes (Ramsay, 2001), but not in ovine adipocytes (Newby et al., 2001).

3. DEVELOPMENT OF LIPID SYNTHESIS The overall pathways for adipocyte lipid synthesis and degradation were understood decades ago (Bauman, 1976); a simplified scheme is indicated in fig. 4. Glucose or acetate are obtained from the plasma, transformed to acetyl-CoA, then synthesized to FAs. The degradation of lipoproteins by lipoprotein lipase (LPL) provides an additional source of FAs. The FAs are esterified to TG, the major storage lipid. Degradation of TG occurs by lipolysis, yielding 3 FAs and glycerol. The overall development of these pathways (fig. 5) indicates that LPL activity and TG synthesis develop rapidly after birth in the pig during the suckling period when fat deposition is rapid using milk fat as a source of FAs. The degradative activity also develops rapidly after birth to provide energy during times of environmental stress or infrequent feeding. De novo synthesis of FAs does not substantially increase until after weaning.

Fig. 4. Simplified depiction of adipocyte lipid anabolic and catabolic lipid metabolism. Abbreviations used are: AcCC = acetyl-CoA carboxylase; β-oxidation = mitochondrial oxidation of fatty acids; FA = long-chain fatty acid; FAS = fatty acid synthase; lipolysis = sequential degradation of TG to 3 FAs + glycerol; LPL = lipoprotein lipase; OAA = oxaloacetate; P = phosphate; TCA cycle = mitochondrial tricarboxylic acid cycle; TG = triacylglycerol.

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Fig. 5. Ontogeny of porcine dorsal subcutaneous adipose tissue lipid metabolism. Weaning was at 21 days postpartum with pigs fed a low-fat diet after weaning. The 100% data are: LPL (lipoprotein lipase) = 6.6 μmol fatty acid released/60 min/106 cells; FA (de novo fatty acid synthesis from glucose) = 661 nmol glucose incorporated into total lipid/60 min/106 cells; TG (triacylglycerol + diacylglycerol + phospholipid synthesis from glycerol 3-phosphate) = 1.9 μmol glycerophosphate incorporated/60 min/106 cells; lipolysis = 285 μmol fatty acid released/60 min/106cells. Data adapted from: LPL (Steffen et al., 1978); FA (Mersmann et al., 1973c); TG (Steffen et al., 1979); lipolysis (Mersmann et al., 1976).

Lipid and glucose metabolism in various tissues undergo multiple adaptations during the perinatal, suckling, and weaning periods (Girard et al., 1992), as well as during postweaning growth (Saggerson, 1985; Vernon, 1992). There are extensive reviews of the early literature about ruminant (Vernon, 1980, 1981; Noble, 1981) and porcine (Mersmann, 1986; Farnsworth and Kramer, 1987) adipocyte lipid metabolism, including developmental aspects. Adipose tissue lipid metabolism, including influences such as genetics, temperature, or diet on its development, continues to be of interest (Bass et al., 1990; Wood, 1990; Chilliard, 1993) with emphasis on pigs (Le Dividich et al., 1994; Mourot et al., 1995, 1996; Camara et al., 1996; Boone et al., 1999; Gerfault et al., 2000; McNeel and Mersmann, 2000; Robert et al., 2000), cattle (Mendizabal et al., 1999), goats (Bas, 1992), and lambs (e.g. Vernon, 1982; Mendizabal et al., 1997; Purroy et al., 1997; Soret et al., 1998; Payne, 1999; Greathead et al., 2001). 3.1. Sources of fatty acids The supply of FAs to the organism is from the diet and from de novo FA synthesis. The direct source of FAs for the adipocyte is lipoproteins circulating in the blood plasma, and in some species, from de novo synthesis in the adipocyte. Developmental aspects of plasma lipoproteins in cattle and pigs have been reviewed (Chapman and Forgez, 1985). The major lipoprotein sources of FAs for the adipocyte are chylomicrons, the very large, heavily lipid-laden particles produced in the intestine after ingestion of a fatty meal and the very low-density lipoproteins (VLDL). Ruminant species do not produce chylomicrons per se due to their low level of fat intake. Rather, they export VLDL (associated with apolipoprotein B48) from the intestinal mucosal cells (see Chapter 13 by Drackley).

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Chylomicrons and VLDL have a high concentration of TG, but the adipocyte does not directly absorb TG. The TG is cleaved at the endothelial cell surface by the adipocyteproduced enzyme, LPL, and the products of this reaction, the FAs and 2-monoacylglycerols, can be moved to the adipocyte and absorbed. Fatty acids are quickly processed in the cell by a variety of mechanisms because the nonesterified and unbound FA is a detergent and potentially quite toxic to the cell. Formation of the coenzyme A thioester is one pathway for further processing of FAs. Once the CoA derivative is formed (acyl-CoA), the FA can be oxidized or used for various biosynthetic purposes, including synthesis of phospholipids, cholesterol esters, and TG. Nonesterified FAs are bound to several proteins that have specific binding sites for FAs. In the blood plasma, almost all of the nonesterified FA is bound to albumin, whereas in the cell, there are specific FA binding proteins, among which is adipocyte fatty acid binding protein (aP2). Fatty acid binding has been demonstrated in adipose tissues of pigs, sheep, and cattle (St. John et al., 1987; Coleman et al., 1988; Miller et al., 1988). In cattle and sheep, depression of de novo fatty acid synthesis is accompanied by depressions in fatty acid binding protein activity (Coleman et al., 1988; Miller et al., 1988). 3.2. Synthesis of FA (fig. 4) Synthesis of FAs from nonlipid sources occurs in the adipocyte of many, but not all, mammalian species (Vernon and Clegg, 1985; Vernon and Taylor, 1986). Glucose or closely related sugars derived from carbohydrates are the usual precursors of FA carbon in nonruminant species. Lactate and the carbon skeletons derived from nonessential amino acids by transamination may each be important lipogenic precursors, under some circumstances. The ultimate donor molecule for FA synthesis is the two-carbon donor, acetyl-CoA. Glycolytic metabolism of glucose to pyruvate is followed by pyruvate entry into the mitochondrion with subsequent decarboxylation to acetyl-CoA. The mitochondrial acetyl-CoA is not used for FA synthesis because the enzymatic machinery for FA synthesis is located in the cytosol. The acetyl-CoA does not traverse the mitochondrial membrane, but is coupled with the fourcarbon product of the tricarboxylic acid cycle, oxaloacetate, to yield the six-carbon, citrate. Citrate can traverse the mitochondrial membrane and in the cytosol is converted back to acetyl-CoA plus oxaloacetate by an enzyme, ATP-citrate lyase (citrate cleavage enzyme in the older literature). Ruminant species generally use glucose sparingly as a FA precursor because metabolism in the rumen limits the glucose supply to the animal. Ruminants use acetate as the primary carbon precursor for FA synthesis. De novo FA synthesis is initiated by the carboxylation of cytosolic acetyl-CoA by acetyl-CoA carboxylase, the tightly regulated and many times ratelimiting enzyme for FA synthesis. The three-carbon product of this reaction, malonyl-CoA, is then the substrate for fatty acid synthase, the enzyme complex that polymerizes two carbon moieties into long-chain FAs. One carbon is removed from malonyl-CoA during the polymerization process. Acetyl-CoA carboxylase limits the rate of de novo fatty acid biosynthesis from glucose in porcine adipose tissue and from acetate in bovine adipose tissue. However, glucose incorporation into fatty acids in bovine adipose tissue apparently is limited by the glycolytic pathway, probably at 6-phosphofructokinase (Smith, 1984). Ruminant adipocyte substrate utilization has been reviewed (Bauman, 1976; Smith, 1995). The major product of de novo FA synthesis is the 16-carbon saturated FA, palmitic acid (C16:0). (Fatty acids are designated as the number of carbons in the FA chain; C16 = 16 carbons, with the number of double bonds indicated after the colon. This simple nomenclature does not indicate the location of the double bonds.) The adipocyte can extend C16:0 by adding

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two-carbon moieties in a chain-elongation reaction, for example, to yield the 18-carbon saturated FA, stearic acid (C18:0). Double bonds can be inserted into the saturated FAs to produce a series of unsaturated FAs. The positions for desaturation are limited in mammals so that certain FAs are essential and must be obtained from the diet. The enzyme stearoyl-CoA desaturase uses C18:0 as a substrate to produce the monounsaturated FA, oleic acid (C18:1), with one double bond between the 9 and 10 carbon atoms. This enzyme can also transform other saturated fatty acids to their 9 monounsaturated counterparts (Yang et al., 1999). One of the essential FAs, obtained from the diet and ultimately from plant products, is the 18-carbon FA with two double bonds (cis-9, cis-12), linoleic acid (C18:2). The cis-12 double bond is at the number 12 carbon counting from the carboxyl group, but at the 6 carbon counting from the methyl group of the FA. Thus, C18:2 is the primary member of a series of FAs called n-6 or omega-6 FAs. Chain elongation and further desaturation of C18:2 gives rise to arachidonic acid (C20:4). The C20:4 is an important constituent of some membranes and a precursor for many eicosanoid molecules, including prostaglandins, leukotrienes, and thromboxanes. The other essential FA is the 18-carbon FA with three double bonds, α-linolenic acid (C18:3). This FA is the primary member of the n-3 series of FAs; it has double bonds at the 9, 12, and 15 carbons (counting from the carboxyl group) or n-3, 6, and 9 counting from the omega or methyl carbon. Chain elongation and desaturation of this FA produces several eicosanoid molecules plus eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6), both of which are important FA constituents of some membranes, particularly in the mammalian central nervous system. Most of these fatty acids are constituents of animal, plant, and microorganism lipids; thus the diet, regardless of its composition, is a major source of longchain FAs. Individual dietary fats are specifically enriched with individual FAs: C16:0 and C18:0 in mammalian meat and milk products, C18:1 in olive oil or some canola oils, C18:2 in corn oil or safflower oil, C18:3 in flaxseed or linseed oil, and C20:5 and C22:6 in certain oils from fatty fishes. 3.3. Regulation of FA synthesis The development of adipocyte de novo FA synthesis has been documented in ruminant and nonruminant mammals. De novo FA synthesis is reciprocally regulated to accommodate the dietary supply of FAs. The measured rate of placental transport of FAs is limited (Leat and Harrison, 1980; Thulin et al., 1989). However, because transport is a continuous process over the extended period of gestation and fetal de novo FA synthesis rates are low, in utero, FAs are probably supplied primarily by the dam. After birth, the milk supplied to the suckling mammal has a relatively high fat concentration so that de novo FA synthesis is again not needed. After weaning, the extent of de novo FA synthesis is dictated by the synthetic and oxidative requirements for FAs coupled with the FA supply from the diet. Most mammals raised for meat production or for biomedical research are fed a relatively low-fat postweaning diet so that de novo FA synthesis is increased at this time. Decreased feed intake and fasting markedly decrease de novo FA synthesis (Mersmann et al., 1981); the timing of the decrease during fasting is probably dependent on the gut transit time. Within days of transformation from the high-fat suckling diet to the low-fat postweaning diet, porcine adipocytes develop increased capacity to synthesize FAs de novo (Mersmann et al., 1973a,b). Enzyme activities associated with de novo FA synthesis, e.g. ATP citrate lyase, acetyl-CoA carboxylase, fatty acid synthase, and cytosolic enzymes that produce reducing equivalents for the biosynthetic process, e.g. glucose-6 phosphate dehydrogenase, NADP-linked malic enzyme, and NADP-linked isocitrate dehydrogenase, are all increased in

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Fig. 6. Expression of adipocyte enzyme or metabolic data. Lipoprotein lipase activity (LPL) and glycerophosphate acyltransferase activity (GPA) expressed per g adipose tissue or per adipocyte. The maximal activity is indicated as 100%. Data are adapted from: LPL (Steffen et al., 1978) and GPA (Steffen et al., 1979).

the adipocyte. In cattle, there can be a considerable delay between weaning and the expression of ATP-citrate lyase and acetyl-CoA carboxylase (Smith et al., 1984; Martin et al., 1999). The manner in which the carbon flux data or the enzyme activities are expressed is important. As the adipocyte increases in size, there is increased capacity for de novo synthesis of FAs. Thus, depending on the exact chronology of the increase in adipocyte size, expression of these metabolic activities on a per g tissue basis can be misleading because the number of cells per g tissue is decreasing. At some point, the metabolic activity per g tissue will decrease, when in actuality the activity per adipocyte is increasing (fig. 6). It is important to express adipocyte metabolic activity per adipocyte to understand the biology of the adipocyte. To understand the biology of the animal, it would be desirable to know the activity for the entire adipose tissue depot and ultimately for the entire animal. Total depot activity is most readily obtained from small laboratory mammals and for selected depots, e.g. the perigonadal, the perirenal, or the inguinal fat depots. Each of these depots can be dissected in toto and because the size is not great, measurement of metabolic activity in a sample of the depot can be extrapolated to the entire depot. Extrapolation is difficult in larger mammals because dissection of an entire depot is usually difficult, and because of the size of the depot, adipocyte development is not uniform across the depot. This is particularly true for the subcutaneous depot that is prevalent in most agricultural species, and for the mesenteric depot that is prominent in ruminant species. Modes of expression of adipose tissue metabolic data are discussed for development of porcine adipose tissue (Hood and Allen, 1973a; Mersmann and Brown, 1973). Many mammals have both a hepatic and an adipocyte capacity for de novo FA biosynthesis, e.g. rats, cattle, and sheep. Because there is little depot fat in the newborn of most mammalian species, the predominant site of FA synthesis is initially the liver. As the organism develops,

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with a continuous increase in adipose tissue mass, the adipocytes become more important for total body FA synthesis. In the pig, there is little or no capacity for hepatic de novo FA synthesis at any age so that the adipocyte is the primary tissue source of FAs. In the human (and the chicken) the reverse is true, i.e. there is little adipocyte capacity for de novo synthesis of long-chain FAs. The extent of de novo FA synthesis is inversely regulated by the dietary fat concentration, so that as the fat is increased in the diet, there is a progressive decrease in de novo FA synthesis. Thus, when nonruminant mammals are fed a particular fat source in the diet, the FA composition of the fat depots will more or less reflect the composition of the diet. In ruminants, the FAs emerging from the rumen are highly saturated and it is difficult to substantially increase the unsaturated FA concentration of adipose tissue depots (Rule et al., 1995). 3.4. Lipoprotein lipase The alternative source of adipocyte FAs is from hydrolysis of plasma lipoprotein TG. Lipoprotein lipase is synthesized by the adipocyte, and migrates to the surface of the adjacent capillary endothelial cell where it hydrolyzes lipoprotein to release FAs. Triacylglycerol cannot be transported into the adipocyte, but the FAs can. Adipocyte LPL is regulated such that it is active in the fed animal to supply the adipocyte with FAs; the LPL activity decreases in the fasted animal. Insulin is the primary mediator of this regulation of adipocyte LPL. In the newborn pig there is limited adipocyte LPL activity, but this increases rapidly so that after several days there is adequate LPL to hydrolyze the lipoproteins synthesized from the lipids provided by the high-fat milk diet (fig. 5). There is a tendency for the LPL activity to be greater in adipose tissue from animals fed high-fat diets compared to low-fat diets, so that in pigs fed grain diets after weaning, the LPL activity is lower than before weaning. Skeletal muscle LPL generally is regulated in an opposite direction to adipocyte LPL; the muscle enzyme is increased during fasting to increase the supply of FAs to the muscle for oxidation. Measurement of LPL activity, LPL protein, and LPL mRNA in the same animal allows interpretation of the regulatory events controlling LPL function (Tavangar et al., 1992). This type of study has not been done in agricultural species, but enzyme activity or the mRNA have been documented in cattle (de la Hoz and Vernon, 1996; Hocquette et al., 2001), pigs (Steffen et al., 1978; McNeel and Mersmann, 2000), and sheep (Andersen et al., 1996; Bonnet et al., 1998, 2000). 3.5. Triacylglycerol synthesis (fig. 4) As previously indicated, cells cannot accumulate large amounts of nonesterified FAs. The adipocyte may be considered a sink or reservoir for FA storage with the TG molecule being the major storage molecule. Synthesis of TG is a sequential process leading to esterification of long-chain FAs to each of the three hydroxyl groups of glycerol. The glycerol molecule, serving as the backbone for triacylglycerol synthesis, as well as synthesis of phospholipid molecules, is derived from glucose via glycolysis. The six-carbon sugar derivative is split into two three-carbon moieties, with one of these, dihydroxyacetone phosphate, providing the initial source of carbons for the glyceride–glycerol moiety. Dihydroxyacetone phosphate is hydrogenated to glycerol-3 phosphate by the enzyme glycerophosphate dehydrogenase. (The activity or mRNA concentration of this enzyme is often used as an indicator for adipocyte differentiation.) Two acyl groups are sequentially added to the glycerophosphate by

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the enzyme, α-glycerophosphate acyltransferase. The resulting product is phosphatidic acid. The phosphatidic acid is dephosphorylated by the enzyme, phosphatidate phosphohydrolase, to yield diacylglycerol. Finally, the diacylglycerol is acylated to yield the triacylglycerol molecule representing the major storage molecule in the adipocyte. Both phosphatidic acid and diacylglycerol are also precursors of phospholipids, so the regulation of TG synthesis is partly regulated by the demand for the intermediates. Phosphatidate phosphohydrolase is generally considered to be a key regulatory enzyme for these pathways, but diacylglycerol acyltransferase has recently emerged as an enzymatic site for regulation. If FAs are supplied to the adipocyte, there must be adequate TG synthesis capacity to esterify the FAs. In a mammal like the pig, with an extremely rapid postnatal increase in adipocyte hypertrophy, there is an early postnatal demand for esterification of FAs (fig. 5). Enzymes or other aspects of this pathway have seldom been measured in agricultural species (Rule, 1995); however, there are reports for cattle (Lin et al., 1992; Wilson et al., 1992; Smith et al., 1998), pigs (see Mersmann, 1986; Rule et al., 1988a,b, 1989), and lambs (Andersen et al., 1996). 3.6. Endocrine regulation of anabolic metabolism Regulation of mammalian adipocyte lipid anabolic processes is primarily via adrenergic and insulin receptors (Etherton and Walton, 1986; Mersmann, 1991; table 1). There are two major types of adrenergic receptors, the α- and β-adrenergic receptors (αAR and βAR, respectively). In many situations, stimulation of αAR produces actions opposing stimulation of βAR. The insulin receptor tends to work in opposition to the βAR to provide the major adipocyte regulatory system. Thus, insulin stimulates the adipocyte anabolic lipid metabolism pathways and βAR agonists inhibit these same pathways. Rodent adipocytes are particularly sensitive to insulin stimulation of anabolism in vitro, with rates of de novo FA synthesis being increased 10 or more times by insulin. Anabolic processes in adipocytes from many other mammalian species, including pigs (Romsos et al., 1971; Etherton and Chung, 1981; Walton and Etherton, 1986, 1987; Mersmann and Hu, 1987; Mersmann, 1989a; Budd et al., 1994; Mills, 1999), are stimulated by insulin, but the magnitude is much less than in rodent adipocytes. The bovine adipocyte is relatively insensitive to insulin (Smith et al., 1983; Vasilatos et al., 1983). After a meal, when insulin is elevated, it stimulates lipid synthesis, whereas after fasting the insulin concentrations are lowered to cause decreased lipid synthesis. The βAR agonists decrease anabolic adipocyte lipid metabolism (Rule et al., 1987; Coleman et al., 1988; Mersmann, 1989b, 1995, 1998, 2002a; Miller et al., 1989; Mills et al., 1990; Etherton and Smith, 1991; Budd et al., 1994; Moody et al., 2000; Bergen, 2001). Adipocyte anabolism may also be controlled by other hormones. Decreased thyroid function leads to increased fat accumulation, as does increased adrenocorticoid production. In contrast, increased somatotropin leads to decreased adipocyte anabolic lipid metabolism (Vernon, 1991; Vernon et al., 1991; Etherton and Louveau, 1992; Harris et al., 1993; Etherton et al., 1995 Bergen, 2001). Estrogens and androgens have effects on adipocyte function as well, leading to the increased fat deposition in females compared to males, and in some species, sexdependent differential sites of fat deposition. Most of these additional endocrine regulations do not enter into the day-to-day control of adipocyte lipid metabolism. Rather they provide tonic control or long-term modulation. It should be noted that exogenous sex steroids (Hancock et al., 1991), somatotropin (Sejrsen et al., 1989; Beermann and DeVol, 1991; Beermann, 1994), and selected βAR agonists (Moloney et al., 1991) have been used to modify animal growth and carcass composition (NRC, 1994; Steele, 1991; Steele et al., 1994).

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3.7. Conjugated linoleic acid There is recent evidence that administration of exogenous conjugated linoleic acid can reduce carcass fat deposition in mice, rats, and pigs. The mechanism(s) is not clear, but there is evidence for increased energy expenditure, increased fat oxidation, decreased preadipocyte proliferation and differentiation, reduced FA synthesis, reduced LPL activity, increased lipolysis (triacylglycerol degradation), and reduced monounsaturated FA production because of reduced stearoyl-CoA reductase activity (Mersmann, 2002b).

4. DEVELOPMENT OF LIPID DEGRADATION The process of adipocyte lipid degradation, or lipolysis, is initiated by stimulation of a membrane-bound receptor (Belfrage, 1985). Typically this would be a β-adrenergic receptor (βAR) that is coupled to a Gs protein that is coupled to adenylyl cyclase (table 1). Thus, the sequential activation of the receptor, the Gs protein, and adenylyl cyclase yields increased synthesis of cAMP from ATP. An increase in intracellular cAMP leads to activation of protein kinase A by attachment of cAMP to the regulatory subunit of the kinase with subsequent cleavage of the catalytic subunit. The kinase catalytic subunit, freed from the regulatory subunit, then phosphorylates hormone-sensitive lipase to activate it. Hormone-sensitive lipase is the rate-limiting enzyme in the process of lipolysis and for the most part is in the nonphosphorylated state until activated by protein kinase A. Hormone-sensitive lipase cleaves the first two FAs from the TG substrate, whereas the last FA is cleaved by another enzyme, monoacylglycerol lipase. The latter lipase is continuously active and does not participate in the regulation of lipolysis. The products of lipolysis are the three FAs that were esterified to TG plus glycerol. The FAs may be transported to the plasma or re-enter the adipocyte intracellular FA pool where they may be utilized for oxidation or esterification to form complex lipid esters, including the synthesis of TG (fig. 4). Glycerol is not recycled in the adipocyte because glycerol kinase, the enzyme responsible for phosphorylation of glycerol and its re-entry into the pathway for synthesis of TG, is present at extremely low concentration in adipocytes. Typically the lipolytic rate in nonstimulated adipocytes is low, but after maximal stimulation with a βAR agonist, the rate is increased several-fold. The extent of stimulation depends on the species, the age of the animal, and the nutritional status. Many of these aspects of the regulation lipolysis have been reviewed (Mersmann, 1990, 1991; Chilliard et al., 2000).

Table 1 Major endocrine effects on adipocyte lipid metabolism a Anabolicb

Catabolic

Hormone

FA

LPL

TG

Lipolysis

Insulin β-adrenergic GH Glucocorticoids

   

   

   

   

a These effects are more or less evident, depending on the species. Broken arrows indicate effects are marginal or reports are mixed regarding the effect. b Abbreviations: FA = de novo fatty acid synthesis; LPL = lipoprotein lipase; TG = triacyglycerol synthesis.

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For example, in adipocytes from young rats, lipolysis is stimulated as much as 6 to 10 times, whereas in adipocytes from older rats the increase may be only 3 to 6 times. The degree of lipolytic stimulation by a βAR agonist tends to be greater in rat adipocytes than in adipocytes from cattle, chickens, or pigs, wherein the maximal rate is usually only 2 to 5 times that of the unstimulated rate. The lipolytic rate is increased in the fasted compared to the fed state in most species to provide substrate, i.e. FAs for oxidative metabolism. There are three subtypes of βAR: β1AR, β2AR, and β3AR. These are three distinct proteins, coded by three different genes. Within a mammalian species the homology of the three subtypes is approximately 50%, whereas across species the homology for a single subtype is ≥75%. Species-specific differences in the protein structure of a βAR subtype can lead to major differences in the function of the receptor and in the potency and efficacy of agonists and antagonists for the receptor. Thus, an antagonist that is specific for the rat β2AR, ICI 118,551, is not specific for the porcine β2AR, and a specific agonist for the rat β3AR, BRL 37,344, is not specific for the porcine β3AR, but is specific for the porcine β2AR, where it acts as an antagonist (Liang and Mills, 2002). Propranolol is an antagonist for the cloned mouse β3AR, but a partial agonist for the cloned bovine and human β3AR (Piétri-Rouxel et al., 1995). Thus, it cannot be assumed that the βAR subtype specificity of an agonist or antagonist in one species will be maintained in another species; the specificity must be tested using cloned receptors from the individual species of interest (Mills and Mersmann, 1995; Mersmann, 2002a). The βAR subtypes are differentially distributed in the various tissues of a particular species, e.g. rat heart has >90% β1AR, rat lung has >85% β2AR, and rat adipocytes have >90% β3AR. Receptor distribution in a particular tissue also varies across species, e.g. there are approximately 65% β1AR in porcine heart compared to the 90% in rat heart and there are approximately 75% β1AR in porcine adipocytes compared to the 90% β3AR in rat adipocytes (McNeel and Mersmann, 1999; Liang and Mills, 2002). In a few cases, there is evidence for a change in adrenergic receptors in a tissue during development. For example, in rat adipocytes the α2AR increased 4-fold between 6 and 20 weeks of age, whereas the βAR decreased 25% in the same time span (Kobatake et al., 1991). In human adipocytes isolated from infants <2 months old, there was more α2AR activity than in adipocytes isolated from adults (Marcus et al., 1987). Also, the rodent preadipocyte has few or no β3AR with a shift to ≥90% β3AR during differentiation to adipocytes (Feve et al., 1991). Ultimately the response of the adipocyte to adrenergic stimulation, in a particular species, will depend on the βAR subtypes present, the ratio of α2AR to βAR on the adipocyte (stimulation of α2ARs inhibits lipolysis), the stage of development of the adipocyte (possibly leading to shifts in receptor subtypes), and the concentration of norepinephrine and epinephrine, the physiological agonists for βAR, to which the adipocyte is exposed (Mersmann, 1998, 2002a). Depending on the species, there are other membrane-bound receptors that may regulate hormone-sensitive lipase through the cAMP-protein kinase A system. Rat adipocytes have receptors for adrenocorticotropin, glucagon, somatotropin, thyrotropin, etc. Stimulation of any of these receptors increases the lipolytic rate. For the most part, these receptors appear to be minor factors in the regulation of lipolysis in most nonrodent species (Mersmann, 1990, 1991; Lanna and Bauman, 1999). Lipolysis is regulated, not only by receptors that stimulate the process, but also by inhibitory receptors. Stimulation of the α2-adrenergic receptor (α2AR) inhibits lipolysis because the α2AR is coupled to the Gi protein, an inhibitory factor. The physiological adrenergic hormone, epinephrine, has both βAR and αAR activity. Thus, the effect of epinephrine on adipocyte lipolysis depends on the relative populations of α2AR and βAR on the adipocyte.

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There are few α2ARs on adipocytes from some species, e.g. rats and pigs, so that this mechanism of inhibition is not operative (Mersmann, 1990). Perhaps the most important physiological negative modulator for lipolysis is insulin (table 1). Increased insulin inhibits lipolysis (Mersmann and Hu, 1987; Mersmann, 1990). Thus, after a meal when insulin concentration is elevated, lipolysis is decreased. At least part of the mechanism for insulin inhibition of lipolysis is stimulation of the activity of cAMP-phosphodiesterase to decrease the concentration of intracellular cAMP, and consequently decrease the activation of lipolysis through protein kinase A and hormone-sensitive lipase. Another negative control of lipolysis is provided by the adenosine receptor (A1R). This receptor also couples to Gi proteins to inhibit lipolysis. Adenosine, the agonist, is produced extracellularly by adipocytes from cAMP. The process is very active and to measure substantial rates of lipolysis with adipocytes isolated from many species, it is necessary to either inhibit the A1R or to destroy the adenosine by addition of adenosine deaminase. The extent of adenosine formation in vivo is not yet clear, but for many adipocyte preparations in vitro, inhibition of adenosine formation leads to a considerable enhancement of the lipolytic rate (Carey, 1995). Although subject to several qualifications, the lipolytic activity may be approximated in vivo by changes in the plasma FA and/or glycerol concentration. The assumption is that the source of most of the nonesterified FA and glycerol in the plasma is adipocyte lipolysis. These types of measurements can estimate the extent of stimulation or inhibition by an acutely infused compound, but do not reflect the actual rate. They also can be used to estimate a dose-response for the compound upon infusion in vivo. Likewise, the nonesterified FA concentration in the plasma can be used to assess the effects of chronic treatment with a particular compound on lipolytic activity in vivo. Such approaches have been used to estimate the response of adipose tissue to various βAR and αAR agonists and antagonists, and other metabolic hormones in cattle, pigs, and sheep (Mersmann, 1987, 1989c, 1995, 1998, 2002a). Function of the lipolytic process in adipocytes from newborn or young mammals is impaired relative to function in mature adipocytes. Thus, the βAR-stimulated rate increases as adipocytes increase in size. Lipolysis is impaired in adipocytes isolated from newborn pigs, but increases several-fold within the first few days of postnatal life (fig. 5; Mersmann, 1986). The ability to mobilize FAs immediately after birth is species-specific and depends on the amount of stored adipose tissue, the chronological development of the enzymatic machinery for lipolysis, the establishment of the appropriate receptor populations, and the development of coupled receptor-driven metabolism. The development of both anabolic and catabolic adipocyte lipid metabolism primarily occurs after birth in many mammalian species, including cattle, pigs, and sheep. The minimal capacity to mobilize FAs after birth is critical in individuals exposed to cold or that do not have sufficient milk intake.

5. FUTURE PERSPECTIVES Although the goal in modern meat animal production is to produce animal protein with minimal fat content, the animal raised under conditions where the environment is not optimal and constant requires fat depots for insulation, to provide oxidative substrates, and to produce selected endocrine materials and growth factors. Mechanisms to decrease adipose tissue lipid metabolism anabolic processes and to increase catabolic processes have been sought. Somatotropin and βAR agonists are two exogenous compounds that mechanistically operate in this fashion. However, in the neonate, the goal cannot be to decrease the anabolic activity or increase the catabolic activity because the neonate is in a precarious metabolic state with

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limited capacity to mobilize fatty acids for oxidative fuels or to synthesize fatty acids for esterification to triacylglycerol. In fact, it might be appropriate to seek to temporarily increase the rate of fatty acid biosynthesis and lipolysis in neonates. Ideally, if selected genes can be regulated, i.e., turned on and off, it might be possible to enhance selected gene expression at particular stages of growth and to diminish expression at others.

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Mersmann, H.J., 1991. Regulation of adipose tissue metabolism and accretion in mammals raised for meat production. In: Pearson, A.M., Dutson, T.R. (Eds.), Growth Regulation in Farm Animals. Advances in Meat Research, Vol. 7. Elsevier Applied Science, London. pp. 135–168. Mersmann, H.J., 1995. Species variation in mechanisms for modulation of growth by beta-adrenergic receptors. J. Nutr. 125, 1777S–1782S. Mersmann, H.J., 1998. Overview of the effects of beta-adrenergic receptor agonists on animal growth including mechanisms of action. J. Anim. Sci. 76, 160–172. Mersmann, H.J., 2002a. Beta-adrenergic receptor modulation of adipocyte metabolism and growth. J. Anim. Sci. 80, E24–E29. Mersmann, H.J., 2002b. Mechanisms for conjugated linoleic acid-mediated reduction in fat deposition. J. Anim. Sci. 80, E126–E134. Mersmann, H.J., Brown, L.J., 1973. Adaptation of swine (Sus domesticus) adipose tissue to increased lipogenesis: expression of data. Int. J. Biochem. 4, 503–510. Mersmann, H.J., Hu, C.Y., 1987. Factors affecting measurements of glucose metabolism and lipolytic rates in porcine adipose tissue slices in vitro. J. Anim. Sci. 64, 148–164. Mersmann, H.J., Allen, C.D., Chai, E.Y., Brown, L.J., Fogg, T.J., 1981. Factors influencing the lipogenic rate in swine adipose tissue. J. Anim. Sci. 52, 1298–1305. Mersmann, H.J., Brown, L.J., Beuving, R.M., Arakelian, M.C., 1976. Lipolytic activity of swine adipocytes. Amer. J. Physiol. 230, 1439–1443. Mersmann, H.J., Goodman, J.R., Brown, L.J., 1975. Development of swine adipose tissue: morphology and chemical composition. J. Lipid. Res. 16, 269–279. Mersmann, H.J., Houk, J.M., Phinney, G., Underwood, M.C., 1973a. Effect of diet and weaning age on in vitro lipogenesis in young swine. J. Nutr. 103, 821–828. Mersmann, H.J., Houk, J.M., Phinney, G., Underwood, M.C., Brown, L.J., 1973b. Lipogenesis by in vitro liver and adipose tissue preparations from neonatal swine. Amer. J. Physiol. 224, 1123–1129. Mersmann, H.J., Underwood, M.C., Brown, L.J., Houk, J.M., 1973c. Adipose tissue composition and lipogenic capacity in developing swine. Amer. J. Physiol. 224, 1130–1135. Miller, M.F., Cross, H.R., Wilson, J.J., Smith, S.B., 1989. Acute and long-term lipogenic response to insulin and clenbuterol in bovine intramuscular and subcutaneous adipose tissues. J. Anim. Sci. 67, 928–933. Miller, M.F., Garcia, D.K., Coleman, M.E., Ekeren, P.A., Lunt, D.K., Wagner, K.A., Procknor, M., Welsh, T.H. Jr., Smith, S.B., 1988. Adipose tissue, longissimus muscle and anterior pituitary growth and function in clenbuterol-fed heifers. J. Anim. Sci. 66, 12–20. Miller, W.H. Jr., Faust, I.M., Hirsch, J., 1984. Demonstration of de novo production of adipocytes in adult rats by biochemical and radioautographic techniques. J. Lipid. Res. 25, 336–347. Mills, S.E., 1999. Regulation of porcine adipocyte metabolism by insulin and adenosine. J. Anim. Sci. 77, 3201–3207. Mills, S., Mersmann, H.J., 1995. Beta-adrenergic agonists, their receptors, and growth: special reference to the peculiarities in pigs. In: Smith, S.B., Smith, D.R. (Eds.), The Biology of Fat in Meat Animals: Current Advances. Amer. Soc. Anim. Sci., Champaign, IL. pp. 1–34. Mills, S.E., Liu, C.Y., Gu, Y., Schinckel, A.P., 1990. Effects of ractopamine on adipose tissue metabolism and insulin binding in finishing hogs: interaction with genotype and slaughter weight. Domest. Anim. Endocrinol. 7, 251–263. Mitchell, A.D., Scholz, A.M., Mersmann, H.J., 2001. Growth and body composition. In: Pond, W.G., Mersmann, H.J. (Eds.), Biology of the Domestic Pig. Cornell University Press, Ithaca, NY, pp. 225–308. Moloney, A.P., Allen, P., Enright, W.J., 2002. Body composition and adipose tissue accretion in lambs passively immunised against adipose tissue. Livest. Prod. Sci, 74, 165–174. Moloney, A., Allen, P., Joseph, R., Tarrant, V., 1991. Influence of beta-adrenergic agonists and similar compounds in growth. In: Pearson, A.M., Dutson, T.R. (Eds.), Growth Regulation in Farm Animals. Elsevier Applied Science, London, pp. 455–513. Moody, D.E., Hancock, D.L., Anderson, D.B., 2000. Phenethanolamine repartitioning agents. In: D’Mello, J.P.F. (Ed.), Farm Animal Metabolism and Nutrition. CABI Publishing, New York pp. 65–96. Morrison, R.F., Farmer, S.R., 2000. Hormonal signaling and transcriptional control of adipocyte differentiation. J. Nutr. 130, 3116S–3121S.

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Ontogeny and metabolism of brown adipose tissue in livestock species S. B. Smith and G. E. Carstens Department of Animal Science, Texas A & M University, College Station, TX 77483-2971, USA

We have reported several aspects of brown adipose tissue (BAT) development and response to environmental stimuli. The metabolism of BAT resembles that from mature ruminant white adipose tissue, in that acetate is the primary precursor for lipogenesis. There is a precipitous decline in rates of de novo lipogenesis during the last trimester, indicating that the contribution of fatty acid biosynthesis to lipid filling is more important earlier in fetal development. The mixture of brown and white adipocytes in subcutaneous adipose tissues from newborn calves suggests that brown adipocytes may involute into white adipocytes in this species. However, there is growing evidence in rodent species that brown and white adipocytes differentiate and develop independently. Clearly, brown adipose tissue from sheep and cattle rapidly dedifferentiates and/or is lost via apoptosis early postnatally. This phenomenon is especially rapid in warm ambient temperatures, but may be delayed by feeding dams diets high in polyunsaturated fatty acids. In Wagyu × Angus crossbred calves, brown adipose tissue function is remarkably refractory to profound reductions in dietary protein intake in dams, indicating the high priority animals place in ensuring adequate thermogenic capacity in their newborn.

1. INTRODUCTION Adverse climatic conditions during the early postnatal period can disrupt thermal balance in newborn lambs and calves leading to hypothermia and/or death. Numerous reports have demonstrated that calf mortality increases during inclement weather. In an epidemiological study involving more than 87,000 Bos taurus calves, Azzam et al. (1993) found that calf mortality increased progressively as ambient temperature decreased or as precipitation amount on the day of birth increased. Moreover, the stress of maintaining homeothermy during severe cold exposure for extended periods may interact with other etiological factors associated with neonatal calf mortality and morbidity through depletion of energy reserves, induction of physical weakness, and/or delay in absorption of immunoglobulins.

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A major thermoregulatory mechanism for survival of neonatal ruminants during cold stress is heat production by brown adipose tissue (BAT). Maintenance of homeothermy during the early postnatal period necessitates an acute and sustained thermogenic response by the newborn calf. Maximal thermogenic response to cold (i.e. summit metabolism), which includes both shivering and nonshivering thermogenesis, is 4- to 5-fold higher than thermoneutral metabolism in neonatal lambs and 3- to 4-fold higher in neonatal calves (Stott and Slee, 1985; Okamoto et al., 1986; Robinson and Young, 1988a,b). Approximately half of the cold-induced summit metabolism in newborn lambs is derived from nonshivering thermogenesis (Stott and Slee, 1985). Therefore, newborn lambs and calves must possess highly active BAT during the early postnatal period when the demand for thermogenesis is greatest. Most of the adipose tissue in the newborn ruminant species is BAT, although small amounts of white adipose tissue (WAT) are present (Alexander et al., 1975; Alexander, 1978; Martin et al., 1997, 1999). Alexander et al. (1975) estimated that the quantity of BAT in newborn calves was ~1.5–2.0% of body weight. This review will focus primarily on BAT in lambs and calves. Little mention will be made of piglets because distinctive brown adipocytes do not appear to be present in porcine adipose tissue.

2. MORPHOLOGY OF BROWN ADIPOSE TISSUE IN NEWBORN LIVESTOCK SPECIES Brown adipocytes from newborn calves do not display the typical multilocular feature that is characteristic of brown adipose tissue in other species. Instead, bovine brown adipocytes contain a large central lipid vacuole with few peripheral lipid inclusions. This is consistent with Alexander et al. (1975), who examined perirenal brown adipocytes in newborn calves at lower magnification and described adipocytes as dominated by a large lipid vacuole with smaller lipid inclusions in the marginal cytoplasm of some of the cells. Napolitano (1963) stated that the major criterion used to characterize brown adipocytes morphologically should be the appearance and differentiation of mitochondria rather than the occurrence of multilocular lipid droplets. Subcutaneous adipose tissue overlying the sternum from Wagyu × Angus crossbred calves contains unilocular adipocytes with few cytoplasmic inclusions (Martin et al., 1997). Unlike perirenal adipocytes from these same calves, only a small number of mitochondria with poorly developed cristae were present. Thus, sternum adipose tissue represents a WAT depot in Wagyu × Angus calves. These results are similar to those of Alexander et al. (1975) for newborn calves, although the earlier work described adipocytes containing a few small lipid droplets in addition to the large central vacuole.

3. SPECIFIC GENE EXPRESSION IN BROWN ADIPOSE TISSUE 3.1. Uncoupling protein-1 Brown adipose tissue’s thermogenic capacity is attributed to uncoupling protein-1 (UCP1) located in the inner mitochondrial membrane. The principle function of UCP1 is to dissipate the proton gradient created by mitochondrial respiration, which uncouples mitochondrial respiration from synthesis of ATP and allows energy to be dissipated as heat. The concentration of UCP1 is a key biochemical marker of the thermogenic capacity of BAT (Himms-Hagen, 1986). Brander et al. (1993) demonstrated that UCP1 mRNA was present in intrascapular

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BAT, but was not present in rat omental, liver, kidney, skeletal muscle, or heart. Casteilla et al. (1989) examined perirenal, pericardial, peritoneal, and subcutaneous adipose tissue depots in newborn calves, and found UCP1 mRNA in all tissues except subcutaneous adipose tissue. UCP1 mRNA was highest in perirenal adipose tissue, followed by pericardial tissue (~50% of perirenal) and peritoneal tissue (~15% of perirenal). More recently, Landis et al. (2002) reported the presence of UCP1 mRNA in s.c. adipose tissue of Brahman and Angus fetal calves (last trimester), indicating that s.c. adipose tissue contained a population of brown adipocytes during late fetal development. 3.2. β-Adrenergic receptors Expression of the β3-adrenergic receptor (β3-AR) gene is especially high in bovine perirenal BAT during fetal and early postnatal development (Casteilla et al., 1989, 1994). β3-Adrenergic receptors have been documented in porcine (white) adipocytes (Mersmann, 1996), indicating that this receptor type is not unique to BAT. However, β3-AR gene expression may be prerequisite to BAT differentiation in fetal calves (Casteilla et al., 1994).

3.3. Brown unknown gene Moulin et al. (2001a,b) reported the existence of a gene that is expressed early in the differentiation of stromal–vascular cells from rat brown adipose tissue. The gene, which they termed BUG (brown unknown gene), is expressed at high levels in rat interscapular adipose tissue, cardiac muscle, brain, and kidney, but at low levels in white inguinal adipose tissue, muscle, liver, and spleen. It also is expressed at higher levels in interscapular adipose tissue from obese rats, which exhibits depressed UCP1 gene expression (Moulin et al., 2001a). The authors concluded that BUG gene expression must be depressed to obtain high rates of UCP1 gene expression. Their results also indicate that stromal–vascular preadipocytes from interscapular (brown) adipocytes represent a cell line that is distinct from preadipocytes from inguinal (white) adipose tissue. To our knowledge, the expression of BUG in BAT from ruminant species has not been described.

4. ONTOGENIC DEVELOPMENT OF BROWN ADIPOSE TISSUE Detectable quantities of perirenal BAT in lambs first appear at 70 days of gestation (Alexander, 1978). Alexander (1978) also reported that the mass of perirenal adipose tissue in fetal Merino sheep increased by approximately 34% over the last 3 weeks of gestation. Vernon et al. (1981) documented a 40% increase in adipocyte volume of fetal lambs in the last 4 weeks of pregnancy, indicating that most, if not all, of the increase in perirenal BAT mass in fetal lambs was due to adipocyte hypertrophy. In fetal calves, some portion of BAT hypertrophy is due to increased adipocyte volume (Landis et al., 2002), at least during the period between 96 and 48 days before parturition (fig. 1). During this period there is a large numerical increase in brown adipocyte volume. However, subsequent to 48 days before birth, total apparent perirenal brown adipocyte number more than doubles (Landis et al., 2002). Thus, hyperplastic growth of adipocytes contributes substantially to the fetal growth of bovine perirenal BAT. Growth of BAT in lambs, relative to fetal weight, is allometric (100 mg/kg of fetal weight) from 70 to 120 days of gestation, and isometric (6 mg/kg) thereafter until term (ovine gestation

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Fig. 1. Adipocyte density and mean volume of perirenal brown adipose tissue. Each data point represents the mean for 3 fetal calves per breed type. The overall SEM for each breed type is affixed to the symbols. The SEM are not large enough to be visible for adipocytes/g. Adapted from Figure 4 of Landis et al. (2002).

length is 150 days) (Alexander, 1978). Lipid locules were first identified in perirenal adipocytes on day 70 of gestation, and by day 80 to 90, mitochondria began to proliferate and take on the morphological features of brown adipocytes. Rapid accumulation of lipid in perirenal adipocytes occurs during the allometric growth phase, whereas increased mitochondrial biogenesis and sympathetic innervation of adipocytes occur during the isometric growth phase. Therefore, even though prenatal growth of BAT is most rapid during mid-gestation, functional development of brown adipocytes does not occur until late gestation. 4.1. Morphological changes during ontogeny Based on histological examinations of bovine BAT during late gestation, fetal brown adipocytes contain fewer mitochondria that are also less developed compared to brown adipocytes from newborn calves (Landis et al., 2002; fig. 2). Mitochondria from fetuses sampled as late as midway through the last trimester are spherical, with cristae that do not traverse the entire mitochondrion, whereas brown adipocytes from late term and newborn BAT are nearly unilocular, with small lipid inclusions peripheral to larger central lipid vacuoles, and the mitochondria are more abundant and more fully differentiated (fig. 3). These observations corroborate Nedergaard et al. (1986), who described similar morphological changes in brown adipocytes during fetal development of rats. At 96 days before birth, some BAT cells have accumulated very little lipid, whereas others already are nearly unilocular (figs. 2A and D). Brown adipocytes gradually accumulate lipid, with the multilocular appearance persisting in Brahman BAT at 24 days before birth (fig. 2E).

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Fig. 2. Transmission electron micrographs of perirenal brown adipocytes. Perirenal brown adipose tissue was obtained by cesarean section at 96 and 24 days before birth, and at birth, from Angus (left column) and Brahman (right column) fetuses. Thick arrows indicate lipid vacuoles and thin arrows point to mitochondria. A nucleus is indicated at the bottom left. Scale bar at the bottom right indicates magnification. Adapted from Figure 2 of Landis et al. (2002).

At birth, brown adipocytes become essentially unilocular, with only a few small lipid vacuoles set apart from the large, central vacuole (figs. 2C and F). At the earliest sampling period, BAT mitochondria are smaller, spherical, and quite dense (figs. 3A and D). By 24 days before birth, the mitochondria become quite large, and cristae, although distinct, are extensive (figs. 3B and E). Within 7 days of parturition, the morphology of the mitochondria changes from primarily spherical to markedly elongated, with extensively differentiated cristae. This morphology is apparent at birth (figs. 3C and F).

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Fig. 3. Transmission electron micrographs of mitochondria from perirenal brown adipocytes. Perirenal brown adipose tissue was obtained by cesarean section at 96 and 24 days before birth, and at birth, from Angus (left column) and Brahman (right column) fetuses. Arrows point to cristae and mitochondria are labeled M in some panels. Scale bar at lower right indicates magnification. Adapted from Figure 3 of Landis et al. (2002).

4.2. Gene expression during ontogeny Fetal bovine perirenal UCP1 mRNA is not detectable until day 211 of gestation, and only in small quantities thereafter until day 259, when levels increase markedly (Casteilla et al., 1989). We recently documented a depression and then recovery in UCP1 gene expression during the last 14 days of gestation (fig. 4; Landis et al., 2002). The decline in UCP1 gene expression coincided with the change in mitochondrial morphology from spherical to elongated, and therefore may have biological significance.

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Fig. 4. UCP1 gene expression in perirenal and tailhead s.c. adipose tissues. Total RNA was extracted from the perirenal (brown adipose tissue, BAT) and tailhead s.c. adipose tissue (white adipose tissue, WAT) samples for slot blot analysis of uncoupling protein-1 (UCP1). Each data point represents the mean for 3 fetal calves per breed. The overall SEM for each breed type is affixed to the symbols. Relative concentrations of UCP1 mRNA are expressed as the ratio of UCP1:18S ribosomal RNA × 100. Adapted from Figure 5 of Landis et al. (2002).

The activity of 5′-deiodinase first appears in fetal bovine perirenal adipose tissue at 2 months of gestation, increases rapidly until activity peaks at 7 months, and declines thereafter until birth (Giralt et al., 1989). The activity of 5′-deiodinase is responsible for deiodination of T4 to T3, which is thought to be required for optimal synthesis of UCP1 in cold-adapted rats (Bianco and Silva, 1988). Giralt et al. (1989) noted that 5′-deiodinase peaked at approximately the same time that Casteilla et al. (1989) first detected UCP1 mRNA, and hypothesized that endogenous production of T3 may be involved in prenatal induction of UCP1 expression in BAT. Trayhurn et al. (1993) demonstrated small amounts of UCP1 protein in goat s.c. adipose tissue at 2.5 days of age. Consistent with this, our laboratory (Martin et al., 1999) demonstrated that s.c. adipose tissue from newborn Brahman and Angus calves contained adipocytes with distinctive brown adipocyte morphology. More recent results (Landis et al., 2002) demonstrated UCP1 mRNA in s.c. adipose tissue during the last trimester of gestation, although the abundance of UCP1 mRNA dropped precipitously during the last 30 days prior to parturition (Fig. 4). Casteilla et al. (1994) examined the expression of adrenergic receptor (β1- and β3-AR) genes in bovine perirenal BAT during fetal and early postnatal development. The β3-AR mRNA was first measurable during mid-gestation, and its concentration increased dramatically between 6 months of fetal life and birth. In contrast, β1-AR mRNA was expressed at low levels throughout fetal life. The appearance of β3-AR mRNA preceded the expression of UCP1 mRNA (Casteilla et al., 1989), suggesting that β3-AR gene expression may be prerequisite to BAT differentiation (Casteilla et al., 1994). Postnatally, β3-AR gene expression

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declined after approximately 3 months of age; the decline was much slower than the more rapid postnatal decline of UCP1 mRNA (Nougues et al., 1993; Trayhurn et al., 1993). Response to β3-adrenergic stimulus apparently persists after birth. Acute treatment of rats with a selective β3-agonist increased BAT GDP-binding of mitochondria (a measure of UCP1 activity) after only 60 min of treatment (Milner et al., 1988). In adult dogs, treatment with a β3-agonist increased UCP1 concentration and the expression of UCP1 mRNA (Champigny et al., 1991). Administration of the β3-agonist ICI-D7114 to newborn lambs during the first several weeks of life delayed the apparent involution of BAT to WAT (Nougues et al., 1993). The β3-agonist-treated lambs continued to express UCP1 mRNA in perirenal and pericardial fat depots at 25 days of age, whereas control lambs of the same age did not. 4.3. Fatty acid metabolism during ontogeny The incorporation of acetate, glucose, and palmitate into glycerolipids of perirenal adipose tissue decreases markedly during late gestation, especially in Brahman BAT (Landis et al., 2002) (fig. 5). Vernon et al. (1981) demonstrated reductions in fatty acid synthesis from acetate and glucose in fetal ovine perirenal BAT that were similar in magnitude to those

Fig. 5. Lipogenesis from acetate, glucose, and palmitate in perirenal brown adipose tissue as a function of average fetal age. The incorporation rate is expressed as nmol substrate incorporated/106 cells/h. Each data point represents the mean for 3 fetal calves per breed type. The rate of palmitate esterification in Brahman fetuses at 96 days before birth was 1,570 nmol/106 cells/h (off scale in the figure). The overall SEM for each breed type is affixed to the symbols. The SEM for glucose incorporation are not large enough to be visible. There was a significant age effect for acetate incorporation into glycerolipids. There was a significant age × breed type effect for palmitate and glucose incorporation into glycerolipids. Adapted from Figure 6 of Landis et al. (2002).

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observed in fetal calves. Although de novo fatty acid biosynthesis is barely detectable by parturition in bovine BAT, glycerolipid synthesis from palmitate remains elevated at that time. Thus, palmitate esterification accounts for 98% of total glycerolipid synthesis in vitro in the newborn calves, and is a primary contributor to lipid filling throughout gestation. Acetate has been well documented as the principal source of carbon for de novo fatty acid biosynthesis in adipose tissue of young and adult ruminant species (Ballard et al., 1972; Ingle et al., 1972; Smith and Prior, 1986). Both acetate and glucose contribute significantly to de novo lipogenesis in newborn calves (Martin et al., 1999). At the beginning of the third trimester in fetal calves, the rate of lipogenesis from acetate is approximately 10-fold greater than lipogenesis from glucose. Some portion of the glucose carbon would have been recovered as glyceride–glycerol (consistent with the high rates of palmitate incorporation into lipids), so that actual rates of de novo fatty acid biosynthesis from glucose would be very low relative to fatty acid biosynthesis from acetate. Thus, fetal bovine adipose tissue preferentially uses acetate as the carbon source for de novo fatty acid biosynthesis early in the third trimester of gestation.

5. ENVIRONMENTAL EFFECTS ON BROWN ADIPOSE TISSUE 5.1. Metabolism and thermogenesis during cold exposure Cold-induced secretion of norepinephrine (NE) increases the specific thermogenic activity of BAT. In addition to its role in acute activation of BAT thermogenesis, NE is also involved in long-term modulation of BAT growth and development during cold stress by enhancing differentiation of BAT precursor cells, mitochondrial proliferation, and transcription of UCP1 via β- and α1-AR pathways (Géloën et al., 1988). Type II thyroxine 5′-deiodinase, which is an important enzyme regulating the thermogenic capacity of BAT, is increased in BAT during cold exposure (Puig-Domingo et al., 1989). Previous studies suggested that the improved survival of lambs born to cold-exposed ewes was due to an enhanced rate of BAT thermogenesis. Stott and Slee (1985) found that lambs born to cold-exposed ewes exhibited significantly higher NE-induced thermogenic rates (in vivo assessment of BAT thermogenesis) than lambs from warm-exposed ewes. Likewise, Symonds et al. (1992) found that lambs from cold-exposed ewes were 15% heavier at birth, and possessed 21% more perirenal BAT that was also 40% more active thermogenically compared to lambs from control ewes. Newborn lambs from cold-exposed ewes were clearly more coldtolerant as thermogenic rates were 16% greater in a warm (28°C) environment and 40% greater in a cold (14°C) environment, relative to lambs from control ewes. We investigated the postnatal changes in perirenal BAT in neonatal calves, and found that cytochrome c oxidase activity of perirenal BAT was highest at birth, and decreased substantially after 7 days of warm exposure (Carstens, 1994). Mitochondrial protein concentrations in warm-exposed calves were only 20% of that found in newborn calves (Carstens, 1994), and bovine perirenal UCP1 was reduced markedly in warm-exposed calves (Martin et al., 1997). Trayhurn et al. (1993) reported a similar postnatal decline in cytochrome c oxidase activity in neonatal goats (325, 50, and 3 μmol oxidized/min/g of BAT in newborn, 7-day-old, and 21-day-old goats, respectively). Cytochrome c oxidase activity was also lower in cold-exposed calves than newborn calves, but was still 2.7-fold higher than in warm-exposed calves at 7 days of age (Carstens, 1994). Thus, cold exposure during the early postnatal period delays the apparent involution of BAT to WAT, resulting in higher BAT thermogenic rates during the neonatal period.

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5.2. Involution during warm exposure Postnatally, in neonatal ruminants BAT is involuted into WAT within 2 to 3 weeks if the neonate is not exposed to cold (Alexander et al., 1975). In bovine perirenal BAT, there is no apparent atrophy or degeneration of brown adipocytes postnatally (Alexander et al., 1975). Instead, there appears to be a progressive accumulation of lipid, accompanied by the dedifferentiation and loss of mitochondria, as perirenal adipocytes acquire the morphology of white adipocytes. More recently, Trayhurn et al. (1993) demonstrated the presence of UCP protein (measured by Western analysis) in s.c. (hindlimb and neck) and internal (perirenal, pericardial, and omental) adipose tissues of goats. Trayhurn et al. (1993) detected UCP protein in s.c. adipose tissue of 7-day-old goats, and in perirenal BAT of 21-day-old goats. Uncoupling protein is more persistent than its mRNA; uncoupling protein mRNA was not detectable by day 2.5 in goat perirenal BAT, and was undetectable in other adipose tissue depots at birth. We examined the effects of postnatal cold exposure on NE-induced BAT thermogenesis in Holstein calves in vivo (table 1). Peak metabolic (PM) rates were 26% lower in warmexposed calves than in newborn calves, yet PM rates were similar between newborn and cold-exposed calves, demonstrating that cold exposure delayed the apparent postnatal involution of BAT. This observation is supported by the fact that cytochrome c oxidase activity and total mitochondrial protein in BAT were 2.7- and 2.5-fold higher in cold-exposed than in warm-exposed calves. We measured UCP1 mRNA in a small number of BAT samples from newborn calves that had been exposed to 4°C for 7 days postnatally. As observed for cytochrome c oxidase activity and mitochondrial protein, there was a dramatic loss of UCP1 mRNA postnatally even during cold exposure (fig. 6). We also recently examined the effects of postnatal cold exposure on BAT thermogenesis in newborn lambs (table 2). Brown adipose tissue mass tended to be greater in the lambs held for 48 h at 28°C than in lambs held at 6°C. Similarly, total and mitochondrial BAT protein concentrations were increased significantly by cold exposure. This observation is supported by the fact that cytochrome c oxidase activity and UCP1 mRNA were approximately 3-fold and 5-fold higher, respectively, in BAT from cold-exposed lambs than in warm-exposed lambs (table 2).

Table 1 Effects of cold exposure on postnatal changes in thermoneutral (TM) and NE-induced peak metabolic rates (PM), and BAT cytochrome c oxidase activity and mitochondrial protein in newborn Holstein, and 7-day-old calves exposed to 4°C (cold-exposed) or 22°C (warm-exposed) temperatures from birth to 7 days of age

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Fig. 6. Uncoupling protein-1 (UCP1) mRNA in newborn and 7-day cold-adapted calves. Lane 1, bovine longissimus muscle RNA. Lanes 2 and 3, RNA from BAT of newborn calves. Lanes 4 and 5, RNA from BAT of calves that had been subjected to 4°C for 7 days postnatally. No bands are visible in lane 5. Lane 6, DNA from a PCR reaction that used the calf UCP1 cDNA as template. The upper band is the UCP-PCR probe. The lower band corresponds to unincorporated primers. Lane 7, Eco RI-excised calf UCP partial cDNA (1.4 kb). (S.B. Smith and G.E. Carstens, unpublished data.)

The reduction in BAT mass, and concomitant elevation in BAT protein content, were the result of cold-induced mobilization of BAT lipid stores, which caused a remarkable delipidation of BAT in response to cold exposure (fig. 7). Mitochondria from warm-exposed lambs underwent extensive dedifferentiation, and by this stage were structurally similar to mitochondria from fetal calves (fig. 3), i.e. they were larger with less extensive cristae. There also was some apparent dedifferentiation of mitochondria in the cold-exposed lambs. The loss of mitochondrial integrity could have been the result of dedifferentiation (involution). Alternatively, brown adipocytes in warm-exposed lambs may have been undergoing apoptosis, to be replaced subsequently with white adipocytes.

6. APOPTOSIS OF BROWN ADIPOCYTES It is not clear if BAT from ruminant species undergoes dedifferentiation and direct conversion to WAT, or whether brown adipocytes experience apoptosis, to be replaced by newly differentiated white adipocytes. In bovine perirenal BAT, there is no apparent atrophy or degeneration of brown adipocytes postnatally (Alexander et al., 1975). Instead, there appears to be a progressive accumulation of lipid, accompanied by the dedifferentiation and loss of

Table 2 Effects of cold exposure (28°C vs 6°C) on perirenal adipose tissue composition, cytochrome c oxidase activity, mitochondrial protein, and UCP mRNA in 48-hour-old lambs

Birth weight, kg BAT, g/kg body weight BAT protein, mg/g Cytochrome c oxidase activity, μmol/g/min Mitochondrial protein, mg/g BAT UCP mRNA:28S rRNA ratio

Warm-exposed

Cold-exposed

P<

4.18 ± 0.17 4.40 ± 0.27 97.6 ± 2.8 57.7 ± 5.0 13.06 ± 1.4 0.10 ± 0.05

4.06 ± 0.15 2.69 ± 0.14 131.2 ± 4.0 153.6 ± 7.5 26.65 ± 1.72 0.64 ± 0.11

NS 0.10 0.001 0.001 0.001 0.05

n = 20 lambs/treatment. NS = not statistically different (P > 0.05). UCP:28S ratio = (Laser densitometer area for UCP mRNA: Laser densitometer area for 28S rRNA) × 100. Areas were calculated from slot blots of 5 μg total RNA, hybridized either to a 32P-labeled 300-bp PCR-generated UCP probe or a 32P-labeled cDNA for the rat 28S ribosomal subunit. (S. B. Smith and G. E. Carstens, unpublished data.)

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Fig. 7. Brown adipose tissue from newborn lambs exposed to 28°C (left) or 6°C (right) for 48 h postnatally. Thick arrows indicate lipid droplets and thin arrows indicate mitochondria. Adipocytes from warm-exposed lambs are multilocular, whereas adipocytes from cold-exposed lambs are nearly devoid of lipid. Mitochondria are less numerous in BAT from warm-exposed lambs. Cristae structure is becoming disrupted in BAT from warm-exposed lambs, indicating dedifferentiation of brown adipocytes. Scale bars are indicated for the lowest and highest magnifications. (S.B. Smith and G.E. Carstens, unpublished data.)

mitochondria, as perirenal adipocytes acquired the morphology of white adipocytes. These data provided evidence to suggest that, prenatally, adipocytes from all depots may initially differentiate as BAT, and that they subsequently involute to acquire WAT morphological characteristics. Our histological examination of Angus and Brahman s.c. adipose tissue (Martin et al., 1999; fig. 8) tends to support this postulate. Although most adipocytes have already acquired WAT characteristics by parturition, we located several adipocytes with distinctly

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Fig. 8. Subcutaneous adipose tissue from Angus (left column) and Brahman (right column) newborn calves. Cells with brown adipocyte morphology (arrows) and white adipocyte morphology (arrowheads) are apparent. A capillary endothelial cell (CE) is centrally located in the Brahman sample. Mitochondrial size and cristae density (bottom panels) are indicative of brown adipocytes, with no apparent difference between breed types. Scale bars are indicated for the lowest and highest magnifications. Adapted from Figure 5 of Martin et al. (1999).

brown adipocyte morphology, i.e. extensive, highly differentiated mitochondria surrounding a smaller lipid vacuole. This could be interpreted to mean that s.c. adipose tissue initially differentiated as BAT, and we are observing the involution of BAT to WAT initiated prenatally. However, we cannot rule out the possibility that brown adipocytes were lost to the total population of adipocytes via apoptosis, a process not detectable in transmission electron photomicrographs. Lindquist and Rehnmark (1998) demonstrated that transferring cold-adapted mice to 28°C caused a rapid increase in the rate of apoptosis in interscapular BAT. Murine brown

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adipocytes exposed to NE in culture exhibited a 50% decline in DNA fragmentation (i.e. apoptosis) (Lindquist and Rehnmark, 1998). The latter results demonstrate the involvement of sympathetic innervation of BAT in maintaining the differentiated state. These data also suggest that apoptosis in BAT may have occurred in our investigation of lambs held at warm vs cold temperatures (Smith et al., 2004). The maintenance of the BAT differentiated state by NE has been demonstrated previously. Norepinephrine induces the expression of UCP1 and may increase of number of brown adipocytes (i.e. be mitogenic; Nedergaard et al., 1995). Conversely, cold exposure maintains the BAT viability by reducing the rate of DNA and protein degradation (Desautels and Heal, 1999).

7. NUTRITION AND BROWN ADIPOSE TISSUE THERMOGENESIS 7.1. Dietary protein restriction Malnutrition of the dam during late gestation has been shown to reduce neonatal calf survival (Hight, 1966; Corah et al., 1975). The inability of the neonate to maximize thermogenesis in response to cold stress during the early postnatal period may be caused by prepartum protein and/or energy malnutrition. Previous studies have demonstrated that prepartum protein (Carstens et al., 1987) and energy (Ridder et al., 1991) restriction of nulliparous heifers reduced thermoneutral metabolism in newborn calves. Carstens et al. (1987) reported that prepartum protein restriction reduced thermoneutral metabolic rates by 11.4%, even though birth weights were unaffected by prepartum protein treatment. Unlike previous reports, the thermoneutral metabolic rate of Wagyu × Angus newborn calves was not affected by prepartum protein restriction (Martin et al., 1997). Consistent with the lack of a treatment effect on peak metabolic rates, prepartum protein restriction did not affect perirenal adipose tissue mass or composition. Nor did prepartum protein restriction alter UCP1 gene expression in BAT. Alexander (1978) fed high- and low-energy diets to pregnant ewes, beginning on day 90 of gestation, and found that prepartum energy restriction reduced the proportional weight of perirenal adipose tissue (the primary BAT depot in newborn ruminants) by 17% in single and 24% in twin fetuses at 125 days of gestation. Furthermore, Tyzbir (1984) demonstrated that prepartum protein restriction of rats reduced BAT mass by 40–50% as well as BAT mitochondrial thermogenic capacity in newborn rat pups, even though birth weights were not affected by prepartum protein restriction. In contrast, the NE-induced peak metabolic rate was the same in calves born to adequate- and restricted-protein heifers in the investigation of Martin et al. (1997). The results of Martin et al. (1997) may have been confounded by the unusual breed type of calves used in that study (Wagyu × Angus crossbred calves). Wagyu calves have lower birth weights than Angus calves (Smith et al., 1992) and, unlike Angus or Brahman purebred calves, subcutaneous adipose tissue of Wagyu crossbred calves contains no detectable brown adipocytes at birth (Martin et al., 1997, 1999). Wagyu calves represent a distinct genetic line resulting from crossbreeding of native Japanese cattle in the mid-nineteenth century (Smith et al., 2001). Thus, the lack of effect of protein malnutrition on fetal calf thermogenesis reported by Martin et al. (1997) should not be considered typical for this species. 7.2. Essential fatty acid supplementation Several studies have shown that brown fat thermogenic rates were higher and sympathetic activity of brown fat increased (higher norepinephrine turnover rates) in rats fed diets containing

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safflower oil (high in polyunsaturated fatty acids; PUFA) compared to rats fed diets containing beef tallow (high in saturated fatty acids; SFA) (Takeuchi et al., 1995a; Matsuo et al., 1995). Additionally, Takeuchi et al. (1995b) found that serum T3 levels were higher in rats fed a safflower-oil diet compared to those fed a beef tallow-fat diet. Lammoglia et al. (1999) demonstrated that prenatal supplementation of cracked safflower seeds to pregnant cows affected cold tolerance of newborn calves (fig. 9). Calves were exposed to cold ambient temperatures starting at 4 h of age. Rectal temperatures in the cold were significantly higher in calves born to cows supplemented with safflower seeds than calves born to cows fed the control diet containing no added fat. Taken together, these observations suggest that maternal supplementation of a bypass source of PUFA to pregnant ewes may have the potential to enhance fetal BAT development. Additionally, studies using fish oils have indicated that the n-3 PUFA eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA) also are effective in stimulating BAT thermogenesis (Sadurskis et al., 1995; Oudart et al., 1997; Saha et al., 1998; Kawada et al., 1998). Polyunsaturated fatty acids, and especially n-3 PUFA, stimulate nonshivering thermogenesis in rodents, and they may do so by increasing NE turnover rate in BAT. Exposure of BAT to NE reduces the extent of apoptosis in response to warm exposure, which in turn should cause elevated thermogenesis relative to animals fed beef tallow or no added n-3 PUFA. Therefore, n-3 PUFA may increase thermogenesis by depressing apoptosis, or even stimulating brown adipocyte differentiation. This is in contrast to results with splenic lymphocytes (Avula et al., 1999) and tumor cells (Das, 1999), in which n-3 PUFA promoted apoptosis, and indicates a tissue-specific effect of n-3 PUFA on BAT. We recently fed pregnant ewes 2%, 4%, or 8% rumen-protected fat. The fat sources were high in either saturated/monounsaturated fatty acids or n-3 PUFA (formaldehyde-protected soy/linseed lipid). The PUFA-fed ewes had higher plasma concentrations of 18:2, 18:3n-3, and EPA, and lower concentrations of 16:0, 16:1, and 18:1, than ewes fed the saturated/ monounsaturated fatty acid diet. The BAT of lambs born to PUFA-fed ewes had higher

Fig. 9. Effects of feeding supplemental fat (safflower seeds) during late gestation on cold tolerance of newborn calves. Adapted from Figure 1 of Lammoglia et al. (1999).

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concentrations of 18:2, EPA, and DHA than lambs born to ewes fed the saturated/monounsaturated fatty acid diet. However, BAT mass, cytochrome c oxidase activity, and GDP binding were not affected by level or source of dietary fat. Cold-induced rectal temperature responses of lambs were not affected by source of prenatal fat. Therefore, unlike results with calves (Lammoglia et al., 1999), prenatal PUFA supplementation did not affect BAT thermogenic activity or cold tolerance of newborn lambs. The effects of DHA or EPA on BAT thermogenesis in lambs cannot be tested until a rumen-bypass source of these fatty acids is developed. 7.3. Copper supplementation Although few studies have investigated the impact of prenatal dietary copper on thermometabolism, clinical evidence of a link between copper deficiency and cold intolerance exists in lambs. Copper plays an essential role in several copper-dependent enzyme systems that regulate thermometabolism, including cytochrome c oxidase and dopamine-β-hydroxylase. Cytochrome c oxidase is the terminal enzyme in the electron transport system linking substrate oxidation to oxidative phosphorylation (ATP synthesis) in mitochondria. Another copper-dependent enzyme, dopamine-β-hydroxylase, regulates the synthesis of NE from dopamine in the sympathetic nervous system. We examined the effects of prenatal dietary copper level on thermometabolism in lambs (Carstens et al., 1999). Twin-bearing ewes were assigned to low- or high-copper treatments during the last trimester of gestation. Even though liver copper concentrations in newborn lambs were reduced 57% by the low-copper treatment (132 vs. 306 ppm copper DM), these lambs would not be classified as being copper-deficient. Despite the fact that the low-copper lambs were not copper-deficient, their rectal temperatures at 2 h of age were 3.3°F lower than lambs born to high-copper ewes (fig. 10). We subsequently found that NE turnover rates in BAT of lambs at 12 h of age were decreased by the low-copper treatment (0.16 vs 0.3 ng NE/mg BAT/h). This suggests that the low-copper treatment decreased dopamineβ-hydroxylase enzyme activity which impaired the thermogenic function of BAT. Additional evidence to support this idea is the finding that low-copper lambs also had lower plasma T3 levels compared to high-copper lambs (fig. 10), even though plasma T4 levels were unaffected by prenatal copper treatment. An important regulatory aspect of BAT thermogenesis is the activation of 5′-deiodinase by NE release from the sympathetic nervous system in response to cold stress. Locally synthesized T3 is a potent regulator of uncoupling protein gene expression in BAT. Because more than 60% of circulating T3 levels in newborn lambs are derived from the conversion of T4 to T3 in peripheral tissues by 5′-deiodinase enzyme (Klein et al., 1980), the fact that plasma T3 levels were depressed in low-copper lambs suggests that 5′-deiodinase activity may have been impaired by the low-copper treatment indirectly through a reduction in NE stimulation.

8. FUTURE PERSPECTIVES Research continues to describe the development and metabolism of brown adipose tissue in lambs and calves. This is important in respect to both the welfare of the newborn animals and the economic impact of neonatal mortality to the livestock industry. Ideally, production strategies such as supplemental n-3 PUFA or copper would improve BAT functionality and thereby increase newborn lamb or calf survival. Conversely, strategies to increase BAT mass in the neonate may lead to increased adiposity in the mature animal if brown adipocytes dedifferentiate into white adipocytes during development.

Fig. 10. Effect of prenatal copper treatment on rectal temperature (left panel) and plasma triiodothyronine concentrations (right panel) in newborn lambs. From Figure 1 of Carstens et al. (1999).

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REFERENCES Alexander, G., 1978. Quantitative development of adipose tissue in foetal sheep. Aust. J. Biol. Sci. 31, 489–503. Alexander, G., Bennett, J.W., Gemmell, R.T., 1975. Brown adipose tissue in the new-born calf (Bos taurus). J. Physiol. 244, 223–234. Avula, C.P., Zaman, A.K., Lawrence, R., Fernandes, G., 1999. Induction of apoptosis and apoptotic mediators in Balb/C splenic lymphocytes by dietary n-3 and n-6 fatty acids. Lipids 34, 921–927. Azzam, S.M., Kinder, J.E., Nielsen, M.K, 1993. Environmental effects on neonatal mortality of beef calves. J. Anim. Sci. 71, 282–290. Ballard, F.J., Filsell, O.H., Jarrett, I.G., 1972. Effects of carbohydrate availability on lipogenesis in sheep. Biochem. J. 226, 193–200. Bianco, A.C., Silva, J.E., 1988. Cold exposure rapidly induces virtual saturation of brown adipose tissue nuclear T3 receptors. Amer. J. Physiol. 255, E496–E503. Brander, F., Keith, J.S., Trayhurn, P., 1993. A 27-mer oligonucleotide probe for the detection and measurement of the mRNA for uncoupling protein in brown adipose tissue of different species. Comp. Biochem. Physiol. 104B, 125–131. Carstens, G.E., 1994. Cold thermoregulation in newborn calves. Vet. Clin. N. Amer.-Food Anim. Pr. 10, 69–106. Carstens, G.E., Eckert, J.C., Greene, L.W., Smith, S.B., 1999. Effects of prenatal dietary copper on endocrine control of brown fat thermogenesis in newborn lambs. In: Proceedings of IX Symposium Ruminant Physiology. S. Afr. J. Anim. Sci. 29, 429–430. Carstens, G.E., Johnson, D.E., Holland, M.D., Odde, K.G., 1987. Effects of prepartum protein nutrition and birth weight on basal metabolism in bovine neonates. J. Anim. Sci. 65, 745–751. Carstens, G.E., Mostyn, P.C., Chapman, S.A., Randel, R.D., 1995. Prenatal and postnatal changes in brown adipose tissue thermogenesis in the neonatal calf. In: Energy Metabolism of Farm Animals. EAAP Publ. No. 76. Pudoc, Wageningen, The Netherlands, pp. 101–104. Casteilla, L., Champigny, O., Bouillaud, F., Robelin, J., Ricquier, D., 1989. Sequential changes in the expression of mitochondrial protein mRNA during the development of brown adipose tissue in bovine and ovine species: sudden occurrence of uncoupling protein mRNA during embryogenesis and its disappearance after birth. Biochem. J. 257, 665–671. Casteilla, L., Muzzin, P., Revelli, J.P., Ricquier, D., Giacobino, J.P., 1994. Expression of β1- and β3-adrenergic-receptor messages and adenylate cyclase β-adrenergic response in bovine perirenal adipose tissue during its transformation from brown to white fat. Biochem. J. 297, 93–97. Champigny, O., Ricquier, D., Blondel, O., Mayers, R.M., Briscoe, M.G., Holloway, B.R., 1991. β3 adrenergic receptor stimulation restores message and expression of brown-bat mitochondrial uncoupling in adult dogs. Proc. Natl. Acad. Sci. USA 88, 1074–1077. Corah, L.R., Dunn, T.G., Kaltenbach, C.C., 1975. Influence of prepartum nutrition on the reproductive performance of beef females and the performance of their progeny. J. Anim. Sci. 41, 819−824. Das, U.N., 1999. Essential fatty acids, lipid peroxidation and apoptosis. Prostagland. Leuk. Essent. Fatty Acids 61, 157–163. Desautels, M., Heal, S., 1999. Differentiation-dependent inhibition of proteolysis by norepinephrine in brown adipocytes. Amer. J. Physiol. 277, E215–Ε222. Géloën, A., Collet, A.J., Guay, G., Bukowiecke, L.J., 1988. β-adrenergic stimulation of brown adipocyte proliferation. Am. J. Physiol. 254, C175–C182. Giralt, M., Casteilla, L., Viñas, O., Mampel, T., Iglesias, R., Robelin, J., Villarroya, R., 1989. Iodothyronine 5′-deiodinase activity as an early event of prenatal brown-fat differentiation in bovine development. Biochem. J. 259, 555–559. Hight, G.K., 1966. The effects of undernutrition in late pregnancy on beef cattle production. NZ J. Agr. Res. 9, 479–490. Himms-Hagen, J., 1986. Brown adipose tissue and cold-acclimation. In: Trayhurn, T., Nicholls, D.G. (Eds.), Brown Adipose Tissue. Edward Arnold, Baltimore, MD, pp. 214–268. Ingle, D.L., Bauman, D.E., Garrigus, U.S., 1972. Lipogenesis in the ruminant: in vivo site of fatty acid synthesis in sheep. J. Nutr. 102, 617–624. Kawada, T., Kayahashi, S., Hida, Y., Koga, K., Nadachi, Y., Fushiki, T., 1998. Fish (Bonito) oil supplementation enhances the expression of uncoupling protein in brown adipose tissue. J. Agr. Food Chem. 46, 1225–1227.

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Klein, A.H., Oddie, T.H., Fisher, D.A., 1980. Iodothyronine kinetic studies in the newborn lamb. J. Dev. Physiol. 2, 29–36. Lammoglia, M.A., Bellows, R.A., Grings, E.E., Bergman, J.W., 1999. Effects of prepartum supplementary fat and muscle hypertrophy genotype on cold tolerance in newborn calves. J. Anim. Sci. 77, 2227–2233. Landis, M.D., Carstens, G.E., McPhail, E.G., Randel, R.D., Green, K.K., Slay, L., Smith, S.B., 2002. Ontogenic development of brown adipose tissue in Angus and Brahman fetal calves. J. Anim. Sci. 80, 591–601. Lindquist, J.M., Rehnmark, S., 1998. Ambient temperature regulation of apoptosis in brown adipose tissue. J. Biol. Chem. 273, 30147–30156. Martin, G.S., Carstens, G.E., Taylor, T.L., Eli, A.G., Tarrant, M., Britain, K., Smith, S.B., 1999. Metabolism and morphology of brown adipose tissue from Brahman and Angus newborn calves. J. Anim. Sci. 77, 388–399. Martin, G.S., Carstens, G.E., Taylor, T.L., Sweatt, C.R., Eli, A.G., Lunt, D.K., Smith, S.B., 1997. Prepartum protein restriction does not alter norepinephrine-induced thermogenesis or brown adipose tissue function in newborn calves. J. Nutr. 127, 1929–1937. Matsuo, T., Shimomura, Y., Saitoh, S., Tokuyama, K., Takeuchi, H., Suzuki, M., 1995. Sympathetic activity is lower in rats fed a beef tallow diet than in rats fed a safflower diet. Metabolism 44, 934–939. Mersmann, H.J., 1996. Evidence of classic beta β3-adrenergic receptors in porcine adipocytes. J. Anim. Sci. 74, 984–992. Milner, R.E., Wilson, S., Arch, J.R.S., Trayhurn, P., 1988. Acute effects of a β-adrenoceptor agonist (BRL 26830A) on rat brown adipose tissue mitochondria: increased GDP binding and GDP-sensitive proton conductance without changes in the concentration of uncoupling protein. Biochem. J. 249, 759–763. Moulin, K., Arnaud, E., Nibbelink, M., Viguerie-Bascands, N., Pénicaud, L., Casteilla, L., 2001a. Cloning of BUG demonstrates the existence of a brown adipocyte distinct from a white one. Int. J. Obes. 25, 1413–1441. Moulin, K., Truel, N., André, M., Arnaud, E., Nibbelink, M., Cousin, B., Dani, C., Pénicaud, L, Casteilla, L., 2001b. Emergence during development of the white-adipocyte cell phenotype is independent of the brown-adipocyte phenotype. Biochem. J. 356, 659–664. Napolitano, L., 1963. The differentiation of white adipose cells: an electron microscopy study. J. Cell Biol. 18, 663–679. Nedergaard, J., Connolly, E., Cannon, B., 1986. Brown adipose tissue in the mammalian neonate. In: Trayhurn, P., Nicholls, D.G. (Eds.), Brown Adipose Tissue. Edward Arnold, London, pp. 152–213. Nedergaard, J., Herron, D., Jacobsson, A., Rehnmark, S., Cannon, B., 1995. Norepinephrine as a morphogen? Its unique interaction with brown adipose tissue. Int. J. Dev. Biol. 39, 827–837. Nougues, J., Reyne, Y., Champigny, O., Holloway, B., Casteilla, L., Ricquier, D., 1993. The β3-adrenoceptor agonist ICI-D7114 is not as efficient on reinduction of uncoupling protein mRNA in sheep as it is dogs and smaller species. J. Anim. Sci. 71, 2388–2394. Okamoto, M., Robinson, J.B., Christopherson, R.J., Young, B.A., 1986. Summit metabolism of newborn calves with and without colostrum feeding. Can. J. Anim. Sci. 66, 937–944. Oudart, H., Groscolas, R., Calgari, C., Nibbelink, M., Leray, C., Le Mayo, Y., Malan, A., 1997. Brown fat thermogenesis in rats fed high-fat diets enriched with n-3 polyunsaturated fatty acids. Int. J. Obes. 21, 955–962. Puig-Domingo, M., Guerrero, J.M., Vaughan, M.K., Little, J.C., Reiter, R.J., 1989. Activation of cerebrocortical type II 5′-deiodinase activity in Syrian hamsters kept under short photoperiod and reduced ambient temperature. Brain Res. Bull. 22, 975–979. Ridder, T.A., Young, J.W., Anderson, K.A., Lodman, D.W., Odde, K.G., Johnson, D.E., 1991. Effects of prepartum energy nutrition and body condition on birthweight and basal metabolism in bovine neonates. J. Anim. Sci. 69, Suppl. 1, 450. Robinson, J.B., Young, B.A., 1988a. Metabolic heat production of neonatal calves during hypothermia and recovery. J. Anim. Sci. 66, 2538–2544. Robinson, J.B., Young, B.A., 1988b. Recovery of neonatal lambs from hypothermia with thermal assistance. Can. J. Anim. Sci. 68, 183–190. Sadurskis, I., Dicker, A., Cannon, B., Nedergaard, J., 1995. Polyunsaturated fatty acids recruit brown adipose tissue: increased UCP content and NST capacity. Amer. J. Physiol. 269, E351–E360.

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Saha, S.K., Ohinata, H., Ohno, T., Kuroshima, A., 1998. Thermogenesis and fatty acid composition of brown adipose tissue in rats rendered hyperthyroid and hypothyroid – with special reference to docosahexanoic acid. Jpn. J. Physiol. 48, 355–364. Smith, S.B., Carstens, G.E., Randel, R.D., Mersmann, H.J., Lunt, D.K., 2004. Brown adipose tissue development and metabolism in ruminants. J. Anim. Sci. 82, 942–954. Smith, S.B., Prior, R.L., 1986. Comparison of lipogenesis and glucose metabolism between ovine and bovine adipose tissues. J. Nutr. 116, 1279–1286. Smith, S.B., Sanders, J.O., Lunt, D.K., 1992. Evaluation of birth and weaning characteristics of halfblood and three-quarter blood Wagyu-Angus calves. McGregor Field Day Report, Texas Agric. Exp. Station Tech. Rep. 92-1, pp. 60–64. Smith, S.B., Zembayashi, M., Lunt, D.K., Sanders, J.O., Gilbert, C.D., 2001. Carcass traits and microsatellite distributions in offspring of sires from three geographical regions of Japan. J. Anim. Sci. 79, 3041–3051. Stott, A.W., Slee, J., 1985. The effect of environmental temperature during pregnancy on thermoregulation in the newborn lamb. Anim. Prod. 41, 341–347. Symonds, M.E., Bryant, M.J., Clarke, L., Darby, C.J., Lomax, M.A., 1992. Effect of maternal cold exposure on brown adipose tissue and thermogenesis in the neonatal lamb. J. Physiol. 455, 487–502. Takeuchi, J., Matsuo, T., Tokuyama, K., Shimomura, Y., Suzuki, M., 1995a. Diet-induced thermogenesis is lower in rats fed a lard diet than in those fed a high oleic sunflower oil diet, a safflower oil diet or a linseed oil diet. J. Nutr. 125, 920–925. Takeuchi, J., Matsuo, T., Tokuyama, K., Suzuki, M., 1995b. Serum triiodothyronine concentration and Na+,K+-ATPase activity in liver and skeletal muscle are influenced by dietary fat type in rats. J. Nutr. 125, 2364–2369. Trayhurn, P., Thomas, M.E.A., Keith, J.S., 1993. Postnatal development of uncoupling protein, uncoupling protein mRNA and GLUT4 in adipose tissues of goats. Amer. J. Physiol. 265, R676–R682. Tyzbir, R.S., 1984. Altered brown adipose tissue mitochondrial function in neonates born to rats overfed foods of various protein contents. J. Nutr. 114, 234–237. Vernon, R.G., Robertson, J.P., Clegg, R.A., Flint, D.J., 1981. Aspects of adipose-tissue metabolism in foetal lambs. Biochem. J. 196, 819–824.

13

Interorgan lipid and fatty acid metabolism in growing ruminants J. K. Drackley Department of Animal Sciences, University of Illinois, Urbana, IL 61801, USA

Lipid metabolism is a dynamic and critically important function in growing ruminants. Preruminants consume a high-fat diet and deposit much of the dietary lipid in adipose tissue. During postweaning growth, dietary fatty acid intake is low and long-chain fatty acid synthesis increases in adipose tissue depots as the animal approaches physiological maturity. Deposition of long-chain fatty acids synthesized within adipose or taken up from blood lipoproteins is extensive in near-mature fattening ruminants. Lipoprotein lipase is a key regulatory enzyme in determining interorgan disposition of long-chain fatty acids from circulating chylomicrons and very low-density lipoproteins in growing ruminants. Considerable research in recent years has characterized the regulation of fatty acid esterification in adipose tissue. The role of molecules such as leptin and tumor necrosis factor α, which are synthesized and secreted by adipose tissue, is only beginning to be elucidated in growing ruminants. The type of dietary fat affects lipid metabolism in liver of preruminants, but surprisingly little is known about the developmental changes in hepatic metabolism of fatty acids in growing ruminants. Although much recent progress has been made in understanding regulation of interorgan lipid metabolism in growing ruminants, many fundamental questions remain.

1. INTRODUCTION Lipid metabolism plays a dynamic role during growth in ruminant animals. Lambs and calves are born with minimal body lipid, accrete body lipid rapidly during suckling of fat-rich milk, undergo minimal fat deposition during the growth phase after weaning, and then again change to fat deposition as the animal approaches physiological maturity. During the suckling or milk-feeding period, the young ruminant (preruminant) functions as a monogastric animal and largely deposits the long-chain fatty acids (LCFA) from milk fat into adipose tissue for storage. As skeletal and muscle growth near completion, adipose storage of triacylglycerol (TG) increases from a combination of both de novo lipogenesis (primarily from ruminally derived acetate) and deposition of absorbed LCFA. Use of LCFA for fuel in well-fed ruminants is

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relatively minimal, except in heart tissue and in skeletal muscle during exercise. Interorgan transport of LCFA is accomplished both by circulation of free or nonesterifed fatty acids (NEFA) and by the various classes of plasma lipoproteins. Metabolism of LCFA and other lipids assumes obvious importance to the growing ruminant as a source of membrane components, signaling molecules, and a reserve of readily available energy. Lipid metabolism occupies a central position in the determination of energetic efficiency of growth and as a result has major impact on the profitability of meat animal production. Moreover, content and composition of carcass lipid has become an increasingly important consideration for consumers of ruminant animal products. Consequently, ruminant lipid metabolism remains of major importance. This chapter highlights some aspects where recent research has improved our understanding of lipid metabolism in growing ruminants. The major focus is on the interorgan relationships of lipid metabolism during growth, and is not intended to be a comprehensive and exhaustive review of the literature. A number of excellent comprehensive and authoritative reviews on various aspects of lipid metabolism in ruminants are available (e.g. Noble, 1978; Bell, 1980; Vernon, 1980; Noble and Shand, 1982; Bauchart, 1993; Chilliard, 1993; Jenkins, 1993) and key reviews are cited where applicable. For a general discussion of lipid metabolism in domestic animals, see Drackley (2000).

2. DIGESTION AND ABSORPTION OF DIETARY LIPIDS 2.1. Preruminants Preruminants fed milk or milk replacer consume a relatively high-fat diet. Bovine milk contains 29–31% fat on a dry solids basis; milk of goats and sheep may contain about 34% and 40% of the solids as fat, respectively (Jenness, 1985). Most commercial milk replacers contain between 12% and 20% fat on a dry solids basis (Davis and Drackley, 1998). Dietary fat in milk or milk replacers consists primarily of TG, which are digested in the small intestine and packaged into chylomicrons for distribution throughout the body. The LCFA of dietary origin are delivered to tissues for oxidative use (primarily heart and skeletal muscle) or for deposition in adipose tissue. The remaining components of the chylomicron particle (cholesterol, cholesterol esters, phospholipids, and apoproteins) participate in additional cycles of lipoprotein metabolism to distribute cholesterol and essential fatty acids (EFA) throughout the body for cell membrane and steroid hormone biosynthesis. Preruminants digest the lipids in milk with high efficiency (typically greater than 97%; Toullec and Mathieu, 1969). A number of other fat sources in milk replacers can be well digested (>90%) by preruminants if properly emulsified, including tallow, lard, and coconut oil (Toullec and Mathieu, 1969; Davis and Drackley, 1998). The milk fat of ruminants contains a relatively large proportion of short- and medium-chain fatty acids, probably as a strategy to maintain fluidity of the fat in the face of the mostly saturated LCFA that reach the maternal duodenum as a result of ruminal biohydrogenation of polyunsaturated LCFA consumed from the herbivorous diet. Milk fat is entrapped in the casein coagulum in the abomasum, and is released as the casein clot undergoes initial digestion. Within the abomasum, fat digestion is initiated by action of an acid lipase secreted into the saliva by glands in the glossoepiglottic or pharyngeal area, which is highly homologous to the gastric lipases of many other species (Gargouri et al., 1989). About one-third of fatty acids in milk TG are hydrolyzed from the glycerol backbone in the abomasum (Edwards-Webb and Thompson, 1978). Activity of the acid lipase has been

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thought to be greater toward the short- and medium-chain fatty acids (Edwards-Webb and Thompson, 1978), although it has been shown recently that the acid lipase has selectivity both for short-chain fatty acids and for fatty acids on the sn-3 position of milk TG (Villenueve et al., 1996). Furthermore, specificity of the enzyme in these regards appears to vary inversely with the same characteristics in mothers’ milk; i.e. enzyme specificity is highest in goat kids, in which the content of short-chain fatty acids is lower than in cows’ milk, and enzyme specificity is lower in the calf where cows’ milk is rich in short-chain fatty acids (Jenness, 1985). The metabolic significance of this preduodenal digestion is not certain but several possibilities exist. Short-chain fatty acids are absorbed into the portal vein and are extensively cleared by the liver. In this regard they are considered “obligate fuels” for the liver (see Chapter 9 by Odle et al., this volume). In preruminants consuming mothers’ milk, a firm coagulum is formed in the abomasum, which slows the flow of casein and long-chain fatty acids from the abomasum. However, lactose and the whey proteins are expelled from the coagulum, and are available for intestinal digestion much more quickly after a meal. Perhaps the initial digestion of milk fat, particularly the hydrolysis of short-chain fatty acids, provides a readily available fuel for the liver to spare amino acids derived from digestion of whey proteins for protein synthesis, and to provide the ATP necessary to drive that protein synthesis. Moreover, the initial hydrolysis of short-chain fatty acids from milk fat may increase emulsification of fat droplets in the intestine (Armand et al., 1994) and may increase subsequent hydrolysis by pancreatic lipases in the small intestine (Borel et al., 1994). While the role of gastric or preduodenal lipases in total fat digestion has been shown to be larger than previously considered (Gooden, 1973), this may be especially true in the young preruminant in which pancreatic lipase activity is immature. The whey protein β -lactoglobulin may bind and remove fatty acids from the acid lipase to prevent end-product inhibition (Perez et al., 1992), although the extent to which this occurs is questionable given that β -lactoglobulin is not retained in the abomasal coagulum. While intestinal hydrolysis of the partially hydrolyzed dietary lipids traditionally has been considered to occur by action of the colipase-dependent pancreatic lipase, recent evidence has demonstrated that the pancreas of most mammalian species also secretes a bile salt-activated lipase (Wang and Hartsuck, 1993). This enzyme recently has been purified, characterized, and sequenced from bovine pancreas (Tanaka et al., 1999). The enzyme exerts considerable catalytic activity toward TG in the presence of bile salts, and has basal activity toward shorter-chain TG even in the absence of bile salts. The enzyme also is active in catalysis of phospholipids; indeed, the enzyme activity was first identified in the bovine as a lysophosholipase (van den Bosch et al., 1993). In addition, the enzyme also hydrolyzes cholesterol esters, phosphatidylcholine, and fatty acid esters of vitamins A and E (Wang and Hartsuck, 1993). The latter roles may be the more physiologically relevant in preruminants; alternatively, the apparent redundancy of having two distinct pancreatic enzymes active toward TG may be yet another example of ensuring that almost all of the dietary TG presented to the young animal is hydrolyzed for absorption. A number of fat sources are used in milk replacers (milk substitutes) fed to young preruminants. Commonly used sources include tallow, lard, and coconut oil (Davis and Drackley, 1998). Vegetable oils such as corn oil, soybean oil, and sunflower oil are not widely used because of early research demonstrating that calves grew poorly and developed diarrhea (scouring) when fed milk replacers containing vegetable oils (Toullec and Mathieu, 1969; Jenkins et al., 1985, 1986). Canola oil did not seem to cause scouring (Jenkins et al., 1986). Subsequent research by the same group suggested that the scouring in earlier studies may have been a result of improper emulsification so that lipid particle size was too large (Jenkins, 1988). Consequently, the degree to which animal-derived fats could be replaced with more

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highly unsaturated vegetable sources, if properly emulsified, without compromising calf health or performance would appear to be an unresolved issue. Free fatty acids also are less acceptable as lipid sources for young preruminants than TG of the same fatty acid composition (Jenkins et al., 1985). Although emulsification and droplet size may have been concerns in that study, free fatty acids clearly inhibit feed intake in young calves (Spanski et al., 1997) as well as in adult cows (Drackley et al., 1992; Christensen et al., 1994; Bremmer et al., 1998). This effect is specific for unsaturated rather than saturated fatty acids (Drackley et al., 1992; Christensen et al., 1994; Bremmer et al., 1998), and is dosedependent (Overton et al., 1998; Drackley et al., 2000). Presence of the unsaturated free fatty acids in the duodenum interacts with receptors that in turn signal through glucagon-like peptide-1 and perhaps cholecystokinin (Drackley et al., 2000; Benson and Reynolds, 2001) to decrease feed intake. Because dietary TG are not hydrolyzed until more distal regions of the jejunum, past the site of greatest density of these neuroendocrine cells, unsaturated TG do not inhibit feed intake as potently as do the same LCFA provided as free acids (Bremmer et al., 1998). This explains why decreases of dry matter intake have been more pronounced with postruminal administration of much smaller quantities of free fatty acids than when much larger quantities of unsaturated TG were administered postruminally (e.g. Gagliostro and Chilliard, 1991; Drackley et al., 2000). Digestibility of the free fatty acids is high (Bremmer et al., 1998) and similar to that of TG in young calves, at least when some TG is present to furnish 2-monoglycerides in the intestine (Spanski et al., 1997). 2.2. Ruminants Digestion and absorption of LCFA in ruminants have been addressed in a number of reviews (Noble, 1978; Moore and Christie, 1984; Bauchart, 1993; Jenkins, 1993; Doreau and Chilliard, 1997) and will be discussed only briefly here. Lipid digestion in ruminants begins in the rumen (see Jenkins, 1993, for review). A number of ruminal microorganisms are actively lipolytic, resulting in extensive hydrolysis of most dietary complex lipids. The resulting free fatty acids undergo varying degrees of biohydrogenation, with production of the saturated stearic acid, as well as smaller quantities of trans-unsaturated monoenes and dienes that leave the rumen for absorption. The microbial population also synthesizes a variety of fatty acids, principally odd-chain and branched-chain acids of 15 to 17 carbons, by elongating shorter-chain fatty acids (<14 carbons) from the diet. The microbes also synthesize phospholipids. Lipids in digesta reaching the postruminal tract consist mainly of mostly saturated free fatty acids adsorbed to the surface of feed particles and bacteria, with the remainder being predominantly phospholipids and sterol esters as components of microbial cells. Because of the low pH in the abomasum and duodenum of ruminants (pH 2.0–2.5), the free fatty acids exist in the protonated state, which facilitates their adsorption to the surface of feed particles. The strong detergent properties of bile salts secreted in the upper duodenum serve to desorb LCFA from particulate matter, with formation of a liquid crystalline phase. Subsequent formation of lysophosphatidylcholine (lysolecithin) from phosphatidylcholine (lecithin, from bile or from acid-mediated disruption of rumen microbial cells) by pancreatic phospholipase A2 promotes formation of micelles. Stable micelles formed from LCFA, lysolecithin, and bile salts function to move lipid across the unstirred water layer of the small intestinal epithelium, where absorption of free LCFA and lysolecithin can occur by diffusion. Stearic acid is the predominant lipid to reach the small intestine. Ruminants are able to absorb saturated LCFA such as palmitic acid and stearic acid with substantially greater efficiency than nonruminants (Moore and Christie, 1984). One reason for this adaptation is the

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reliance on lysolecithin as the major micelle stabilizer. Swelling amphiphiles are substances that can expand the volume of bile salt micelles and their hydrophobic interior in the aqueous environment of the small intestinal lumen (Small, 1968). Of all the naturally occurring swelling amphiphiles (monoglycerides, medium-chain fatty acids, long-chain unsaturated fatty acids, phospholipids, and lysophospholipids), lysolecithin is the most efficient at increasing the solubility of stearic acid. For example, lysolecithin increases the partitioning of stearic acid into the micelle by 115%, compared with only 36% for 1-monolein (Freeman, 1984). A second factor contributing to greater absorption of long-chain saturated fatty acids in ruminants is the lower pH in the upper small intestine, ranging from about 3.0 in the duodenum to about 6.0 in the mid-jejunum. The relatively low pH helps minimize formation of calcium soaps of palmitate and stearate, which has long been known to decrease absorption of these fatty acids in nonruminants (Cheng et al., 1949). In ruminants, the major bile salt is taurocholate rather than glycocholate; the lower pKa of taurocholate (2.0) is likely an advantage since it is less likely to become insoluble in the acid conditions of the ruminant small intestine than is glycocholate (pKa 4.7; Harrison and Leat, 1975). True digestibility of LCFA generally is quite high in ruminants (Moore and Christie, 1984). Based on results of Palmquist (1991), true digestibility of LCFA may decline with increasing LCFA intake. As discussed by Bauchart (1993), this decrease suggests that pancreatic phospholipase activity and bile lipids (phospholipids and bile salts) may become limiting for absorption of large dietary loads of LCFA. 2.3. Changes during the weaning transition from preruminant to ruminant During the weaning transition, the young animal changes to a relatively low-fat diet, in which dietary lipids may constitute only 2–6% of dry matter. These lipids typically consist of galactolipids and phospholipids from forages and TG from cereals and oilseeds (Noble, 1978). During the weaning transition, the dietary supply of glucose largely ceases as rumen microbial fermentation of dietary carbohydrates is established. The resultant short-chain or volatile fatty acids (VFA), particularly acetate, become the major oxidative fuel for most tissues of the ruminant. Acetate also becomes the major precursor for lipogenesis. As the young ruminant begins to consume solid food, the ruminal tissue function, capacity, and microbial activity increases (see Davis and Drackley, 1998), leading eventually to the pattern discussed for ruminants. During the transition period when the young animal is receiving both a liquid diet and dry feed, lipids in the liquid (milk) diet continue to reach the abomasum and small intestine via closure of the esophageal groove. Few data are available that quantify LCFA absorption during the transition from preruminant to ruminant. Spanski et al. (1997) found that apparent total tract digestibility of LCFA by calves fed a control (starter) diet averaged about 82% and did not differ among measurements at 6, 8, and 10 weeks of age. Digestibility of LCFA when a liquid supplement was provided that contained either lard TG or a mixture of lard TG and free fatty acids from lard was not appreciably different from LCFA digestibility of the control diet. An enigma exists for transitioning ruminants, as well as for pigs, in that digestibility and utilization of LCFA is markedly less after weaning to dry feed than for LCFA use from the liquid diet before weaning. Indeed, these changes seem to occur as abruptly as the weaning process itself. Before weaning preruminants consume a diet in which a major portion of dietary lipid is provided in milk or milk replacer, which may contain from 15% to 30% fat on a dry solids basis. After weaning, dry starter diets may contain only 3% to 6% dietary fat, yet digestibility and use are much lower even with the lower fat content. For example, Fallon et al. (1986) showed that average daily gain (ADG) of body weight decreased as a fat

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supplement (calcium soaps of palm oil fatty acids) was increased from 0 to 20% of the starter formulation. What factors suddenly become limiting for use of fat? A portion of the decreased performance might be attributed to interference by fat with ruminal fermentation; however, differences persist even when fats that should be relatively inert in the young rumen are fed (Fallon et al., 1986). Increasing fat content of milk replacer or starter decreases development of starter intake (Kuehn et al., 1994). In contrast, increasing fat in a liquid diet (as in veal production) increases ADG, whereas increasing fat in starter decreases ADG (Doppenberg and Palmquist, 1991). Because of ruminal hydrolysis of dietary lipids, substantial quantities of free fatty acids reach the upper small intestine when young ruminants consume starter feeds; the presence of free fatty acids in the duodenum may exert negative feedback on appetite as described earlier. Likely of major importance is the nature of the fat in the feed, i.e. a highly emulsified milk fat or fat in milk replacer vs. fat incorporated into a solid feed matrix. A limiting factor may be that the compounds needed to emulsify lipid and form micelles in the small intestinal lumen may be secreted in insufficient amounts in the young ruminant. For example, as discussed earlier, when TG are no longer being hydrolyzed to 2-monoglycerides in the intestine, lysophosphatidylcholine becomes the predominant stabilizing compound of mixed micelles (Freeman, 1984). Whether biliary secretion of phospholipids to provide substrate for phospholipase-mediated production of lysophospholipids is limiting, or whether phospholipase activity itself might be limiting during this transition, has not been investigated. Gooden (1973) determined that conversion of lecithin to lysolecithin occurred much more rapidly in intestinal contents of ruminating calves compared with 1- to 2-week old calves, although much of the lower rate of conversion in milk-fed calves may have been from the presence of milk TG. The possibility that hepatic synthesis and secretion of bile salts might be inadequate to disperse lipids for micelle formation also does not appear to have been investigated in young ruminants.

3. INTESTINAL METABOLISM AND TRANSPORT OF ABSORBED DIETARY LIPIDS General aspects of intestinal absorption of LCFA, re-synthesis of TG, and synthesis of lipoproteins have been reviewed recently by Phan and Tso (2001). Function of these processes in preruminants and ruminants also has been reviewed (Noble and Shand, 1982; Bauchart, 1993; Doreau and Chilliard, 1997). Absorption of LCFA into intestinal epithelial cells generally has been assumed to occur by simple diffusion into and across the lipid-bilayer membrane, down the concentration gradient maintained by intracellular binding and metabolism. Recently, several putative transporter proteins for LCFA have been identified in other species (Chen et al., 2001). These proteins may facilitate uptake across cell membranes, especially at low extracellular concentrations, and may be subject to metabolic regulation depending on the physiological state of the animal. To date, however, no data are available on the presence and role of such proteins in ruminant tissues. Cytosolic fatty acid binding proteins (FABP) have been identified in most tissues of rodents and other species that have been examined (see Glatz and van der Vusse, 1996, for review). Intracellular FABP activity has been identified in the small intestine of the preruminant calf (Jenkins, 1986). Binding activity in calf intestinal tissue was associated with a protein fraction with molecular weight of approximately 12 kD, which is in the range reported for FABP of other species. Functions of intestinal FABP are not entirely clear, but it is intuitive that FABP binds LCFA as they desorb from the plasma membrane, to keep intracellular

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concentrations of potentially toxic free fatty acids low. In pigs, FABP is induced in the small intestine by colostrum feeding after birth (Reinhart et al., 1992). Evidence has been presented in other tissues and species for possible roles of FABP in directing LCFA to appropriate intracellular sites of metabolism, such as mitochondria or endoplasmic reticulum (see Glatz and van der Vusse, 1996) or nucleus (Wolfrum et al., 2001). Absorbed LCFA are activated by acyl-CoA synthetase located on the outer leaflet of the endoplasmic reticulum (Moore and Christie, 1984). After activation of LCFA to their acyl-CoA esters, they undergo re-synthesis to TG by one of two pathways (fig. 1). In the preruminant that absorbs both free LCFA and 2-monoglycerides, fatty acid esterification proceeds by the monoglyceride pathway, in which the remaining two positions on the glyceride molecule are esterified with acyl-CoA to form TG. In functioning ruminants, no 2-monoglyceride is absorbed; consequently, TG synthesis in the intestine proceeds by the phosphatidate (Kennedy) pathway from glycerol-3-phosphate. While few data are available for ruminants, these pathways are not believed to be major sites for metabolic regulation. Rather, the pathways function to repackage the amount of LCFA presented to the intestine into TG for distribution throughout the body. Teleologically, one can argue that it is prudent for the animal to efficiently take up all LCFA available from the diet at any time, given the possibility that dietary energy sources might not be available at some point in the future. Thus, regulation occurs at disposition and storage sites within the body rather than at the site of assimilation. Triacylglycerol synthesis occurs in concert with synthesis of the TG-rich lipoproteins, chylomicrons and very low-density lipoproteins (VLDL). The general scheme for synthesis and secretion of these lipoproteins has been well described (Tso and Balint, 1986; Davidson and Shelness, 2000; Phan and Tso, 2001), although specific details in ruminants are lacking. The primary structural apolipoprotein (apoprotein) of intestinal TG-rich lipoproteins is apoprotein(apo)-B48, whereas the liver synthesizes primarily apo-B100. Ruminants synthesize apo-B

Fig. 1. Major pathways of esterification of fatty acids to glycerolipids in growing ruminants. Key enzymes involved: (1) glycerophosphate acyltransferase (GPAT), which may be mitochondrial or microsomal; (2) lysophosphatidate acyltransferase; (3) phosphatidate phosphohydrolase; (4) diacylglycerol acyltransferase; and (5) monoacylglycerol acyltransferase. Reactions 1 to 4 constitute the phosphatidate (Kennedy) pathway and are found in adipose tissue, liver, and muscle. Activity of reaction 5 (monoacylglycerol acyltransferase) is largely confined to intestinal enterocytes during digestion of milk triglycerides in preruminants. Pi, inorganic phosphate. Adapted from Drackley (2000) based on Rule (1995).

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proteins similar to those of other species. Chylomicrons and VLDL found in intestinal lymph of ruminants contain only the protein analogous to apo-B48, whereas the VLDL found in plasma contain both apo-B100- and apo-B48-like proteins (Laplaud et al., 1990, 1991). Both forms of the protein are translated from the mRNA produced by a single gene. The shorter form (apo-B48) is produced as a result of a unique post-transcriptional mRNA editing mechanism in intestinal cells (Davidson and Shelness, 2000). The apo-B mRNA of bovine intestine is almost completely edited to the shorter form (95%), whereas less editing (40%) occurs in sheep intestine (Greeve et al., 1993). Apo-B synthesized and secreted by ruminant liver in the form of VLDL contains only the apo-B100-like protein (Laplaud et al., 1991). Whether ruminants synthesize and secrete chylomicrons or just VLDL has been the subject of considerable debate, but clear evidence for the existence of chylomicrons is available in preruminants (Laplaud et al., 1990; Bennis et al., 1992). In functioning ruminants fed typical diets, even those supplemented with fats, it appears that the intestinal cells mainly secrete VLDL (Bauchart, 1993). However, if polyunsaturated LCFA are infused postruminally in large amounts, more of the absorbed LCFA will be transported as TG in chylomicrons rather than VLDL (Harrison et al., 1974). Of interest also is the observation that ruminants, especially calves fed high-fat milk diets, appear to secrete considerable amounts of VLDL into the portal vein rather than into the lymphatics (Bauchart et al., 1989; Laplaud et al., 1990). The ontogeny of ruminant lipoproteins has been characterized by several researchers. No VLDL were detected in plasma of fetal calves near term (Forte et al., 1981). Both low-density lipoproteins (LDL) and high-density lipoproteins (HDL) were present in plasma of fetal calves near term, with LDL being the more abundant lipoprotein class (Forte et al., 1981). Marcos et al. (1991) found that plasma TG decreased steadily from about day 115 to day 265 of gestation in cattle; TG concentrations near term were very low, while apo-B concentrations had decreased less, suggesting that VLDL decreased from mid- to late gestation but that LDL remained unchanged or decreased less. Declining concentrations of LDL were also reported during gestation in fetal lambs (Turley et al., 1996) so that HDL became the major lipoprotein fraction at term (Noble and Shand, 1983). After birth, concentrations of VLDL increase with consumption of colostrum and milk, while LDL concentrations decrease in calves (Jenkins et al., 1988; Marcos et al., 1991). During the suckling period, concentrations of LDL appear to increase in sheep (Turley et al., 1996) and goats (Bennis et al., 1992). With the onset of suckling, HDL increases rapidly and become the predominant lipoprotein class in calves, lambs, and kids (Forte et al., 1981; Noble and Shand, 1983; Jenkins et al., 1988; Marcos et al., 1991; Bennis et al., 1992).

4. TISSUE UTILIZATION OF CIRCULATING TRIGLYCERIDES Triglycerides in circulating chylomicrons and VLDL are hydrolyzed by the enzyme lipoprotein lipase (LPL) found in peripheral tissues. Products of the LPL reaction are free fatty acids (i.e. NEFA) and monoglycerides, which are hydrolyzed by nonspecific lipases associated with peripheral tissues. Activity of LPL creates a locally increased NEFA concentration that increases the likelihood for NEFA uptake by cells, although not all NEFA are taken up by the tissue and NEFA concentration may increase in venous blood draining the tissue bed. The unusual LPL enzyme is a glycosylated protein produced by tissue parenchymal cells (e.g. adipocytes), which is secreted from those cells and becomes active when anchored via heparin sulfate proteoglycans on the inner surface of capillary endothelial cells (see Olivecrona and Olivecrona, 1999, for review). In growing ruminants LPL is found predominantly in adipose tissue, skeletal muscle, and heart. Very low activities of LPL were found in

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small intestine, kidney, spleen, adrenals, ovary, and lung in bovines; the LPL mRNA also was barely detectable in these tissues and was undetectable in brain (Hocquette et al., 1998a). Only trace amounts of LPL activity were found in bovine liver, and the absence of detectable mRNA by Northern analysis indicates that this probably represented LPL from blood retained in the tissue (Hocquette et al., 1998a). Activity and mRNA abundance were higher in heart and masseter muscle than in other oxidative and glycolytic muscles of calves, but LPL activity was lower for bovine muscles in general than for rat muscles in parallel experiments (Hocquette et al., 1998a). Internal adipose depots (perirenal and omental) had greater LPL activity and mRNA abundance than subcutaneous adipose tissue from fattened calves (Hocquette et al., 1998a). Activity of LPL in perirenal adipose tissue was similar in cows and ewes, and LPL is transcribed from similar 3.4 and 3.8 kb mRNA in both species (Bonnet et al., 1998). Extensive research has been conducted on LPL in laboratory animals, farm animals, and humans over the last several decades. Despite this intense scrutiny, details of the regulation of LPL still are not resolved (Olivecrona and Olivecrona, 1999). Because LPL is synthesized by parenchymal cells of the tissue and then transported across the capillary endothelial cells, where the enzyme is anchored on the capillary luminal surface to be active, its synthesis and regulation are understandably complex. Dogma developed on the basis of studies in rats assumes that LPL in adipose tissue is upregulated during positive energy balance, decreases during fasting, and increases on refeeding after a fast, whereas in heart and skeletal muscle the enzyme activity changes less but in opposite direction to that in adipose, i.e. increasing during fasting and decreasing with refeeding (e.g. Sugden et al., 1993; Cortright et al., 1997). In this way, heart and skeletal muscles would receive metabolic priority for use of TG circulating in VLDL derived from liver repackaging of NEFA mobilized from adipose tissue. In both ewes and cows, underfeeding (20% of maintenance requirements) results in decreased LPL activity and mRNA abundance in perirenal adipose tissue. Contrary to the dogma from laboratory animals, however, LPL activity in heart and skeletal muscle also decreases during underfeeding, and increases with refeeding (Bonnet et al., 2000; Faulconnier et al., 2001). Similar changes occur in pigs (Enser, 1973). Bonnet et al. (2000) proposed a linear relationship between the change in LPL activity in cardiac muscle and the ability of liver slices to secrete TG among six species (pig, sheep, guinea pig, rabbit, rat, and chicken), which makes teleological sense in that circulating TG concentrations are maintained during fasting or feed restriction in species that actively secrete VLDL from liver (e.g. chicken, rat, rabbit) but fall in species that do not (e.g. pig, sheep). In ruminants, therefore, during feed restriction the energy needs of heart and skeletal muscle are met more via NEFA and ketone bodies and less from TG. In contrast to the results for ruminants subjected to underfeeding, altering the plane of nutrition above maintenance does not produce the same changes in LPL among tissues. Andersen et al. (1996) fed groups of ewe lambs a diet in amounts to support daily gains of 0.15 or 0.25 kg. Adipose tissue LPL activity was greatest for lambs fed at the highest plane of nutrition, whereas skeletal muscle LPL was highest for lambs grown at the slower rate. For cardiac muscle LPL, rates were greatest for lambs grown at the slowest rate and for a group of lambs fed for compensatory gain (0.33 kg/d) after a period of growth at rates equal to the slowest rate of gain. These data suggest that the concept of reciprocal regulation between adipose and muscle LPL is valid when comparing differences in energy balance above maintenance, but not in more extreme situations where animals are in negative energy balance. The molecular basis for the differences in response of LPL among tissues within species, and across species, is not known. The cDNA for bovine LPL has been cloned (Senda et al., 1987) and the mRNA has been characterized in both sheep and cattle (Hocquette et al., 1998a; Bonnet et al., 1998). In sheep, the 3.4 kb mRNA was predominantly expressed in adipose

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tissue, whereas the 3.8 kb mRNA was predominant in cardiac muscle, with no differential regulation according to nutritional state (Bonnet et al., 2000). Large differences in LPL expression among different muscles (e.g. heart and masseter vs. rectus abdominis) were paralleled by changes in mRNA abundance, suggesting that transcriptional regulation is a major factor in differences among tissues. Activity of LPL and the presence of its mRNA were detected in heart, masseter muscle, and perirenal adipose tissue of bovine fetuses (230–260 days in gestation), but activities and mRNA were lower than in growing calves (Hocquette et al., 1998a). Changes in LPL activity and mRNA in adipose tissues and muscles over the transition from preruminant to ruminant were quantified by Hocquette et al. (2001). Activity of LPL was 2-fold lower in adipose tissue from weaned calves than from milk-fed calves; mRNA abundance was correspondingly lower for weaned calves as well. In contrast, neither LPL activity nor mRNA abundance were different between weaned and milk-fed calves in any of seven skeletal muscles studied, with the notable exception of the masseter muscle. Both LPL activity and mRNA abundance were more than doubled in masseter of weaned calves. Contraction of the masseter, located in the cheek, would increase greatly in weaned calves with the increased frequency and workload of chewing; consequently, LCFA use as oxidative fuel might be expected. Activity, but not mRNA abundance, of LPL tended to decrease in heart from weaned calves. Together, the available data implicate a role for LPL in controlling use of TG-FA by growing ruminants. Indeed, for well-fed growing ruminants, Pethick and Dunshea (1993) calculated that most of the NEFA flux was derived from hydrolysis of lipoprotein TG by LPL. The potential role of LPL in determining fat deposition, especially for intramuscular fat (marbling) in fattening ruminants, is still an unanswered question.

5. LIPOPROTEIN METABOLISM Lipoprotein metabolism in ruminants differs significantly from the more-studied laboratory species such as rats, mice, and guinea pigs. An excellent comprehensive review (Bauchart, 1993) is available to which the interested reader is referred and in which can be found references to other, older reviews. A schematic is presented in fig. 2 to describe the general patterns of lipoprotein metabolism in ruminants. The TG-rich lipoproteins (chylomicrons and VLDL) function to deliver LCFA absorbed from the intestine to peripheral tissues. As discussed earlier (see section 3), intestinally synthesized VLDL predominate in ruminants, whereas chylomicrons would be more important in the milk-fed preruminant. Following secretion into the lymphatics and entry into the venous blood system, chylomicrons and intestinal VLDL acquire additional surface-coat compounds, including phospholipids and apo-C proteins, from circulating HDL. Apo-CI and apo-CII are important for lipid binding and activation of LPL activity, respectively. Chylomicrons and VLDL thus activated by apo-CI interact with LPL in the capillaries of peripheral tissue to catalyze “unloading” of TG in those tissues. Following LPL action, chylomicrons and VLDL are converted to chylomicron remnants and intermediate-density lipoproteins (IDL), respectively. Chylomicron remnants and IDL containing apo-B48, i.e. those of intestinal origin, are found in extremely low concentrations in ruminants, suggesting that they may be more rapidly cleared by the liver. As hydrolysis of TG occurs, the particle size decreases and thus surface components (phospholipid, apo-A and apo-C) become excessive and are transferred back to HDL. Most LDL are formed in circulation from IDL in the ruminant. The LDL make up a small proportion of total lipoproteins (about 10%) in adult ruminants (Bauchart, 1993).

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Fig. 2. Schematic diagram showing major aspects of lipoprotein metabolism in growing ruminants. Chylomicrons (mainly in preruminants) or very low-density lipoproteins (VLDL) secreted from the intestine or liver acquire apoproteins- (apo)-CI and apo-CII from circulating high-density lipoproteins (HDL). Triacylglycerols in chylomicrons or VLDL are hydrolyzed by lipoprotein lipase (LPL) in peripheral tissues, which is activated by apo-CII and allows fatty acid uptake by tissues. Excess surface components (phospholipids, PL; apoproteins C and A; free cholesterol, chol) that arise as chylomicrons and VLDL decrease in size during LPL catalysis of triacylglycerols are transferred to HDL. The chylomicron remnants are cleared by the liver. Remnants of VLDL are called intermediate density lipoproteins (IDL) and are either cleared by the liver or undergo further triacylglycerol hydrolysis to produce low-density lipoproteins (LDL). LDL are degraded in liver or after receptor-mediated uptake in peripheral tissues. HDL take up excess cholesterol (chol) from peripheral tissues and convert it to cholesterol esters by action of lecithin cholesterol acyltransferase (LCAT); lysolecithin is released into plasma and cholesterol esters enter the core of the HDL. Action of LCAT to produce cholesterol esters, and the uptake of excess surface components from chylomicrons and VLDL, results in increasing size and decreasing buoyant density of lipid-poor heavy HDL (HDLH) to lipid-rich light HDL (HDLL). HDL can deliver cholesterol and essential fatty acids to tissues or return cholesterol to liver for conversion to bile salts. Many tissues also possess an HDL receptor that results in clearance of HDL particles. Adapted from Drackley (2000).

Low concentrations have been attributed to the low activities of cholesterol ester transfer protein (Ha and Barter, 1982) and hepatic lipase in ruminants (Cordle et al., 1986), which are involved in the production of LDL in other species. Plasma concentrations of LDL in ruminants are also controlled by expression and activity of tissue LDL receptors. The major sites for receptor-mediated removal of LDL are bone, intestine, and liver (Rudling and Peterson, 1985). High concentrations of LDL receptors are also found in bovine adrenals and corpora lutea (Rudling and Peterson, 1985), which indicates that LDL may be important sources of cholesterol for steroid hormone biosynthesis in these tissues. Because LDL are rich in cholesterol esters and phospholipids containing linoleic acid, tissue uptake of LDL also served to distribute this EFA for membrane formation. Bovine HDL are the main lipoprotein class in ruminants, constituting over 80% of total plasma lipoproteins in both preruminants and ruminants (Bauchart, 1993). The synthesis of HDL remains an enigma even in more well-studied species (Fielding and Fielding, 2001), but precursor particles (apo-AI) are synthesized by liver and small intestine (Fielding and Fielding, 1995).

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Genesis of pre-HDL particles appears to occur in circulation, either from chylomicrons and VLDL or within the interstitial spaces (Fielding and Fielding, 1995). Initial complexes of apo-AI and phospholipids (pre-β-HDL) acquire cholesterol from tissue membranes by as yet undefined mechanisms that may involve some combination of simple diffusion, facilitated diffusion, active transport, or other mechanisms (see Fielding and Fielding, 2001, for review). The lecithin-cholesterol acyltransferase (LCAT) reaction then functions to catalyze transfer of an unsaturated fatty acid (primarily linoleic) from the 2-position of phosphatidylcholine (lecithin) to cholesterol, both polar surface-coat lipids of HDL. The more nonpolar cholesterol ester migrates to the core of the HDL, while the lysolecithin is shed to serum albumin. Continued transfer of cholesterol esters to the core of the HDL causes their expansion in size, forming “light” HDL. The lack of cholesterol ester transfer protein, which would transfer cholesterol esters to LDL or VLDL, in ruminants and the high turnover of surface-coat lipids from VLDL result in the large size of HDL (Bauchart, 1993). The presence of the very large and less dense (i.e. high lipid content) HDL in ruminants results in overlapping density ranges for LDL and HDL, thus making separation of LDL and HDL by density gradient ultracentrifugation procedures impossible in ruminants. Alternative approaches have been developed that rely on combinations of affinity chromatography and centrifugal separations (see Bauchart, 1993 for review). The HDL of ruminants possesses apo-AIV, which is secreted by intestinal cells in TG-rich lipoproteins and then transferred to HDL in lymph and plasma (Bauchart et al., 1989). Originally described as an activator of peripheral LPL, apo-AIV has been implicated in recent years as a regulator of food intake in humans and laboratory animals (see Tso et al., 2001, for review). An intestinal fat load stimulates synthesis and secretion of apo-AIV, which in turn acts to suppress additional feed intake. The potential roles of apo-AIV in regulation of feed intake in ruminants, or its response to supplemental fat, have not been investigated. Because of their predominance in ruminants, HDL function as the main distribution vehicle for cholesterol and EFA. Specific receptors for HDL have been identified and characterized (Graham and Oram, 1987). This receptor recognizes apo-AI but not LDL or apo-E; the apo-E protein is not expressed to any extent in ruminants (Bauchart, 1993). Uptake of HDL by bovine liver, and probably other tissues, is regulated by the density of the HDL receptors, which are almost always occupied with HDL, rather than by concentration of HDL in plasma (Mendel et al., 1986). Cholesterol uptake by tissues is used for membrane synthesis, steroidogenesis, or, in the liver, bile salt synthesis. Uptake of cholesterol from peripheral tissues by HDL followed by clearance of HDL by the liver constitutes a cycle that has been referred to as “reverse cholesterol transport” in other species and is one component of the regulation of cholesterol homeostasis in the body (Fielding and Fielding, 1995). Relatively little is known about cholesterol synthesis and homeostasis in ruminants. The major site of cholesterol synthesis appears to be the small intestine (Nestel et al., 1978), which is in keeping with the low rate of production of VLDL by the ruminant liver (Kleppe et al., 1988; Graulet et al., 1998; Gruffat-Mouty et al., 1999), although the ruminant liver does synthesize some cholesterol. Transport of cholesterol from intestine and liver to other tissues by LDL and HDL furnishes the needs of those tissues for membrane structure and steroid hormone biosynthesis. Although beyond the scope of this chapter, metabolism of the EFA, i.e. those LCFA produced from elongation and desaturation of linoleic acid (18:2n-6) and linolenic acid (18 : 3n-3), is a particularly fascinating topic in ruminants (Noble, 1984). Little transfer of LCFA occurs across the ruminant placenta, so the fetuses and young are born with what would be considered severely deficient status for EFA in other mammalian species (see Noble

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and Shand, 1982, for review). Linoleic and linolenic acids in plasma phospholipids and cholesterol esters are extremely low at birth, but then increase substantially by 3 weeks with little additional change after weaning (Jenkins et al., 1988). After birth, the young receive some EFA from colostrum and milk, but again intakes are insufficient relative to other species because of the low EFA content in ruminant milk. Because functional deficiencies of EFA do not seem to occur in preruminants or ruminants, the ruminant animal must have developed extremely efficient mechanisms for capturing and maintaining EFA within the body. Mechanisms for the conservation and concentration of EFA include lower rates of oxidation compared with more abundant saturated LCFA (Lindsay and Leat, 1977), which may be mediated via the low affinity of mitochondrial dehydrogenases for EFA (Reid and Husbands, 1985). Other mechanisms include the high affinity of phospholipid esterification enzymes for incorporation of EFA (Lindsay and Leat, 1977), the high affinity of lecithin cholesterol acyltransferase for linoleic acid (Noble et al., 1972), and the slow turnover of the phospholipid and cholesterol ester pools in plasma (Palmquist, 1976). Interorgan transport and metabolic conversions of the lipoproteins serve as a vehicle for distribution of EFA to cells throughout the body for incorporation into cell membranes. Other methods by which tissues acquire EFA include uptake of EFA as free fatty acids, uptake of the EFA-acyl group from lysophosphatidylcholine, and local desaturation and elongation of free linoleic acid (see review by Zhou and Nilsson, 2001). The extent to which peripheral tissues of ruminants acquire EFA by uptake or transfer of phospholipids from lipoproteins to cell membranes vs. receptor-mediated uptake of LDL or HDL is not well characterized. Zhou et al. (2002) demonstrated that plasma free arachidonic acid was the major source of arachidonic acid taken up by extrahepatic tissues in rats. Regardless, increasing dietary intake of fats and oils rich in n-6 or n-3 LCFA results in corresponding increases in contents of these LCFA in phosphatidylcholine and cholesterol esters in plasma of preruminants, and corresponding increases in the n-6 and n-3 LCFA in phophatidylcholine and phosphatidylethanolamine in liver and muscle (Jenkins and Kramer, 1990). Similar changes occur in functioning ruminants if dietary sources of EFA are protected from ruminal biohydrogenation (Ashes et al., 1995). It appears that phosphatidylethanolamine in muscle, primarily found in the inner leaflet of plasma membranes, may be particularly important for concentration of n-3 LCFA (Jenkins and Kramer, 1990; Ashes et al., 1995). While a considerable body of research has appeared in the last decade describing interorgan lipoprotein metabolism in growing ruminants, these studies mostly were conducted on calves that were maintained in the preruminant state on milk diets long after what would be conventional in North American production systems. Research on lipoprotein metabolism in growing ruminants is lacking. The significance and role in growth, therefore, is still largely as speculative as it was over twenty years ago (Kris-Etherton and Etherton, 1982).

6. TISSUE FATTY ACID METABOLISM IN GROWING RUMINANTS Excellent comprehensive reviews of lipid metabolism in adipose tissue (Vernon, 1980; Chilliard, 1993) and liver, muscle, and other tissues (Bell, 1980) are available. Discussion here will mainly consider recent research on lipid metabolism in key organs relative to growth of ruminants. 6.1. Skeletal muscle and heart During growth of ruminants, muscle protein accretion is afforded a higher metabolic priority than is lipid deposition. For example, Smith et al. (1992) fed groups of ovariectomized

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Angus–Hereford heifers different amounts of the same diet to supply 0.76, 1.43, 1.74, and 2.05 times the estimated requirements for metabolizable energy for a 128-day feeding period. Retention of dietary N increased linearly with increasing intake, whereas measurements of adipose lipid synthesis increased only for groups fed 1.74 and 2.05 times maintenance. Rates of protein deposition in muscle (and whole carcass) decrease as the animal approaches maturity (Campbell, 1988). Skeletal muscles contain differing proportions of fibers that are primarily oxidative (red muscle), primarily glycolytic (white muscle), or a mixture of both. Metabolic properties may vary even within the same muscle (Brandstetter et al., 1997). So-called red muscle is characterized by a higher density of mitochondria than is found in white muscle. Furthermore, at least two distinct subpopulations of mitochondria exist in ruminant muscle (Piot et al., 2000). The subsarcolemmal mitochondria are located just beneath the muscle membrane or sarcolemma, whereas the intermyofibrillar mitochondria are found inserted into the myofibrillar matrix. Piot et al. (2000) found that enzymes of oxidative metabolism and LCFA oxidation were present in greater specific activity in intermyofibrillar mitochondria than in subsarcolemmal mitochondria. Capacity of heart tissue for LCFA oxidation was much greater than that of skeletal muscles (rectus abdominis and longissimus thoracis) from preruminant calves, due to higher density of both intermyofibrillar and subsarcolemmal mitochondria and to greater specific activities of oxidative enzymes within the mitochondria from heart. In fed, growing ruminants, glucose, acetate, and β-hydroxybutyrate (BHBA) are the major fuels for heart and skeletal muscle, potentially accounting for 31–57%, 15–29%, and 18%, respectively, of fuel for muscle oxidation at rest (Hocquette et al., 1998b; Hocquette and Bauchart, 1999). In contrast, oxidation of NEFA at rest accounts for only about 5% of oxidative needs. Uptake of NEFA is linearly related to arterial concentration up to about 1 mM (Bell and Thompson, 1979). Muscle uptake of NEFA likely is facilitated by muscle-type FABP (Moore et al., 1993). Uptake exceeds the amount oxidized immediately for energy, with the excess stored in an intracellular TG pool (Bell and Thompson, 1979). Although not investigated in ruminants, hormone-sensitive lipase is present in muscle from other species and responds to catecholamine stimulation to initiate intracellular hydrolysis of stored TG for oxidation within the muscle (Langfort et al., 1998). Rate-limiting control of NEFA oxidation lies at the enzyme carnitine palmitoyltransferase-1 (CPT-1), which governs entrance of fatty acyl-CoA into mitochondria. Malonyl-CoA is produced by the muscle form of acetyl-CoA carboxylase and inhibits muscle CPT-1 (Winder, 2001). While not yet characterized in ruminants, in laboratory rodents and humans malonylCoA is produced by the muscle form of acetyl-CoA carboxylase (Chien et al., 2000) and degraded by malonyl-CoA decarboxylase (Young et al., 2001). Regulation of malonyl-CoA concentration in muscle thereby represents an elegant control system that coordinates utilization of glucose and LCFA depending on substrate availability and muscle energy needs (see Winder, 2001, for review). Oxidative capacity per unit of tissue is lower in muscle from cattle than in muscle from rats (Ottemann-Abbamonte et al., 1998; Piot et al., 1998). Oxidation of palmitate by isolated strips of muscle in vitro appeared to be saturated at a palmitate concentration of 0.5 mM for muscle from adult cows, but was not yet saturated at 2.0 mM for rat muscle (OttemannAbbamonte et al., 1998). Piot et al. (1998) attributed the greater oxidative capacity of rat muscle to a greater mitochondrial density; peroxisomal oxidative capacity was not different between rats and preruminant calves but was lower for 15-month-old growing bulls. In that same study, total oxidation capacity of muscle homogenates was about 1.7-fold greater for preruminant calves than for growing bulls. Piot et al. (1998) proposed that the difference

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between preruminant and older ruminant muscle was related both to decreased relative quantity of mitochondria as well as to changes in the oxidative properties of muscle mitochondria. Bartelds et al. (1998) studied the in vivo flux of energy supplying nutrients to the heart in fetal, newborn (1 to 4 days), and 7-week-old lambs. In fetal lambs, NEFA contributed no energy to the heart. In newborn lambs, the supply of NEFA to the heart increased 10-fold, but heart still did not oxidize NEFA for energy. Glucose was the major energy source in both fetal and newborn lambs, accounting for 89% and 69% of oxygen consumption, respectively. By 7 weeks, the flux of NEFA through heart was increased 3-fold, and the supply and use of ketone bodies likewise was increased. Similar developmental data for skeletal muscle are lacking. 6.2. Adipose tissue Adipose tissue represents a complex assortment of anatomical depots, including internal (e.g. perirenal, omental), intermuscular, subcutaneous, and intramuscular. In growing ruminants, adipose tissue functions to accrete excess energy in the form of TG (see Chapter 10 by Mersmann and Smith, this volume). The LCFA that are incorporated into adipose tissue TG during growth and fattening can derive from de novo lipogenesis within the adipose tissue or via uptake from blood. From a biological perspective, adipose tissue functions as a reserve of energy for situations of dietary shortage. Recent research has demonstrated that adipose is not an inert storage vessel, however, but also communicates with other organs and systems of the body by synthesizing and secreting a variety of mediators such as leptin, tumor-necrosis factor α (TNFα), and adipsin (see Vernon et al., 1999; Chilliard et al., 2000). Given its importance in determining eating quality of ruminant meat, and in the production economics of ruminant agriculture, it is not surprising that lipid metabolism in adipose tissue has been widely studied. Lipogenesis is low during the milk-feeding period (Pothoven et al., 1975), then increases during growth as rates of lean tissue growth decline (Pothoven and Beitz, 1973; Smith et al., 1984, 1987). Internal adipose tissues have greater rates of de novo lipogenesis in younger animals, whereas in more mature animals subcutaneous depots have the greater activity (Ingle et al., 1972; Pothoven and Beitz, 1973; Hansen et al., 1995). Increasing feed intake increases lipogenesis in subcutaneous adipose (Mills et al., 1989; Smith et al., 1992); conversely, feed restriction during growth decreases lipogenic rates (Pothoven et al., 1975). The intermediary metabolism of lipogenesis in adipose tissue appears to differ between sheep and cattle in several ways (Smith and Prior, 1986); regulation in sheep and cattle adipose tissue is discussed in detail by Smith (1995). Differential activity of lipogenesis among adipose depots and with stage of growth may relate to adipocyte size. Hood and Allen (1978) found that lipogenic enzyme activities were positively correlated with the cell volume of adipocytes across several anatomical sites in sheep. More recently, Barber et al. (2000) showed that expression of mRNA for LPL and acetyl-CoA carboxylase-α (ACCα), the rate-limiting and regulated step in LCFA synthesis, per 106 cells was highly correlated with the size of adipocytes isolated from seven subcutaneous and internal depots from wethers at slaughter. These data agree with other observations that lipogenesis is lower in adipose tissue with smaller adipose cell diameters, such as intramuscular adipose or in adipose tissue from young animals (Smith, 1995). However, factors other than adipocyte size also must impact metabolic activity among different adipose tissue depots. For example, in fattened sheep lipogenesis from acetate was about 10-fold lower in perirenal adipose than in subcutaneous adipose tissue, but mean cell size and numbers of cells per gram of adipose tissue differed by only 10% and 24%, respectively (Hansen et al., 1995).

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Classic experiments by Hanson and Ballard (1967) demonstrated that acetate and not glucose was the major carbon source for LCFA synthesis in adipose tissues of ruminants. A possible exception is intramuscular adipose depots in which glucose may account for 50–75% of LCFA carbon (Smith and Crouse, 1984). However, overall rates of de novo LCFA synthesis are low in intramuscular adipose, with rates of incorporation of acetate into LCFA only 10–50% of those in subcutaneous depots (Smith and Crouse, 1984; Miller et al., 1991). Although rates of esterification of LCFA to glycerol-3-phosphate are lower in intramuscular adipose than in subcutaneous, differences in LCFA synthesis are much more pronounced (Smith et al., 1998). This suggests that uptake of preformed LCFA from blood TG by action of LPL may be more important for lipid deposition in intramuscular adipocytes. Uptake of preformed LCFA into adipose tissue can be quite substantial in preruminants consuming high-fat milk-based diets. For example, it can be calculated from data of Tikofsy et al. (2001), in which preruminant calves were isocalorically fed milk replacers containing 14.8%, 21.6%, or 30.6% fat, that about 45% of the additional fat intake was deposited in the body. The amount of LCFA originating from the diet in functioning ruminants is much more limited but not insignificant. For example, typical forage- and grain-based diets for growing ruminants contain only 3–4% LCFA, which would supply about 6–10% of the digestible energy intake. Even with near-maximal incorporation of supplemental fat into diets for ruminants, LCFA will usually supply less than 20% of the energy. However, the amount of LCFA supplied may still contribute a substantial portion of fat deposited in adipose tissues. An example of the estimated contribution of dietary LCFA supply to adipose fat deposition is shown in table 1, based on data from Zinn (1992). In this example, cattle fed basal diets based on corn or wheat had digestible LCFA intakes of about 144 g/d, whereas cattle fed corn- or wheat-based diets supplemented with 6% yellow grease would have digestible LCFA

Table 1 Estimation of contribution of dietary fat to adipose lipid deposition and suppression of de novo fatty acid synthesis in fattening beef cattle a Diet

Variable Dry matter (DM) intake, kg/d Dietary fatty acids (FA)b, % of DM FA intake, g/d Estimated digestible FA intakec, g/d Estimated FA deposited in adiposed, g/d Measured fat gain, g/d Estimated FA gaine, g/d Dietary FA as percentage of FA gain Estimated de novo FA synthesis f, g/d % Suppression of de novo synthesis a Data

Basal (no supplemental fat)

Basal plus 6% yellow grease

7.82 2.45 192 144 118 480 432 27 314 –

7.42 7.45 553 415 340 550 495 69 155 51

for intake, diet composition, and fat gain are means from Zinn (1992). dietary ether extract concentration multiplied by assumed 85% fatty acid content. c Assumes dietary fatty acids are 75% digestible (Zinn, 1989). d Digestible energy of fat equals metabolizable energy; assumes that efficiency of use of metabolizable energy for fat deposition (i.e. NEg) is 82% (Zinn, 1989). e Assumes adipose tissue triacylglycerol is 90% fatty acids. f Estimated fatty acid gain minus dietary fatty acids deposited. b Reported

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intakes of about 415 g/d. Assuming that absorbed LCFA are used in fattening cattle with an efficiency of 82% (Zinn, 1989), cattle fed basal and fat-supplemented diets would deposit 118 and 340 g/d of dietary LCFA. Whole-body LCFA gains averaged 432 and 495 g/d, respectively, which indicates that 27% and 69% of the total LCFA deposited originated from dietary LCFA for control and fat-supplemented diets, respectively. By difference, whole-body de novo synthesis of LCFA would need to be decreased by about 51% when the fat-supplemented diet was fed. Page et al. (1997) found that de novo synthesis of LCFA from acetate was decreased by 29% in subcutaneous adipose tissue from steers fed a diet containing 30% whole cottonseed (which supplied an additional 6% lipid to the diet) compared with adipose tissue from control steers, suggesting that decreased lipogenesis of the magnitude estimated here (table 1) is not biologically unrealistic. Other research suggests that dietary lipid supplements also may decrease lipogenesis in sheep. Hood et al. (1980) found that rumen-protected safflower oil, but not rumen-protected tallow or palm oil, decreased lipogenesis from acetate by 43% in perirenal adipose tissue. In subcutaneous adipose tissue, protected safflower oil decreased in vitro lipogenesis by 75%; differences due to protected tallow (−42%) and protected palm oil (−32%) were not statistically significant (Hood et al., 1980). Lipogenesis, measured both in vitro and in vivo, was decreased in sheep fed calcium salts of palm oil (Moibi et al., 2000a). Decreased lipogenesis by dietary lipid was accompanied by a 30% decrease in fatty acid synthase activity in subcutaneous, mesenteric, and perirenal adipose tissues; in contrast, activity of acetyl-CoA carboxylase actually was increased by supplemental fat (Moibi et al., 2000b). On the other hand, lipogenesis in isolated adipocytes was not different between control sheep and those fed a diet containing 5% soybean oil (Jenkins et al., 1994). Although the data are not conclusive, together these observations suggest that increased dietary LCFA, even if not protected from ruminal biohydrogenation, may suppress de novo lipogenesis from acetate in ruminant adipose tissue. This differs from the situation in rodents, in which only unsaturated LCFA have been shown to be effective at suppressing lipogenesis (see Clarke, 2001, for review). Regardless of the source of LCFA available to adipose tissue, the ultimate pathway for accretion of lipid stores is the series of enzymatic steps involved with attachment of LCFA to glycerol-3-phosphate (fig. 1). Considerable progress has been made in the last decade in characterizing the esterification pathway (see review by Rule, 1995, and references therein). Esterification activity is greater in bovine subcutaneous adipose tissue than in liver (Wilson et al., 1992). Evidence indicates that phosphatidate phosphohydrolase may be the rate-regulating step in adipose but that glycerol-3-phosphate acyltransferase may be more likely to control esterification in liver (Wilson et al., 1992; Smith et al., 1998). Changes in palmitate esterification and activity of glycerol-3-phosphate acyltransferase parallel changes in fat deposition and carcass fat thickness with different rates of gain and degrees of maturity in both bovine and ovine adipose tissue (Bouyekhf et al., 1992; West et al., 1994; Andersen et al., 1996). Palmitate esterification in vitro was decreased by >48% by the catecholamines clenbuterol, norepinephrine, and isoproterenol as well as by cAMP in subcutaneous adipose tissue from ewes fed at maintenance; however, for tissue from ewes fed on a high-energy diet for 6 weeks, only isoproterenol and cAMP inhibited esterification but to a lesser degree than for maintenance-fed lambs (Bouyekhf et al., 1993). A period of 72 h of starvation decreased LCFA esterification and phosphatidate phosphohydrolase activity by about 50% in bovine subcutaneous adipose tissue, but not in intramuscular adipose tissue (Smith et al., 1998). Changes in adipose esterification rates in parallel with positive energy balance and during fattening suggest a major control by insulin. However, insulin has only modest effects on esterification activity in vitro (Jacobi and Miner, 2002), suggesting that removal of

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antagonistic effects during positive energy balance may be the more dominant regulatory influence. Other factors may be involved in stimulating esterification. For example, acylationstimulating protein is an 8.9 kD protein generated within adipose tissue from components of the alternative complement pathway (see Cianflone et al., 1999 for review). This protein acts in an autocrine fashion to promote uptake and esterification of LCFA by adipocytes from humans and laboratory rodents as well as cell lines. Recently, Jacobi and Miner (2002) demonstrated that human acylation-stimulating protein increased acetate incorporation into LCFA by 15–30% and oleate incorporation into TG by 10–25% in bovine adipose tissue, and that this stimulation was not affected by feed restriction. It will be of interest to isolate the bovine counterpart of acylation-stimulating protein and to determine its role in growth under different dietary regimes. For example, in other species, a factor carried by chylomicrons after a meal is important in activating the synthesis of acylation-stimulating protein (Cianflone et al., 1999); does increased synthesis of acylation-stimulating protein during fat feeding in ruminants contribute to the general increase in body fat deposition under those conditions? In growing ruminants consuming adequate feed, the primary state is lipid accretion so that lipid mobilization is a less important process. This contrasts with lactating ruminants, which regularly undergo periods of intense lipid mobilization after parturition to support lactation (McNamara, 1994). Situations do occur with growing ruminants that demand lipid mobilization from adipose tissues to meet energy needs, for example, animals grazing poor-quality pasture (due to environmental conditions or poor management). Agents that increase intracellular cAMP concentrations, such as epinephrine and norepinephrine, are the primary stimuli for increased lipolysis in sheep and cattle (Etherton et al., 1977) and exert lipolytic activity via binding to β-adrenergic receptors (Houseknecht et al., 1996). The adrenergic receptors, through protein kinase A, lead to phosphorylation and activation of hormone-sensitive lipase (HSL). In laboratory animals, proteins called perilipins also are phosphorylated by β-adrenergic activation (Clifford et al., 2000). When phosphorylated, perilipins appear to increase access of activated HSL to the lipid droplet, and thereby promote lipolysis. Stimulatory effects of catecholamines on lipolysis in ruminants are enhanced by fasting (DiMarco et al., 1991). Glucagon does not stimulate lipolysis in adipose tissue from either sheep or cattle (Etherton et al., 1977). Somatotropin increases responsiveness of adipose tissue to catecholamine-stimulated lipolysis (for review see Etherton and Bauman, 1998), but somatotropin, insulin-like growth factors I and II, prolactin, and placental lactogen are without effect on lipolysis in ruminants (Houseknecht et al., 1996). Prostaglandins (specifically PGE2) also are not involved with regulation of lipolysis in bovine adipose tissue, as demonstrated by the lack of effect of both exogenous PGE2 and indomethacin, which blocks prostaglandin synthetase, on basal and epinephrine-stimulated lipolysis (DiMarco et al., 1991). Although insulin is known to suppress lipolysis, direct effects of insulin to inhibit basal or stimulated lipolysis have been difficult to demonstrate in vitro (DiMarco et al., 1991). Basal and stimulated lipolytic rates of adipose tissues increase with growth and fattening (Smith et al., 1984; Rule et al., 1992) but are not affected by diet (high forage vs. high grain) at equal energy intake (Smith et al., 1984). Lipolytic rates generally are higher in subcutaneous adipose than in internal depots (Etherton et al., 1977; Rule et al., 1992), although differences among depots seem to diminish with increased age or body size (Rule et al., 1992). As animals grow and fatten, lipolysis becomes less complete, resulting in greater release of NEFA than of glycerol (Smith et al., 1984); this may indicate greater re-esterification activity within adipocytes that contributes to increased fat deposition. Hansen et al. (1995) found that glycerol release was greater from subcutaneous adipose than from perirenal adipose from sheep at market weight, but that fatty acid release and tissue fatty acid pool size were greater in

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perirenal than in subcutaneous adipose. Consequently, lipolysis was less complete in the internal depot than in the subcutaneous depot. An exciting and expanding field concerns the impact of various factors produced by adipocytes that may act in autocrine, paracrine, or endocrine fashion to alter metabolism (Vernon et al., 1999; Chilliard et al., 2000). One of these, leptin, has received tremendous attention in both the biomedical arena (for review see Reidy and Weber, 2000) and in animal agriculture (for reviews see Hossner, 1998; Houseknecht et al., 1998; Chilliard et al., 2001; Ingvartsen and Boisclair, 2001). As adipocytes fill with lipid, leptin synthesis and secretion increases. Leptin acts centrally to decrease feed intake and increase thermogenesis, and acts directly to increase lipolysis in adipose tissue and NEFA oxidation in peripheral tissues. Another autocrine or paracrine factor produced by adipocytes is TNFα, which acts to increase lipolysis and suppress LPL (Gasic et al., 1999). Expression of TNFα mRNA appears to change in bovine subcutaneous adipose tissue with degree of fatness (Drackley et al., unpublished observations) as in other species. The role of these adipose-derived regulatory factors in counteracting or decreasing the efficiency of fattening in ruminants will be an area of considerable interest in the future. 6.3.

Liver

Because the liver of ruminant animals is not a major site of de novo synthesis of fatty acids (Hanson and Ballard, 1967), its role lies in processing and redistributing exogenous and endogenous LCFA taken up from the diet and from plasma lipoproteins, as well as in metabolism of NEFA mobilized from adipose tissue. In growing ruminants, the latter usually is a minor process. Glycolytic and lipogenic enzymes are present in the milk-fed calf, but specific activities decrease sharply after weaning (Pearce and Unsworth, 1980). The rate of VLDL synthesis and secretion in liver of ruminants is lower than in rats (Kleppe et al., 1988; Graulet et al., 1998), which is associated with the low rate of hepatic de novo fatty acid synthesis as observed in other species (Pullen et al., 1990). In fasted preruminant calves, considerable uptake of VLDL by the liver has been noted (Bauchart et al., 1989). Furthermore, heavy, lipid-poor HDL were also taken up by the liver of fasting calves, with concomitant production and release of lighter, lipid-enriched (primarily with cholesterol esters) HDL particles (Bauchart et al., 1989); no appreciable flux of LDL was noted. Uptake of VLDL by the liver changed to secretion as dietary tallow intake increased in calves (Auboiron et al., 1995). Addition of supplemental methionine, which might be postulated to be limiting for hepatic synthesis of apo-B, slightly increased secretion of VLDL (Auboiron et al., 1995). Secretion of VLDL by calf liver slices occurred at 6- to 18-fold lower rates than in rat liver slices (Graulet et al., 1998), despite similar rates of synthesis of apoB in liver slices (Gruffat-Mouty et al., 1999). This lower rate of VLDL synthesis or secretion may be attributable to a low rate of synthesis of TG in the microsomal compartment responsible for VLDL assembly, with no limitation in the ability to synthesize and store cytosolic TG (Graulet et al., 1998). Hepatic lipid metabolism in preruminants may be sensitive to the LCFA profile of the diet, as replacement of tallow with hydrogenated coconut oil (rich in medium-chain saturated fatty acids and deficient in unsaturated LCFA) or soybean oil (high in polyunsaturated LCFA) caused accumulation of TG in the liver of calves (Jenkins and Kramer, 1986; Leplaix-Charlat et al., 1996; Piot et al., 1999). This suggests that changes in the profile of dietary LCFA that are markedly different from that of bovine milk fat cause alterations in hepatic lipid metabolism.

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Accumulation of TG in liver of preruminant calves fed milk replacer containing soybean oil appeared to result from secretion of VLDL that were enriched with cholesterol esters rather than TG, which consequently decreased hepatic secretion of TG (Leplaix-Charlat et al., 1996). Because of the low oxidation of linoleic acid (Lindsay and Leat, 1977; Reid and Husbands, 1985), high dietary linoleate supply could have decreased hepatic LCFA oxidation and so contributed to TG accumulation. Liver uptake of very large and light HDL enriched with cholesterol esters was observed, and appeared to be increased by including cholesterol in the diet with soybean oil. This corresponded with simultaneous secretion of heavy HDL. Liver slices from calves fed coconut oil-enriched milk replacers had lower rates of LCFA oxidation and greater rates of LCFA esterification than did liver slices from calves fed an equal amount of tallow (Graulet et al., 2000). Liver from coconut oil-fed calves also had decreased rates of apo-B synthesis (Gruffat-Mouty et al., 2001); consequently, greater rates of TG synthesis coupled with lower rates of VLDL export likely explain the accumulation of TG in liver of calves fed coconut oil. Since milk fat contains more short- and medium-chain fatty acids, which are extensively metabolized by the liver, than does tallow, and less PUFA than does soybean oil, it would be of interest to compare the effects caused by coconut oil and soybean oil with milk-fat controls rather than tallow controls to discern more about the native metabolism in liver of preruminant calves. Regulation of hepatic LCFA metabolism in ruminants has been reviewed extensively (Bell, 1980; Bauchart, 1993; Grummer, 1993; Bauchart et al., 1996; Hocquette and Bauchart, 1999; Drackley et al., 2001). Liver removal of NEFA increases as arterial concentration of NEFA increases due to changing intake in growing beef steers (Lapierre et al., 2000). Increased uptake of NEFA mobilized from adipose tissue may lead to increases in TG formation and ketone body synthesis, although these changes are much smaller in magnitude than those observed during the negative energy balance of early lactation in dairy cows. For example, a 9-day starvation period in growing steers only increased liver TG concentration from 0.47% to 1.38% of wet weight, whereas plasma NEFA concentration increased to 1.03 mM (Lyle et al., 1984). Ketone body concentrations in that study were only modestly elevated (BHBA = 6.9 mg/dl). Consequently, consideration of hepatic TG accumulation or excessive ketone body production is not of major concern in growing ruminants. Oxidation of LCFA in liver of preruminants and ruminants occurs in mitochondria and peroxisomes. Peroxisomes are subcellular organelles that possess a pathway for β-oxidation of fatty acids in which the first oxidation step is not coupled to ATP production but which results in the dissipation of heat (see Reddy and Hashimoto, 2001, for review). Peroxisomal β-oxidation is believed to function to oxidize fatty acids that are poor substrates for mitochondrial oxidative enzymes and to help process fatty acids when cellular uptake greatly exceeds the cells’ need for energy. Peroxisomal β-oxidation in liver of ruminants was first studied by Grum et al. (1994), who found that hepatic tissue capacity for peroxisomal β-oxidation constituted about 50% of the total β-oxidation capacity in liver from mature dairy cows, which was much greater than capacity measured in retired female breeder rats (26%). Subsequent studies showed that peroxisomal β-oxidation constituted from 44% to 48% of total β-oxidation in liver from sheep at market weight (Hansen et al., 1995). Absolute rates of peroxisomal β-oxidation and the contribution to total β-oxidation capacity in isolated liver tissue were lower in preruminant calves than in 15-month-old bulls (Piot et al., 1998). The potential role and importance of hepatic peroxisomal β-oxidation during growth is uncertain. Surprisingly little is known about the development of hepatic tissue capacity for metabolism of LCFA during the transition from preruminant through weaning to functioning ruminant. Ontogenic studies of key regulatory enzymes and pathways for fatty acid oxidation

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and esterification, as well as for VLDL assembly and secretion, would seem to be of interest and importance to a full understanding of the metabolism of ruminant growth.

7. FUTURE PERSPECTIVES Regulation of interorgan lipid metabolism during growth will continue to be an important topic for biological scientists studying ruminant animals. Although much has been learned about metabolism of LCFA and other lipids during the last decade, there are numerous areas that lack fundamental understanding. Notable among these is the inability to alter rates of development and filling of intramuscular adipocytes, which is critically important to eating qualities of meat. An understanding of genetic and physiological mechanisms that control adipose development among different depots would be of enormous benefit in development of strategies to minimize lipid accumulation in internal and subcutaneous depots while maximizing marbling. The role of exogenous (dietary) LCFA, delivered by intestinally derived chylomicrons and VLDL, as substrates for TG synthesis in intramuscular adipose also remains of interest given the apparently low rate of de novo lipogenesis in this tissue. Another area where information is unexpectedly incomplete is the developmental changes in lipid metabolism in key organs, especially the liver, as ruminants make the transition from preruminant through weaning to functioning ruminant. Additional progress will certainly be made in the near future as techniques of molecular biology are increasingly applied to questions concerning ruminant growth. The increasing ease of measuring specific mRNA concentrations, as well as transcription rates, will likely lead to a more complete understanding of how lipid metabolism is regulated through transcriptional and post-transcriptional means. Furthermore, high-throughput techniques such as microarray analysis of global changes in gene expression can be applied strategically, both to test hypotheses and to search for novel genes that are regulated differently between physiological states, genetic backgrounds, disease states, or applications of nutritional or pharmacological treatments. Finally, impacts of the external animal environment on lipid metabolism during growth is an area that should see additional exploration. Increasing knowledge of the multiway communication among the neuroendocrine system, the central nervous system, the immune system, and intermediary metabolism of numerous organs (see e.g. Johnson, 1997; Kelley, 2001; and Chapter 4 by Johnson and Escobar, this volume) offers exciting possibilities to improve both efficiency of ruminant animal production and animal well-being. Such efforts are critical to the long-term sustainability of ruminant animal production.

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Tso, P., Balint, J.A., 1986. Formation and transport of chylomicrons by enterocytes to the lymphatics. Amer. J. Phyiol. 250, G715–G726. Tso, P., Liu, M., Kalogeris, T.J., Thomson, A.B.R., 2001. The role of apolipoprotein A-IV in the regulation of food intake. Annu. Rev. Nutr. 21, 231–254. Turley, S.D., Burns, D.K., Rosenfeld, C.R., Dietschy, J.M., 1996. Brain does not utilize low density lipoprotein-cholesterol during fetal and neonatal development in the sheep. J. Lipid Res. 37, 1953–1961. van den Bosch H., Aarsman, A.J., de Jong, J.G., van Deenem, L.L., 1993. Studies on lysophospholipases. I. Purification and some properties of a lysophospholipase from beef pancreas. Biochim. Biophys. Acta 296, 94–104. Vernon, R.G., 1980. Lipid metabolism in the adipose tissue of ruminant animals. Prog. Lipid Res. 19, 23–106. Vernon, R.G., Barber, M.C., Travers, M.T., 1999. Present and future studies on lipogenesis in animals and human subjects. Proc. Nutr. Soc. 58, 541–549. Villeneuve, P., Pina, M., Graille, J., 1996. Determination of pregastric lipase specificity in young ruminants. Chem. Phys. Lipids 83, 161–168. Wang, C.S., Hartsuck, J.A., 1993. Bile salt-activated lipase: a multiple function lipolytic enzyme. Biochim. Biophys. Acta 1166, 1–19. West, T.R., Riley, M.L., Rule, D.C., 1994. Palmitate esterification and glycerophosphate acyltransferase activity in adipose tissue of growing lambs. J. Anim. Sci. 72, 81–86. Wilson, J.J., Young, C.R., Smith, S.B., 1992. Triacylglycerol biosynthesis in bovine liver and subcutaneous adipose tissue. Comp. Biochem. Physiol. B 103, 511–516. Winder, W.W., 2001. Energy-sensing and signaling by AMP-activated protein kinase in skeletal muscle. J. Appl. Physiol. 91, 1017–1028. Wolfrum, C., Borrmann, C.M., Borchers, T., Spener, F., 2001. Fatty acids and hypolipidemic drugs regulate peroxisome proliferator-activated receptors alpha- and gamma-mediated gene expression via liver fatty acid binding protein: a signaling path to the nucleus. Proc. Natl. Acad. Sci. USA 98, 2323–2328. Young, M.E., Goodwin, G.W., Ying, J., Guthrie, P., Wilson, C.R., Laws, F.A., Taegtmeyer, H., 2001. Regulation of cardiac and skeletal muscle malonyl-CoA decarboxylase by fatty acids. Amer. J. Physiol. 280, E471–E479. Zhou, L., Nilsson, A., 2001. Sources of eicosanoid precursor fatty acid pools in tissues. J. Lipid Res. 42, 1521–1542. Zhou, L., Vessby, B., Nilsson, A., 2002. Quantitative role of plasma free fatty acids in the supply of arachidonic acid to extrahepatic tissues in rats. J. Nutr. 132, 2626–2631. Zinn, R.A., 1989. Influence of level and source of dietary fat on its comparative feeding value in finishing diets for feedlot steers: metabolism. J. Anim. Sci. 67, 1038–1049. Zinn, R.A., 1992. Comparative feeding value of supplemental fat in steam-flaked corn- and steam-flaked wheat-based finishing diets for feedlot steers. J. Anim. Sci. 70, 2959–2969.

PART IV Carbohydrate and energy metabolism

14

Environmental and hormonal regulation of energy metabolism in early development of the pig P. Herpin, I. Louveau, M. Damon and J. Le Dividich INRA, Joint Research Unit for Calf and Pig Production, 35590 Saint-Gilles, France

This chapter is concerned with the development and the environmental regulation of the energy metabolism in the neonatal pig, a species devoid of brown adipose tissue. The newborn pig is poorly insulated and maintenance of its homeothermic balance is essentially related to its ability to produce heat. The skeletal muscle is the major contributor to the energy metabolism, with shivering being the main mechanism of cold-induced thermogenesis. The piglet does not express UCP1. UCP2 and UCP3 are both expressed in skeletal muscle, but there is no evidence that they play a role in the cold-induced thermogenesis. Major factors involved in the postnatal improvement in the cold-induced thermogenesis include changes in muscle structure and cardiovascular, biochemical and hormonal adjustments. Muscle maturation is suggested by the marked postnatal increase in myofibrillar mass and the transitory expression of α-cardiac heavy chain myosin and triad proliferation. Moreover, muscle mitochondria are functional at birth, and both the increase in mitochondrial mass and their ultrastructural change account for the increased oxidative potential of the muscle. Cardiovascular adjustments include the redistribution of the cardiac output towards the skeletal muscle at the expense of the skin, liver and intestine. The effects of the shift in the energy source from carbohydrate to fat at birth on the development of metabolic pathways including gluconeogenesis, lipogenesis and fatty acid oxidation are also examined. Emphasis is given to the key role of CPT-1 in the regulation of fatty acid oxidation. Finally, the actions of thyroid, HPA and, to a lesser extent, somatotropic axes on the regulation of the energy metabolism are considered. It is concluded that the newborn pig is immature at birth in several respects. As a whole, factors involved in the improvement of its postnatal thermogenic capacity are all suggestive of the enhancement of this maturity within the first postnatal days.

1. INTRODUCTION The neonatal period is attended by important modifications in several physiological functions associated with dramatic changes in energy metabolism and nutrition. Before birth, the maternal 353

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organism provides an appropriate environment so that the foetus does not have to actively regulate body temperature. At birth the newborn pig experiences a sudden and dramatic cold stress since the gap between the dam’s uterus and the ambient temperature (Ta) may be as high as 10–12°C. The foetus receives a continuous supply of substrates for growth and metabolism among which glucose is the main energy substrate (Père et al., 1995). At birth, the piglet is abruptly switched to intermittent feeding of high-fat, low-carbohydrate colostrum and milk. Because the ability to alter metabolic rate is a prerequisite for survival, successful adaptation to this critical period implies that the piglet is able to activate its thermoregulatory mechanisms and to provide energy to its heat-producing tissues. This, in turn, requires profound changes in major metabolic pathways including oxidative, lipogenic and gluconeogenic pathways associated with changes in nutrition and hormonal status. During the past years, several reviews have been devoted to development of the metabolic responses to cold (Mount, 1968; Curtis, 1974) and the metabolic patterns in the neonatal pig (Mersmann, 1974). Data presented in this chapter rely mostly on recent results which provide new insights on the adaptation of the energy, fatty acids (oxidation, storage) and glucose metabolism during the neonatal period and their environmental and hormonal control. The actions of the thyroid, hypothalamic–pituitary–adrenal (HPA) and, to a lesser extent, somatotropic axis will be developed even though other hormones like insulin play an important role in the control of metabolism.

2. OVERVIEW OF THE ENERGY METABOLISM IN THE NEONATAL PIG 2.1. General aspects Maintenance of homeothermia results from a dynamic balance between heat loss and heat production. The newborn pig is poorly insulated, being virtually hairless and devoid of subcutaneous fat, and although cold exposure induces some vasoconstriction, it does not reduce the cardiac output to the skin (Lossec et al., 1999). This poor ability of the piglet to conserve heat is reflected by the fact that each 1°C coldness is associated with a 2 kJ/h/kg BW increase in heat production, which is 2.6-fold higher than at the time of weaning (Le Dividich et al., 1998). In fact, the weight-specific requirement for energy of the neonatal pig is maximal at birth. This is due to the high rate of heat production associated with thermoregulation, physical activity related to the establishment of the nursing order, and a high potential for growth. At birth, the typical ambient temperature (Ta) of 24–20°C is 10–12°C below the lower critical temperature of the piglet and is close to the temperature of 18°C at which the metabolic rate is maximal (Berthon et al., 1993). This period of cold stress is commonly associated with a temporary fall in rectal temperature, the extent of which and the time taken for recovery being dependent on body weight and Ta. In practice, this period of hypothermia may be a cause of mortality by weakening the piglet and predisposing it to crushing by the sow and starvation that is illustrated by the usual high level of mortality in the first 48 h after birth. However, cold resistance improves markedly in the early postnatal period in fed piglets but not in those consuming little or no colostrum (Le Dividich et al., 1991a; Herpin et al., 1994). Because thermal insulation does not change substantially during the first postnatal days (Berthon, 1994), maintenance of the homeothermic balance is largely dependent on the ability of the newborn to produce heat. The newborn pig responds quickly and vigorously to cold stress as exemplified by the 30% increase in metabolic rate at 18°C compared with 31°C within the first 20 min after birth (Noblet and Le Dividich, 1981), with the difference increasing to 100%

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Table 1 Minimal (mmR) and maximal (MmR) metabolic rate, lower critical temperature (LCT) and ambient temperature at which maximal metabolic rate (TMmR) is reached in pigs aged from 2–48 h Age, h

mmR, kJ/h/kg BW MmR, kJ/h/kg BW LCT, °C TMmR, °C

2

24

48

12.9 36.6 34.2 17.8

16.9 43.3 33.1 12.8

20.2 >46.8 30.2 <10

Adapted from Berthon et al. (1993, 1994).

in the first 90 min (Herpin et al., 1994). Also (table 1), maximal and minimal metabolic rate are increased by 56% and 28% respectively, during the first 48 h of life (Berthon, 1994). Together, these indicate that mechanisms responsible for heat production are active soon after birth. However, maintenance of a high metabolic rate during cold stress is closely dependent on both the availability of energy substrates and on the ability of the piglets to utilize these as an energy source. Once the piglet displays an efficient thermoregulatory behaviour and once a regular milk intake is established, cold stress is usually of minor importance during the remaining suckling period. The energy metabolism is then regulated essentially by the amount of milk intake (Marion and Le Dividich, 1999). 2.2. Evidence for the major importance of muscle in energy metabolism To cope with the neonatal cold challenge, skeletal muscle plays a central role in neonatal energy metabolism (fig. 1). Indeed, the calculated contribution of skeletal muscle to total

Fig. 1. Total body O2 consumption and contribution of muscle to total body O2 consumption in relation to age and ambient temperature (TN, thermoneutral; C, cold). Adapted from Lossec et al. (1998b).

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oxygen consumption averages 34–40% at thermal neutrality and 50–64% in the cold. The skeletal muscle is the major contributor to regulatory thermogenesis, accounting for 97% of the cold-induced increase in heat production at 5 days of life (Lossec et al., 1998b). In addition, this stimulation of muscle energy metabolism is accompanied by a redistribution of cardiac output towards skeletal muscles at the expense of digestive tract and liver blood flows (Lossec et al., 1999) which, however, might be detrimental to the digestive processes through the possible shortening of energy supply in the cold.

3. MECHANISMS OF HEAT PRODUCTION 3.1. Overview of heat production mechanisms During cold exposure, maintenance of the homeothermic balance is achieved through the development of specific heat-producing mechanisms. Two main mechanisms are usually reported: shivering and non-shivering thermogenesis (NST). NST has been defined as “heatproducing mechanisms due to processes that do not involve muscular contractions, such as those involved in ion pumping or mitochondrial loose coupling mediated by uncoupling protein-1 (UCP1) in brown fat”. Shivering is defined as an involuntary rhythmic contraction of skeletal muscle myofibrils involving no voluntary movements or external work. Heat production during shivering involves biochemical mechanisms close to those associated with the contraction of the skeletal muscle myofibril: heat is produced during both the hydrolysis of ATP and the associated processes of ATP re-synthesis, and all energy substrates can support shivering. Tremendous amounts of heat can be produced during shivering but this mechanism is not very efficient because it occurs at the periphery of the body, and therefore enhances thermolysis, and it impairs physical movements. In addition, heat produced during physical activity and meal consumption can contribute significantly to the extra thermoregulatory heat produced in the cold. 3.2. Contribution of feeding to extra heat production A major role of colostrum is the provision of immunoglobulins. However, owing to the low body energy stores at birth, it is obvious that colostrum is of utmost importance in the provision of energy in the first day of life. This is evidenced by the fact that in cold conditions both body temperature and heat production are positively related to the amount of colostrum intake (Noblet and Le Dividich, 1981). Moreover, ingestion of colostrum results in metabolic heat production (Gentz et al., 1970). This metabolic response, referred to as postprandial thermogenesis, represents the energy cost associated with digestion, absorption and processing of nutrients. However, its contribution to thermoregulation is marginal, accounting for about 10% of the extra heat produced in the cold (Herpin et al., 1994), probably because of the high (0.91) efficiency of colostral metabolizable energy (ME) for growth (Le Dividich et al., 1994). In contrast, the efficiency of milk ME for growth is lower (0.73; Marion and Le Dividich, 1999), suggesting a possible higher contribution of the thermal effect of milk to the extra heat produced in the cold. 3.3. Shivering or non-shivering thermogenesis The nature of heat production mechanisms in cold-exposed newborn mammals has long been an open question. It has been known for years that the newborn pig shivers vigorously from

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birth (Mount, 1968) and is at variance with most other newborn mammals in that it contains very little adipose tissue of any type at birth, and appears to have no brown fat. The unlikely presence of brown fat and existence of NST are suggested by (i) the absence of calorigenic response to the injection of noradrenaline during the first week of life (LeBlanc and Mount, 1968; Brück et al., 1969) and (ii) the immunoblotting studies of Trayhurn et al. (1989) showing that piglets do not express UCP1 in various tissues. However, some doubts subsist, because small quantities of adipose tissue resembling brown adipose tissue have been detected in 3-month-old pigs (Dauncey et al., 1981). In skeletal muscle, the situation is even more intriguing because loose-coupled mitochondria have been detected in skeletal muscle of 2-month-old cold-adapted pigs (Herpin and Barré, 1989). Interestingly, UCP homologues have been recently identified in numerous mammalian tissues including skeletal muscle (review in Ricquier and Bouillaud, 2000), which reinforces interest in the molecular mechanisms underlying cellular thermogenesis. These proteins (UCP) allow the dissipation of part of the proton electrochemical gradient generated by the electron transfer chain across the inner mitochondrial membrane and can thus increase heat production by uncoupling respiration from ATP synthesis (Boss et al., 1998b). This role is probably significant since the proton leak, in part sustained by UCPs, contributes up to 50% to skeletal muscle basal respiration rate and nearly 30% to standard metabolic rate in the rat (Brand et al., 1994). UCP1 is exclusively expressed in brown adipose tissue and plays a vital role in protection against cold. The UCP homologues, UCP2 and UCP3, are respectively 59% and 57% identical to UCP1 in their amino acid sequences. UCP2 is widely expressed in human and rat tissues whereas UCP3, which shares 73% identity with UCP2, is highly expressed in muscle (Fleury et al., 1997). Both proteins are able to uncouple respiration when they are recombinantly expressed in yeast (Fleury et al., 1997; Gong et al., 1997) and in myoblasts (Boss et al., 1998a). 3.3.1. Search for uncoupling proteins in piglet skeletal muscle Despite the absence of UCP1 in pigs, UCP2 and UCP3 are expressed in skeletal muscle (Damon et al., 2000a). However, in agreement with most recent studies, cold stress has no effect on UCP3 expression in pig skeletal muscle (Damon, unpublished observation). Results that do not support a classic thermogenic uncoupling role for UCP2 or UCP3 include the absence of an abnormality in thermoregulation in UCP3 or UCP2 knockout mice (Arsenijevic et al., 2000; Gong et al., 2000) and a rise in their skeletal muscle transcript levels during fasting, food restriction or chronic exercise. Thus, the physiological importance of UCP2 and UCP3 in regulatory cold-induced thermogenesis is still a matter of debate in the pig, as in other species, and the presence of UCP2 and UCP3 in pig muscle is not conclusive of the existence of NST. As far as we know, the most recent investigations suggest that UCP3 has the potential to act as a molecular determinant in the regulation of resting metabolic rate by 3,5,3′-triiodothyronine (T3) (De Lange et al., 2001), and to play a role in the export of fatty acids from mitochondria (Himms-Hagen and Harper, 2001). In piglet muscle, the up-regulation of UCP3 mRNA by acute T3 treatment is less marked than in rats (Damon et al., 2000a). 3.3.2. Search for the existence of non-shivering thermogenesis in the piglet To establish the possible existence of non-shivering thermogenesis, it is necessary to measure simultaneously the magnitude of shivering and the level of heat production at temperatures

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Fig. 2. Changes in heat production (  ) and shivering intensity (integrated EMG,  ) with ambient temperature at 2 h and 5 days of age. Adapted from Berthon et al. (1994).

ranging from thermoneutrality to cold (Barré et al., 1985). As illustrated in fig. 2, this allows comparison of the lower critical temperature (LCT) with the temperature threshold for shivering (STT). The lack of delay between the cold-induced increase in heat production (LCT) and the onset of shivering (STT), and the strict linearity of the relationship between metabolic rate and shivering intensity within the range of ambient temperature studied (fig. 2), confirm the absence of NST and the main role of shivering in neonatal thermogenesis (Berthon et al., 1994). This result is observed at 2 h as well as at 5 days of life and is not modified after 2 days of cold exposure (Berthon et al., 1994). One interesting point in fig. 2 is the reduction of shivering intensity between 2 h and 5 days of age. In the absence of regulatory non-shivering thermogenesis, this reduction of shivering intensity with age for a given metabolic rate suggests that the thermogenic efficiency of shivering, i.e. the heat power of shivering, increases with postnatal age in the cold. In other words, more heat is produced per unit increase in the electrical activity of shivering at 5 days than at 2 h of life (Berthon et al., 1994), which should definitely contribute to the improvement of piglet thermostability after birth. In pigs aged 5–6 weeks and exposed to cold for 3 weeks, shivering is progressively replaced by NST. In the absence of brown fat, skeletal muscles are potential candidates for such adaptations, especially when one considers that skeletal muscle mass contributes substantially to total body weight. Two mechanisms that are likely to contribute to heat production have been identified in skeletal muscles from cold-acclimated weaned piglets. First, it is now well accepted that cellular O2 consumption and ion pumping are intimately linked (Gregg and Milligan, 1982), and it has been shown that Na+/K+-ATPase-dependent respiration represents about 20% of whole-muscle O2 consumption. The activity of this pump is increased by 75% in the cold and this increase accounts for 70% of the total increase in muscle respiration (Herpin et al., 1987a; Harrison et al., 1994), which is probably associated with a similar increase in heat production. Second, heat can also be directly produced at the level of ATP synthesis. Indeed, a loose coupling between oxidation and phosphorylation is observed in subsarcolemmal mitochondria from rhomboideus muscle (Herpin et al., 1987b). This muscle is located between the shoulders, i.e. at the same place that brown fat, with its mitochondrial uncoupling, occurs in cold-acclimated rats. However, the biological significance and importance of those mechanisms is difficult to assess and the existence of non-shivering thermogenesis has not been confirmed in vivo.

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Fig. 3. Overview of the structures and metabolic pathways involved in muscle contraction and heat production during shivering.

3.4. Mechanisms involved in the postnatal improvement of shivering efficiency All the components of skeletal muscle energy metabolism can be potentially involved in the postnatal increase in the capacity to produce heat during shivering. Indeed, to produce heat, muscle fibres need to exhibit both an optimal coupling between excitation and contraction, an optimal power of contraction, an adequate supply of substrates and oxygen, and finally an efficient ATP synthesis (fig. 3). 3.4.1. Mechanisms and structures involved in muscle contraction During shivering, repetitive contractions of muscle fibres produce heat by myosin cross-bridge cycling, Ca2+ cycling and Na+/K+ transport. Obviously, the power of contraction of muscle fibres is directly related to myofibril mass and, unfortunately, piglet fibres definitely lack myofibrils at birth (Bradley et al., 1980; Handel and Stickland, 1987; Herpin et al., 2002). Recent results show that myofibril volume density is quite low in longissimus lumborum (LL, 32%) and rhomboideus (RH, 40%) muscles at birth, and increases markedly within 5 days (fig. 4). The high rate of muscle protein synthesis in the newborn is reported to be restricted entirely to the myofibrillar protein compartment (Fiorotto et al., 2000). This marked postnatal increase in myofibril mass is probably one of the key events in the development of muscle function and should contribute to the enhancement of muscle contraction potential during shivering. With regards to muscle fibre types, endurance and high aerobic capacities are important features for thermogenesis because of the continuous nature of shivering. Structures enhancing endurance are mitochondria and their fuel source, lipids, and they are mostly present in slow-oxidative (type I) and fast-oxidoglycolytic (type IIa) fibres. Moreover, concerning the economy of contraction, fast muscles consume more energy than slow when working for the same time against the same load (Crow and Kushmerick, 1982) and fast-cycling muscles have

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Fig. 4. (A) Fibre (Vfibre) and (B) myofibril (Vmyofibril) volume density in longissimus lumborum (LL) and rhomboideus (RH) muscles of newborn (open bars) and 5-day-old piglets exposed to thermoneutral (striped bars) or cold (solid bars) conditions. Means sharing a common superscript did not differ significantly (P > 0.05). The effect of muscle type on Vfibre is significant (P < 0.05). Adapted from Herpin et al. (2002).

relatively uneconomical force development owing to their high cross-bridge cycling rate and short contraction time (Alpert and Mulieri, 1986). As a whole, fast-contracting highendurance fibers (type IIa fibres) may produce more heat per unit time than other fibres when used in shivering. Interestingly, the proportion of type IIa fibres is very high in piglet muscles at birth (Lefaucheur and Vigneron, 1986). In addition, transitory expression of α-cardiac myosin heavy chain (MHC), with contractile properties intermediate between type I and type IIa MHC, has been recently demonstrated in muscle fibres of piglets (Lefaucheur et al., 1997). Moreover, the expression of this myosin isoform is markedly increased within the first 5 days of life and is stimulated by chronic cold exposure (Lefaucheur et al., 2002). Although our knowledge of α-cardiac MHC properties is very limited, its marked up-regulation in coldexposed shivering piglets points out its potential role in the enhancement of shivering efficiency after birth. Also, the excitation–contraction coupling apparatus appears to be immature at birth in pigs, as already shown in birds (Eppley and Russell, 1995). Triads correspond to the junctional association of transverse tubules with sarcoplasmic reticulum terminal cisternae in mature skeletal muscle (fig. 3) and thereby play a crucial role in calcium release in excitation– contraction coupling (Flucher, 1992). Electron microscopic examination showed that they proliferate rapidly in LL and RH muscles of cold-exposed piglets after birth (+70% to +90% within 5 days) (fig. 5), suggesting that the efficiency of excitation–contraction coupling

Fig. 5. Number of triad profiles per unit fibre area (Ntriad) in longissimus lumborum (LL) and rhomboideus (RH) muscles of newborn (open bars) and five-day old piglets exposed to thermoneutral (striped bars) or cold (solid bars) conditions. Means sharing a common superscript did not differ significantly (P > 0.05). Adapted from Herpin et al. (2002).

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increased in the cold (Herpin et al., 2002). In other words, for a given electrical stimulation more of the sarcoplasmic reticulum is likely activated during contraction and calcium release and cycling are higher, thereby promoting an increase in ATP hydrolysis and heat production through cross-bridge cycling, calcium reuptake and myofibril contraction (Block, 1994). Therefore, triad proliferation is probably of utmost importance in the adaptive and improved response of skeletal muscle to sustained shivering during the early neonatal period. 3.4.2. Muscle blood supply Blood flow through muscles is highly adaptable and represents an important determinant of oxygen and nutrient supply and consumption in all species (Hoppeler et al., 1981). It should be sufficient to supply oxygen and nutrient during shivering activity and to support heat production mechanisms. When comparing muscle blood flow in 1- and 5-day-old piglets, it appears clearly that muscle cardiovascular adjustments in the cold are limited in the newborn pig (table 2). Measurement of blood flow in a representative muscle compartment, i.e. the hindquarters, in conscious piglets shows that muscle blood flow increases with age and short-term cold exposure. However, changes in blood flow in response to a similar cold challenge were 3 times higher in 5-day-old (+65%) than in 1-day-old (+25%) piglets, suggesting that blood supply to the shivering muscle was considerably improved with age (Lossec et al., 1998b). Measurement of blood flow in individual muscles using coloured microspheres confirms these results (Lossec et al., 1999). Interestingly, a preferential redistribution of cardiac output towards skeletal muscle was only observed at 5 days of life, at the expense of the small intestine, the liver and the skin; this cardiovascular response was more pronounced in the most oxidative skeletal muscles studied (RH vs. LL). This should favour, and probably potentiate, the efficiency of shivering. Indeed, a redistribution of cardiac output to the most thermogenic

Table 2 Cardiac output and its fractional distribution Fco to selected tissues or whole organs in 1- and 5-day-old pigs exposed to thermoneutral (TN) or cold (C) environment 1-day-old

Cardiac output, mL/min/kg Fco for 10 g tissue (%) Longissimus thoracis Rhomboideus Subcutaneous adipose tissue Skin Fco to whole organs, % Heart Liver Small intestine Brain Adrenals Thyroid

5-day-old

TN

C

TN

C

436

535*,a

448

550*

0.42 0.47 0.25 0.25

0.55* 0.64* 0.22 0.25

0.42 0.49 0.21 0.35

0.61* 0.87* 0.22 0.27*

1.87 3.97 13.6 2.72 0.14 0.08

2.45* 3.75 14.0 2.86 0.14 0.09

3.24 6.44 13.7 3.27 0.13 0.08

4.61* 5.18* 10.8* 3.12 0.15 0.11*

a given age. *denotes significant effect (P < 0.05) of cold. Adapted from Lossec et al. (1999).

a At

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organs has already been reported in rats (Foster and Frydman, 1979), pigs (Mayfield et al., 1986) and ducklings (Duchamp and Barré, 1993). Additional investigations using electron microscopy have shown no increase in skeletal muscle capillary bed with age or cold exposure (Herpin et al., 2002). This suggests that the observed postnatal improvement of cardiovascular adjustments required for shivering thermogenesis may be accommodated by existing capillaries. Therefore, present changes are more likely related to the postnatal maturation of the nervous and hormonal regulation of muscle blood flow. 3.4.3. Activity and ultrastructure of muscle mitochondria The plasticity of mitochondrial density and activity according to age or cold acclimation is well documented. During shivering, optimal supply of ATP via oxidative phosphorylation is necessary when glycogen stores are exhausted. In contrast to liver mitochondria (Mersmann et al., 1972), muscle mitochondria are functional from birth in pigs and are changing primarily quantitatively during the first 5 days of life (Berthon et al., 1996a; Schmidt and Herpin, 1997). The oxidative potential of pig muscle increases gradually after birth, but no consistent changes in mitochondrial respiration, respiratory control and phosphorus:oxygen ratio are evidenced during this period and after short-term cold exposure (Schmidt and Herpin, 1997). However, biochemical characteristics of intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondria differ from birth. The higher respiration rate and higher respiratory control ratio shown by IMF compared with those shown by SS mitochondria are principally due to the higher activities involved in substrate oxidation because there is no difference in the proton leak between both populations (Lombardi et al., 2000). Thus, the actual event responsible for the postnatal increase in skeletal muscle oxidative potential is the enhancement of mitochondrial mass (Schmidt and Herpin, 1997), as already reported in various tissues and species during the neonatal period (Mersmann et al., 1972; Eppley and Russell, 1995). Between birth and 5 days of life, mitochondrial mass increased by 49% in glycolytic LL muscle and by 93% in oxidative RH muscle. In LL muscle this increase was only supported by the proliferation of IMF mitochondria, whereas both types of mitochondria (IMF and SS) proliferate in RH muscle. The mechanisms underlying these changes have been elucidated by electron microscopic examination. Within 5 days (fig. 6), there is an increase in both the number of mitochondria and the surface of the inner membrane and cristae of each mitochondrion (Herpin et al., 2002). Indeed, the number of respiratory chain and F1-ATPase units is directly related to this parameter (Hoppeler, 1986). Interestingly, this postnatal change in the surface of the inner membrane is more marked in RH than in LL muscle, and is further enhanced when piglets are exposed to cold for 5 days. As a whole, this should contribute to the enhancement of muscle endurance during contractile activity associated with shivering, and to the postnatal acquisition of muscle metabolic type.

4. SOURCES OF ENERGY The requirement for energy in the newborn pig is met by body energy reserves, colostrum and milk. During the neonatal period the protein accretion is very high, and the potential for protein deposition is probably beyond that allowed by milk intake (Le Dividich and Sève, 2001). The rate of amino acid catabolism is very low during this period and is not enhanced in cold conditions (Herpin et al., 1992). The contribution of energy derived from amino acid catabolism is therefore of marginal importance and will not be discussed.

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Fig. 6. Stereological parameters of mitochondria in longissimus lumborum (LL) and rhomboideus (RH) muscles of newborn (open bars) and 5-day-old piglets exposed to thermoneutral (striped bars) or cold (solid bars) conditions.(A) Number of mitochondria per unit fiber area (Nmitochondria); (B) Mitochondria volume density (Vmitochondria); (C) Surface of outer mitochondrial membranes per unit tissue volume (Somm/Vtissue); (D) Surface of inner mitochondrial membrane and cristae per unit mitochondrial volume (Si+c/Vmitochondria). Means sharing a common superscript did not differ significantly (P > 0.05). The effect of muscle type on Si+c/Vmitochondria is significant (P < 0.05). Adpated from Herpin et al. (2002).

At birth, body energy stores are present as glycogen and fat (fig. 7). Because FFA are poorly transferred across the swine placenta (Thulin et al., 1989), the amount of fat reserves in the newborn pig is low, ranging from 15 to 20 g/kg body weight (BW). Most (45%) of this stored fat is structural fat and is not available for mobilization. Total body glycogen stores range from 30–38 g/kg BW with the major part (~90%) being located in muscle. From an

Fig. 7. Available energy stores at birth and cumulative available energy derived from ingested colostrums. Adapted from Mellor and Cockburn (1986) and Le Dividich et al. (1997).

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energy point of view, glycogen represents >90% of the available stored energy. Nevertheless, available energy derived from body stores is low, accounting for <10% of that of the newborn infant (Mellor and Cockburn, 1986). Soon after birth the pig is fed at intervals with colostrum for 24–36 h and thereafter milk. The first suckling occurs in the 20–30 min following birth and colostrum intake can be very high immediately after birth since the first three sucklings account for ~25% of the total colostrum ingested during the first day (Fraser and Rushen, 1992; Le Dividich et al., 1997). Although highly variable, total consumption of colostrum in the first day of life is in the range of 310–340 g/kg BW and may be as high as 460 g/kg BW (Le Dividich et al., 1997). Fat accounts for 35–50% and 55–65% of total energy of colostrum and milk, respectively, and represents the main source of energy. Colostrum and milk fats are highly digestible (~100%). They are composed of long-chain fatty acids (>C14), but are devoid of medium-chain fatty acid (MCFA). Both the fatty acid profile and amount of colostrum and milk fat can be manipulated by the source of dietary fat provided to the dam in late gestation and throughout lactation (Averette et al., 1999), but there is no evidence for mammary MCFA transfer (Newcomb et al., 1991). Moreover, lactose is the predominant carbohydrate in colostrum and milk, but its content is lower in colostrum than in milk (3.1–3.9% vs. 4.8–5.5%). This abrupt shift in the source of energy substrates from mainly glucose to colostral and milk fat implies that the piglet is rapidly capable of (i) oxidizing fat to provide energy for heat production, (ii) depositing fat for thermal insulation and energy through its mobilization at weaning, and (iii) providing glucose to its glucose-dependent tissues.

5. EFFECTS OF THE SHIFT IN THE ENERGY SOURCE ON LIPID OXIDATION, LIPOGENESIS AND GLUCONEOGENESIS 5.1. Lipid oxidation During the first postnatal hours, piglets rely almost entirely on carbohydrate to meet their thermoregulatory needs (Mount, 1968). However, in usual environmental conditions at birth, 75% of liver glycogen and 41% of muscle glycogen is mobilized by 12 h postpartum (Elliot and Lodge, 1977). Cold exposure hastens the depletion in both tissues (Herpin et al., 1992), and increases the rate of glucose turnover (Lossec et al., 1998a) and of peripheral glucose uptake (Duée et al., 1988; Lossec et al., 1998a). However, colostrum intake increases availability of lipids. The ensuing increase in plasma NEFA (Le Dividich et al., 1991b) and glycerol (Bengtsson et al., 1969) secondary to the increased activity of the adipose tissue hormonesensitive lipase (Horn et al., 1973; Steffen et al., 1978) is associated with a progressive decline in respiratory quotient during the first postnatal day in both thermoneutral and cold environments (fig. 8), providing evidence for an early involvement of lipids as an energy source (Noblet and Le Dividich, 1981; Berthon et al., 1993). At 48 h of age, fatty acids account for ~60% of the energy metabolism, increasing to 90% in the 7-day-old pig fed at maintenance (Marion and Le Dividich, 1999). Biochemical adjustments associated with the improved ability of the piglet to oxidize fat in the skeletal muscle include: 1. An increase in muscle lipid content within the first postnatal days. For example, muscle lipid content nearly doubled within 5 days in LL and RH muscle, in agreement with the increase in the number of lipid droplets per unit tissue area (Herpin et al., 2002). At birth these lipid droplets are scarce, but at 5 days they are wedged between the myofibrils and the IMF mitochondria, a position that is ideal for optimizing the provision of energy for oxidative metabolism and sustained shivering.

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Fig. 8. Respiratory quotient of the neonatal pig in relation to age and ambient temperature. Adapted from Berthon et al. (1993) and Schmidt and Herpin (1997).

2. An increase in NEFA uptake by the shivering muscle at 5 days of life whereas this uptake is negligible in 1-day-old piglets (Lossec et al., 1998b). 3. An increase in mitochondrial (+100%) and peroxisomal (+160%) β-oxidation potential in LL and RH muscle homogenates between birth and 5 days of life (Herpin, unpublished observation). Interestingly, a similar increase in mitochondrial and peroxisomal β-oxidation potential has been previously reported in liver, kidney and heart of young pigs (Yu et al., 1997). Deeper investigations into the molecular and biochemical regulation of fatty acid oxidation in piglet skeletal muscle showed that oleic, linoleic and palmitic acids are readily oxidized from birth by isolated skeletal muscle mitochondria (Schmidt and Herpin, 1998). MCFA (C8–C10), which are now being introduced in colostrum and milk substitutes, are readily oxidized by the liver (for review, see Odle, 1997). However, in vivo studies in respiration chambers (Léon et al., 1998) provide evidence that substitution of MCFA for long-chain fatty acids in colostrum does not improve the energy status of the newborn, even in cold conditions (fig. 9). It is suggested that MCFA are poorly oxidized by skeletal muscle as indicated by in vitro studies using isolated muscle mitochondria (Schmidt and Herpin, 1997). Surprisingly, the mitochondrial potential is not increased with age, which suggests that the enhancement of fatty acid oxidation potential with age is mostly supported by the above-mentioned postnatal proliferation of muscle mitochondria. Complex changes in the expression and activity of carnitine palmitoyltransferase I (CPT I), which is the limiting enzyme of fatty acid β-oxidation, have also been observed (Odle et al., 1995; McGarry and Brown, 1997). In piglets, CPT I activity increases postnatally in SS muscle mitochondria and is modulated by malonyl-CoA in IMF mitochondria (Schmidt and Herpin, 1998). Indeed, between birth and 5 days of life, both the sensitivity of CPT I to malonyl-CoA inhibition and the tissue level of malonyl-CoA decreased, which could partly relieve CPT I inhibition and enhances fatty acid utilization. Further, during cold stress, the decrease in the tissue levels of malonyl-CoA is even more marked in the most oxidative muscle. A surprising result is the difference in CPT I sensitivity to malonyl-CoA between piglets and rats: in piglets, sensitivity to malonyl-CoA is much lower in muscle than in the

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Fig. 9. Effect of substitution of medium- (MCFA) for long- (LCFA) chain fatty acids in colostrum on heat production (a) and respiratory quotient (b) in the newborn pig in relation to environmental temperature. Data adjusted to a common ME intake of 960 kJ/kg BW per 26 h. Adapted from Léon et al. (1998).

liver (Schmidt and Herpin, 1998; Nicot et al., 2001) whereas the opposite has been obtained in rats. At the molecular level, two isoforms with tissue-specific expression and sensitivity to malonyl-CoA inhibition are usually described. Recently, partial liver (CPT1-L) and muscle (CPT1-M) cDNA sequences have been successfully cloned and the co-expression of these two isoforms in skeletal muscle of neonatal piglets has been demonstrated (Damon et al., 2000b). The expression of CPT1-L in pig skeletal muscle could provide a partial answer to the difference of sensitivity to malonyl-CoA between rat and pig muscles (Schmidt and Herpin, 1998). However, co-expression of both isoforms should result in an intermediate and not a reverse sensitivity to malonyl-CoA inhibition. Recent data support another seductive hypothesis. In fact, in yeast expressing pig CPT1-L, kinetic characteristics (Km’s for carnitine and palmitoyl-CoA) were similar to those of human and rat CPT1-L whereas sensitivity to malonyl-CoA inhibition was found to be closer to that of rat and human CPT1-M isoforms (Nicot et al., 2001). It then appears that pig CPT1-L possesses specific biochemical properties despite its high degree of homology with CPT1-L from other mammals. Thus pig CPT1-M could also behave as CPT1-L of other mammals in terms of malonyl-CoA sensitivity. It has been speculated that CPT1-L was prone to hormonal regulation whereas CPT1-M was more regulated by nutritional factors (Cook et al., 2001). Thus co-expression of both isoforms in pig skeletal muscle could allow a fine tuning of lipid utilization. 5.2. Lipogenesis The neonatal pig has a remarkable capacity to deposit large amounts of fat soon after birth. Depending on the colostrum fat content, carcass fat content increases by 25–100% during the first day of life (Le Dividich et al., 1997). During the suckling phase, fat accretion occurs at a mean rate of 30–35 g/day, depending mainly on the amount of ingested milk (Marion and Le Dividich, 1999) and on the milk fat content (Jones et al., 1999). De novo lipogenesis is marginal, albeit being limited not by enzyme activities or insulin-regulated glucose transporter (GLUT 4), but by the substrate availability (Gerfault et al., 2000). Colostrum and milk are the major routes for lipid acquisition (Sarkar et al., 1985). Adipose tissue lipoprotein lipase,

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an enzyme playing a key role in fat storage, contributes to the ability of the suckling pig to deposit large amounts of fat. Its activity, already high at birth, is increased 3–4-fold in the early neonatal period (Steffen et al., 1978; Le Dividich et al., 1997). It is suggested that sow’s milk is designed to promote fat accretion in the young pig. Stored fat is mostly subcutaneous fat that provides thermal insulation to the young pig and energy through its mobilization during the period of low feed intake following weaning (Le Dividich and Sève, 2001). 5.3. Gluconeogenesis The glucose requirement of the neonatal pig is very high, reaching ~15 g/kg BW per day (Flecknell et al., 1980; Pégorier et al., 1984). It is 50% higher than in lambs and human infants. Moreover, the glucose requirement is enhanced ~30% in the cold (Duée et al., 1988; Lossec et al., 1998a). Glucose requirements are met by (i) liver glycogenolysis, since only liver glycogen is able to release glucose into the blood, (ii) colostrum and milk and (iii) gluconeogenesis. Provision of glucose by the first two sources meets 50–60% of the requirements during the first day of life, which underlines the importance of the gluconeogenic pathway in the glucose homeostasis of the piglet. Gluconeogenesis is the process by which glucose is synthesized from various precursors. The liver is the main site of gluconeogenesis. The developmental pattern of hepatic gluconeogenesis has been the subject of several extensive reviews (Girard, 1986; Girard et al., 1992) and will not be discussed in detail in this chapter. In brief, key enzymes involved in the pathway, i.e. pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6diphosphatase and glucose-6-phosphatase (G6Pase), have a substantial activity (35-105% of adult values) at birth. In both fed and unfed pigs, enzyme activity increases markedly during the first postnatal day. Also, the insulin:glucagon molar ratio decreases after birth in both fed and unfed piglets, thus providing an appropriate environment for an active gluconeogenesis. However, the level of plasma NEFA is much higher in the fed pigs. In fact, colostrum ingestion is essential to sustain a high rate of hepatic gluconeogenesis. Based on the amount of glucose available from lactose digestion (glucose + galactose), colostrum provides at least 40–45% of the glucose requirement and ~80–90% on the assumption that all galactose is converted into glucose by the G6Pase. However, piglets fasted from birth (Goodwin, 1957) or fed a low-fat colostrum (Herpin et al., 1992) are unable to sustain normal glycaemia. It is suggested that fatty acid oxidation plays a major role in glucose homeostasis (Girard, 1986) through the supply of ATP and co-factors (NADH and acetyl-CoA) for catalysing key reactions. This is convincingly demonstrated by the in vivo and in vitro studies of Pégorier et al. (1985) and Duée et al. (1985). In contrast, Lepine et al. (1991) failed to find any stimulatory effects of fatty acid oxidation on the rate of glucose production by isolated hepatocytes from piglets.

6. REGULATION OF ENERGY METABOLISM DURING EARLY DEVELOPMENT 6.1. The thyroid axis Thyroid hormones (TH) are known to play a major role in the regulation of metabolic adaptations and growth. They exert their effects primarily through interactions with nuclear TH receptors (TR) which occur as a series of isoforms controlling the transcription of thyroid hormone-responsive genes (Lazar, 1993; Wrutniak-Cabello et al., 2001). The ontogenic

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profile of the thyroid system suggests that TH metabolism is fully developed at birth. Plasma concentrations of both total and free TH, thyroid gland weights and hepatic 5′-deiodinase activity all increase during late gestation (Berthon et al., 1993). It is relevant to notice that receptors are already present in skeletal muscle, but not in the liver, at 80 days of gestation, suggesting that porcine muscle can potentially respond to TH much earlier in development than the liver (Duchamp et al., 1994). During the first 6 h after birth, there is a surge in T3, free T3 and T4 plasma concentrations and, apart from a transient decline at 12 h, TH concentrations remain elevated during the first 2 days and then decline slightly over the next 2 weeks (Slebodzinski et al., 1981; Berthon et al., 1993, 1996b). The finding that the postnatal surge in plasma TH levels precedes the physiological rise in heat production in the newborn suggests a close relationship between perinatal thyroid status and neonatal thermogenic capacity (Berthon et al., 1993). This is exemplified by the findings that (i) hypothyroidism at birth is associated with depressed thermoregulatory capabilities of the newborn (Berthon et al., 1993) and (ii) a single injection of T4 induces an increase in metabolic rate (Slebodzinski, 1979). TH control the oxidative capacities in the newborn through a short-term regulation of mitochondial respiration (Herpin et al., 1996) and a longterm regulation of mitochondriogenesis (Mutvei et al., 1989). The thyroid axis is also regulated by nutrition and TH actions might be complementary to catecholamine actions during cold-induced thermogenesis. Cold-exposed newborn pigs fed a limited amount of milk exhibit high catecholamines but low T3 levels whereas the opposite is observed in piglets fed a high milk intake (Herpin et al., 1995; Berthon et al., 1996b). These adaptations are assumed to optimize the utilization of either body stores (low intake) or exogenous substrates (high intake). The marked effects of food intake on the thyroid axis are also observed during the whole suckling period: a low intake reduces thyroid gland activity, circulating TH concentrations and nuclear TR abundance in muscle (Dauncey, 1990; Morovat and Dauncey, 1995). 6.2. HPA axis Circulating levels of glucorticoids and catecholamines are very high at the time of birth and dramatically decrease thereafter (Kaciuba-Uscilko, 1972; Randall, 1983). Cortisol and catecholamines are potent stimulators of catabolism and one can speculate that these high levels induce mobilization of glycogen stores after birth. However, response of catechalomine to cold exposure during the first 5 days of life is variable, with both no change (Lossec, 1998) and a marked increase (Duée et al., 1988; Le Dividich et al., 1991a) being reported. Moreover, the response to cold is found to be impaired in moderately hypothermic piglets (Mayfield et al., 1989) or, as mentioned above, to be dependent on the level of milk intake (Herpin et al., 1995). A lipolytic response is only detected at 2–4 days of age (Curtis and Rogler, 1970; Persson et al., 1971). Neither norepinephrine nor epinephrine administration elicits a thermogenic response in the neonatal pig (LeBlanc and Mount, 1968; Persson et al., 1971). Clearly, these observations indicate that the actual role of catecholamines in the neonatal thermogenesis requires further investigation. 6.3. Somatotropic axis Even though the regulation of energy metabolism by the somatotropic axis is well documented in the growing pig (Louveau and Bonneau, 2001), there is no evidence from the literature that the somatotropic axis contributes to the cold-induced thermogenesis. However, because of a high potential for protein synthesis and growth of the neonatal pig (Le Dividich

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and Sève, 2001), one might expect that the energy metabolism is to some extent regulated by the somatotropic axis. Plasma GH concentrations are very high at birth and decrease sharply during the next 2–3 days (Scanes et al., 1987; Carroll et al., 1998). Although the significance of these high levels of plasma GH is not completely understood, GH could contribute to the maintenance of protein accretion in the newborn pig, even in negative energy balance (Herpin et al., 1992). Plasma IGF-I concentrations increase significantly during the first 3 weeks after birth (Lee et al., 1991, 1993; Louveau et al., 1996). After 24 h of feeding, IGFBP profile changes with the abundance of plasma IGFBP-3 predominating (Lee et al., 1991). Changes in GH and IGF-I receptor levels are also observed during this period, with GH receptor increasing over the first 10 days of life in liver and IGF-I receptor decreasing in skeletal muscle and other tissues (Breier et al., 1989; Lee et al., 1993; Louveau et al., 1996; Schnoebelen-Combes et al., 1996). These profiles are modulated by thyroid status (Duchamp et al., 1996). In addition, the somatotropic axis appears to be functional and responsive to GH administration in neonatal pigs, although the responsiveness is reduced compared to older pigs (Harrell et al., 1999). The administration of GH at a dose that is commonly used in older pigs has little or no effect on growth rate or plasma IGF-I or IGFBP-3 (Harrell et al., 1999; Dunshea et al., 2001). Perhaps the lack of response in the growth rate is not surprising owing to the already high rate of protein synthesis. Changes in nutritional status during the neonatal period are associated with several changes in the GH–IGF-I axis. Both moderate and severe feed restriction (Dauncey et al., 1994; Louveau and Le Dividich, 2002) in the suckling period decrease plasma IGF-I and IGFBP-3 levels. These data indicate that circulating IGF-I is directly related to energy intake in neonatal pigs as observed in older animals. Even though the regulation of receptors may represent an important mechanism of control within the GH–IGF-I axis, the few available data indicate that the regulation of IGF-I and GH receptors is tissue-specific and dependent on the type of undernutrition during the suckling period (Louveau and Le Dividich, 2002).

7. CONCLUDING COMMENTS AND FUTURE PERSPECTIVES This chapter provides new insights on the development of the energy metabolism in a species devoid of brown fat. Key factors involved in the poor abilities of the newborn pig to withstand cold stress include mainly the relative immaturity of the newborn pig and the availability of energy substrates. Improvement of its thermogenic capacities within the first postnatal days parallels maturation of the skeletal muscle metabolism and function and of the cellular machinery. In the future, in the light of improving survival, it should be relevant to select piglets on physiological traits related to maturity. This is convincingly attested by the first findings (Leenhouvers, 2001) that selection of pigs with different genetic merit for survival leads to piglets with a higher maturity at birth. Attempts made to improve the energy available at birth resulted only in moderate increase in energy stores at birth. However, effects of sow nutrition during pregnancy on fetal muscle development during the critical stages of fetal development warrant future investigation. In addition, we suggest that more research should be focused on factors initiating and controlling quantity and quality of colostrum and milk produced by the sow. However, during the past decades, selection for lean tissue growth has led to less mature pigs at birth (Herpin et al., 1993). Selection of sows for higher litter size has resulted in problems of increased intrapartum deaths, proportion of weak piglets and competition at the udder (Quiniou et al., 2002). Therefore, our efforts will be in vain if the survival of the piglet continues to be challenged unwisely by the pig industry.

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Newcomb, M.D., Harmon, D.L., Nelssen, J.L., Thulin, A.J., Allee, G.L., 1991. Effect of energy source fed to sows during late gestation on neonatal blood metabolite homeostasis, energy stores and composition. J. Anim. Sci. 69, 230–236. Nicot, C., Hegardt, F.G., Woldegiorgis, G., Haro, D., Marrero, P.F., 2001. Pig liver carnitine palmitoyltransferase I, with low Km for carnitine and high sensitivity to malonyl-CoA inhibition, is a natural chimera of rat liver and muscle enzymes. Biochemistry 40, 2260–2266. Noblet, J., Le Dividich, J., 1981. Energy metabolism of the newborn pig during the first 24 h after birth. Biol. Neonate 40, 175–182. Odle, J., 1997. New insights into the utilization of medium-chain triglycerides by the neonate: observations from a piglet model. J. Nutr. 127, 1061–1067. Odle, J., Lin, X., van Kempen, T.A.T.G., Drackley, J.K., Adams, S.H., 1995. Carnitine palmitoyltransferase modulation of hepatic fatty acid metabolism and radio-HPLC evidence for low ketogenesis in neonatal pigs. J. Nutr. 125, 2541–2549. Pégorier, J.P., Duée, P.H., Simoes-Nunes, C., Peret, J. Girard, J., 1984. Glucose turnover and recycling in unrestrained and unaesthetized 48-h-old fasting or post-absorptive newborn pigs. Brit. J. Nutr. 52, 277–287. Pégorier, J.P., Simoes-Nunes, C., Duée, P.H.,. Peret, J. Girard, J., 1985. Effect of intragastric triglycerides administration on glucose homeostasis in newborn pigs. Amer. J. Physiol. 249, E268–E275. Père, M.C., 1995. Maternal and fetal blood levels of glucose, lactate, fructose and insulin in the conscious pig. J. Anim. Sci. 73, 2994–2999. Persson, B., Gentz, J.C.H., Hakkarainen, J., Kellum, M., 1971. Catecholamine-induced lipolysis and its relation to oxygen consumption in the newborn piglet. Pediat. Res. 5, 435–445. Quiniou, N., Dagorn, J., Gaudré, D., 2002. Variation of piglets’ birth weight and consequences on subsequent performance. Livest. Prod. Sci., Special Issue 78, 63–70. Randall, G.C., 1983. Changes in the concentrations of cortocosteroids in the blood of fetal pigs and their dams during late gestation and labor. Biol. Reprod. 29, 1077–1084. Ricquier, D., Bouillaud, F., 2000. Mitochondrial uncoupling proteins: from mitochondria to the regulation of energy balance. J. Physiol. 529, 3–10. Sarkar, N.K., Kramer, J.K.G., Wolynetz, M.S., Elliot, J.I., 1985. Influence of dietary fat on growth and body composition of the neonatal pig. Can. J. Anim. Sci. 65, 175–184. Scanes, C.G., Lazarus, D., Bowen, S., Buonomo, F.C., Gilbreath, R.L., 1987. Postnatal changes in circulating concentrations of growth hormone, somatomedin C and thyroid hormones in pigs. Domest. Anim. Endocrinol. 4, 252–257. Schmidt, I., Herpin, P., 1998. Carnitine palmitoyltransferase I (CPT I) activity and its regulation by malonyl-CoA are modulated by age and cold exposure in skeletal muscle mitochondria from newborn pigs. J. Nutr. 128, 886–893. Schmidt, I., Herpin, P., 1997. Postnatal changes in mitochondrial protein mass and respiration in skeletal muscle from the newborn pig. Comp. Biochem. Physiol. B118 639–647. Schnoebelen-Combes, S., Louveau, I., Postel-Vinay, M.-C., Bonneau, M., 1996. Ontogeny of GH receptor and GH-binding protein in the pig. J. Endocrinol. 148, 249–255. Slebodzinski, A.B., 1979. Metabolic responses to thyroxine in the newborn pig. Biol. Neonate 36, 198–205. Slebodzinski, A.B., Nowak, G., Zamyslowska, H., 1981. Sequential observation of changes in thyroxine, triiodothyronine and reverse triiodothyronine during the postnatal adaptation of the pig. Biol. Neonate 39, 191–199. Steffen, D.G., Brown, L.J., Mersmann, H.J., 1978. Ontogenic development of swine (Sus domesticus) adipose tissue lipases. Comp. Biochem. Physiol. B 59, 195–198. Trayhurn, P., Temple, N.J., Van Aerde, J., 1989. Evidence from immunoblotting studies on uncoupling protein that brown adipose tissue is not present in the domestic pig. Can. J. Physiol. Pharmacol. 67, 1480–1485. Thulin, A.J., Allee, G.L., Harmon, D.L., Davis, D.L., 1989. Utero-placental transfer of octanoic, palmitic and linoleic acids during late gestation in gilts. J. Anim. Sci. 67, 738–745. Wrutniak-Cabello, C., Casas, F., Cabello, G., 2001. Thyroid hormone action in mitochondria. J. Mol. Endocrinol. 26, 67–77. Yu, X.X., Drackley, J.K., Odle, J., 1997. Rates of mitochondrial and peroxisomal β-oxidation of palmitate change during postnatal development and food deprivation in liver, kidney and heart of pigs. J. Nutr. 127, 1814–1821.

15

Hepatic gluconeogenesis in developing ruminants S. S. Donkina and H. Hammonb aDepartment

of Animal Sciences, Purdue University, West Lafayette, IN 47907, USA Institute for Biology of Farm Animals Nutrition Physiology (Oskar Kellner Institute), 18196 Dummerstorf, Germany. bResearch

The transition from preruminating to ruminating status represents one of the most dramatic changes in glucose metabolism in mammals. Within 5 weeks of birth, ruminants must undergo the anatomical and physiological adaptations necessary to permit extensive fermentation of plant materials in the rumen and postabsorptive utilization of the end-products. Several well-characterized metabolic adaptations have been documented that act to spare glucose oxidation with the onset of rumination; however, the endocrine and molecular factors that modulate changes in glucose synthesis and metabolism during this transition are not yet fully characterized. This review focuses on the endocrine and metabolic state of the ruminant fetus at term, the development of metabolic competence in the neonatal ruminant, and changes that occur during the transition to ruminating status.

1. GLUCONEOGENIC SUBSTRATES AND METABOLISM Propionate, lactate, and amino acids furnish most of the carbon used for gluconeogenesis in fed ruminants and glycerol provides some gluconeogenic carbon during feed restriction (Huntington, 1990). In neonatal and developing ruminants, milk lactose supplies approximately 25% of the daily glucose needs (Girard, 1990). In the absence of a functional rumen, amino acids, lactate, and to a limited extent, glycerol from milk are used for gluconeogenesis. Development of the fermentation capacity of the rumen is accompanied by changes in the type of carbohydrates ingested, reductions in the amount of fat in the diet, a decrease in availability of dietary carbohydrate to the developing ruminant, and an increased supply of propionate as a gluconeogenic precursor. In both the preruminant and ruminant states the need for active gluconeogenesis to maintain glucose homeostasis is apparent. Pyruvate is a common entry point in the gluconeogenic pathway for lactate, alanine, and other gluconeogenic amino acids. Pyruvate formed from lactate and amino acids is transported into the mitochondria and carboxylated to oxaloacetate by pyruvate carboxylase (PC) (fig. 1).

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Fig. 1. Metabolism of propionate, lactate, pyruvate, and alanine to glucose in bovine liver. Substrate abbreviations are given in the key. Reactions catalyzed by the key enzymes discussed in the text, cytosol phosphoenolpyruvate carboxykinase (PEPCK-C), mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), pyruvate carboxylase (PC), and pyruvate kinase (PK), are indicated by the shaded backgrounds.

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Propionate, in contrast, is metabolized through part of the TCA cycle to oxaloacetate following activation to propionyl-CoA and metabolism through propionyl-CoA carboxylase, methylmalonyl-CoA racemase, and methylmalonyl-CoA mutase. Oxaloacetate can be metabolized to phosphoenolpyruvate (PEP) by phosphoenolpyruvate carboxykinase (PEPCK) or metabolized in the TCA cycle. In turn, PEP carbon can be metabolized to glucose or recycled to pyruvate via pyruvate kinase (PK). In order for lactate carbon to be metabolized to glucose, the flux through PEPCK and PC must exceed the PK flux, whereas net flux of propionate carbon requires only a greater flux through PEPCK relative to PK. Therefore, an increase in PEPCK activity in the absence of changes in PC activity would favor the use of propionate for gluconeogenesis. The presence of PEPCK activity in the cytosol (PEPCK-C) and mitochondria (PEPCK-M) is one of the important features of gluconeogenesis that permits compartmentalization of the pathway and results in the characteristic pattern of regulation and use of lactate and pyruvate. The distribution of this activity is uniquely species-dependent and most mammals display both a mitochondrial and a cytosolic form of the PEPCK enzyme. Rodents express primarily PEPCK-C and both forms are found in liver of the developing chicken, yet only the mitochondrial form is found in liver from the adult chicken. There are approximately equal activities of PEPCK-M and PEPCK-C in the ruminant (Taylor et al., 1971) and human (Hod et al., 1987) liver. Bovine PEPCK-C and PEPCK-M have been recently cloned and characterized (Agca et al., 2002) and the ratio of mRNA indicates a 10-fold greater expression of PEPCK-C than PEPCK-M in lactating cows. Similar data are not yet available for developing bovine. The stoichiometry of gluconeogenesis dictates that the formation of phosphoenolpyruvate from propionate, pyruvate, and some amino acids requires the independent synthesis of NADH in the cytosol for the subsequent reduction of 1,3-diphosphoglycerate in gluconeogenesis. It has been proposed that PEPCK-C is required for gluconeogenesis from amino acids and PEPCK-M is more suited to gluconeogenesis from lactate (Watford et al., 1981). Pyruvate and amino acids are metabolized to oxaloacetate in mitochondria and are shuttled to the cytosol as malate from which NADH and oxaloacetate are regenerated followed by PEP formation that is catalyzed by PEPCK-C. Lactate can also be metabolized to PEP in mitochondria of species that possess appreciable PEPCK-M activity and subsequently shuttled to the cytosol (Holcomb et al., 1995).

2. GLUCOSE RELEASE FROM HEPATOCYTES Glucose-6-phosphatase is a membrane-bound enzyme that is located on the internal membrane of the endoplasmic reticulum and is involved in the terminal step of gluconeogenesis as well as glycogenolysis. The enzyme catalyzes the conversion of glucose-6-phosphate to glucose to enable release from the cell. In nonruminants the enzyme is expressed in liver, kidney cortex, and jejunum, but only the liver form of the enzyme is upregulated at birth and to weaning in rodents (Chatelain et al., 1998; Kalhan and Parimi, 2000). The hepatocyte glucose transporter, GLUT2, and glucose-6-phosphatase act in concert to control the release of glucose from liver. The symmetry of GLUT2 enables the transport of glucose into or out of the hepatocyte and the directionality depends only on the concentration differential between intracellular free glucose and blood glucose (Burchell, 1994). Glucose-6-phosphate (G-6-P), formed through gluconeogenesis or glycogenolysis, must be dephosphorylated through the action of glucose-6-phosphatase (G-6-Pase), an enzyme that is contained within the endoplasmic reticulum in order to release glucose from the hepatocyte. An endoplasmic glucose transporter GLUT7 was initially proposed that would facilitate the

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transport of G-6-P to the endoplasmic reticulum where G-6-Pase acts to release free glucose into the cytoplasm (Burchell, 1994), but has since been retracted (Burchell, 1998). It is now thought that G-6-Pase acts in combination with a specific G-6-P translocase to channel G-6-P into the endoplasmic reticulum where G-6-Pase is compartmentalized (Van Schaftingen and Gerin, 2002). Recently a second transporter protein for G-6-P has been identified (Hosokawa and Thorens, 2002) which complements the activity of the specific G-6-P translocase. When G-6-P is overexpressed in hepatocytes there is a marked increase in glucose release and a decline in intracellular G-6-P and glycogen concentrations (Seoane et al., 1997; Aiston et al., 1999). Measures of G-6-Pase activity at term indicate that the capacity of the enzyme is fully developed at birth in ruminants (Edwards et al., 1975; Stevenson et al., 1976; Narkewicz et al., 1993). Unfortunately data are not yet available for ruminants describing developmental changes in expression of G-6-P translocases.

3. GLUCONEOGENESIS IN FETAL RUMINANTS The contribution of fetal gluconeogenesis to the glucose needs of the developing ruminant conceptus is equivocal. A portion of the discrepancies regarding the contribution of gluconeogenesis to the glucose economy of the developing fetus lies in recycling errors that are inherent to measuring glucose entry rates using isotope dilution (Kalhan and Parimi, 2000). Available data indicate that uterine glucose requirements during the last trimester of pregnancy account for 20–70% of the glucose needs of pregnant ewes (Prior, 1982), that fetal glucose uptake is reduced when ewes are deprived of feed (Tsoulos et al., 1971; Boyd et al., 1973; Chandler et al., 1985; Leury et al., 1990), and that the rate of fetal glucose utilization during maternal feed restriction is less affected than glucose removed via the umbilical artery (Hay et al., 1984). These observations imply that the hypoglycemic ovine fetus is capable of significant endogenous glucose release and is subject to activation in utero in response to maternal nutrition. Amino acids and lactate are the major gluconeogenic substrates in the fetus, and urea excretion rates indicate that 25% of oxygen consumption by fetal ovine liver is due to amino acid catabolism (Gresham, 1972). Efficient extraction of propionate by maternal liver precludes appreciable propionate supply to the developing ruminant fetus for gluconeogenesis. In some experiments, gluconeogenesis from lactate accounts for 22% of lactate turnover and supplies 49% of fetal glucose (Prior, 1980), but in other experiments fetal gluconeogenesis from lactate was undetectable (Warnes et al., 1977). These contradictory observations reflect variations in maternal nutrition immediately prior to the experimental period (Girard et al., 1992). Experimental evidence suggests that the rate of gluconeogenesis is increased in fetal lamb liver in response to inadequate nutrition of the dam (Leury et al., 1990; Apatu and Barnes, 1991a). Increased fetal urea production during nutritional insufficiency in pregnant ewes is consistent with an increase in fetal gluconeogenesis from amino acids (Hodgson et al., 1982). Therefore gluconeogenesis is active and adaptable in fetal ruminants, unlike rodents, and plays a critical role in the glucose economy of the maternal–fetal unit. Enzyme activities have been used to characterize developmental changes in liver metabolism and provide an estimate of the maximum flux through a single step in the gluconeogenic pathway. The activities of glucose-6-phosphatase, fructose-1,6,-bisphosphatase, pyruvate carboxylase, and PEPCK, key enzymes for gluconeogenesis, are similar in term-fetal, neonatal, and adult sheep (Warnes et al., 1977; Narkewicz et al., 1993). There appears to be sequential development of gluconeogenic enzymes in caprine (Dhanotiya and Bhardwaj, 1988) and ovine fetuses (Stevenson et al., 1976). However, the enzymes of the gluconeogenic path are present

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in fetal liver by 100−128 days of gestation (Warnes et al., 1977; Prior, 1980). There is greater activity of alanine aminotransferase in fetal than in neonatal liver, which may reflect a greater capacity for amino acid metabolism to glucose in utero. Changes in hepatic enzyme activity during the period of rumen development are modest and changes in glucose metabolism reflect decreased glycolytic activity in both muscle and liver (Howarth et al., 1968; Pearce and Unsworth, 1980). The activity of lactate dehydrogenase and alanine aminotransferase are lower in adult ewes compared to neonatal sheep or 3-month-old lambs (Edwards et al., 1975), suggesting decreased capacity to metabolize alanine to glucose with development in neonatal ruminants.

4. NUTRITIONAL CHANGES AT BIRTH At birth the neonate must cope with the loss of umbilical glucose supply and survive a brief period of starvation before receiving colostrum and milk. Liver glycogen, at birth, is approximately 4–6% of liver wet weight. This energy reserve is depleted within a few hours (Hamada and Matsumoto, 1984; Girard, 1990) and supplies glucose for erythrocytes, brain, and kidney medulla. Glucose supplied by milk lactose accounts for approximately 25% of glucose utilization of the neonatal lamb (Girard, 1986), therefore gluconeogenesis is necessary to maintain neonatal glucose homeostasis. Development of supporting pathways, production of cofactors, and substrate supply may affect the rates of gluconeogenesis in utero and during postnatal development. For example the inability to oxidize fatty acids at birth has been characterized in detail and stems from a lack of activity of fatty acyl-CoA synthases, carnitine palmitoyltransferase I (CPT-I), enoylCoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and oxoacyl-CoA thiolase (Girard et al., 1992). The gluconeogenic promoting effects of fatty acids have been recognized for some time in nonruminants (Williamson et al., 1966). A similar regulation is likely in the bovine based on data that indicate that specific long-chain fatty acids promote gluconeogenesis in hepatocytes from ruminating calves (Mashek et al., 2002). Likewise, inhibition of CPT-I activity decreases gluconeogenesis in sheep hepatocytes (Chow and Jesse, 1992). Information is lacking on initiation of fatty acid oxidation in neonatal ruminants, but if a parallel can be drawn from rodent data, the induction of gluconeogenic capacity may be linked to the induction of fatty acid oxidation.

5. HORMONAL CHANGES: INSULIN, GLUCAGON, GLUCOCORTICOIDS Nutrient supply during the prenatal period consists primarily of a carbohydrate-rich energy supply (glucose, lactate), yet during the neonatal period a switch is made to a high-fat, lowcarbohydrate diet (Aynsley-Green, 1988). Newborns develop marked hypoglycemia after birth because glucose derived from lactose in colostrum does not meet postnatal glucose demands (Girard, 1986). Therefore, glycogenolysis and gluconeogenesis increase rapidly in the liver of newborns; however, there are species differences in prenatal development of gluconeogenesis. In the developing rat and pig fetus the gluconeogenic pathway does not mature in utero (Ballard and Oliver, 1963; Swiatek, 1971), whereas in the bovine fetus there is gluconeogenic activity measurable from day 80 of gestation (Prior and Scott, 1977). This might indicate, as discussed above, that the bovine fetus is less dependent on maternal glucose supply than the rat and pig fetus; however, newborn calves experience hypoglycemia as do other species (Aynsley-Green, 1988; Egli and Blum, 1988; Hadorn et al., 1997; Hammon and

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Blum, 1998). In neonatal ruminants and nonruminants, glucose is mostly produced by gluconeogenesis using amino acids (alanine, glycine, glutamine), glycerol, and lactate. In human newborns, research using stable isotopes demonstrates that gluconeogenesis from lactate, glycerol, and alanine occurs at a significant rate within the first 8 h of life and is critical for neonatal survival (Ferre et al., 1986). Insulin and glucagon are integral to normal fetal development in ruminants (reviewed in Blum and Hammon, 1999a). The change in glucose supply that accompanies loss of umbilical nutrient supply is reflected by an increase in glucagon and decreased insulin concentration during the immediate postnatal period (Girard, 1990). Blood profiles in newborn calves are characterized by hypoglycemia, high nonesterified fatty acids, and low triglyceride, phospholipids, and cholesterol (Blum and Hammon, 1999b). Neonatal calves respond to nutritional challenges by increasing glucagon and decreasing insulin in a manner similar to adult animals; however, the glucose–insulin relationship is less developed in neonates. The lack of glucose clearance in response to insulin may prevent hypoglycemia and serve to protect the neonatal calf. Cortisol plays an important role in enhancing fetal capacity for glucose production and glycogen storage (Fowden, 1995; Barnes, 1997). Changes in plasma insulin and glucagon may be related to the stress associated with birth and the concomitant rise in serum cortisol, fetal hypoxia, or both (Girard, 1990). Key genes for gluconeogenesis are also responsive to thyroid hormones (Park et al., 1997); a rise in thyroxine during the first 24 h of life in neonatal sheep (Fisher et al., 1977) may play a role in induction of metabolic competence. Recent data indicate that fetal thyroid hormone production is essential to the development of gluconeogenesis and is especially critical under adverse conditions such as undernutrition (Fowden et al., 2001). Plasma glucagon concentrations rise during the immediate postnatal period due, in part, to a drop in blood glucose that occurs within a few hours of birth. An infusion of somatostatin in lambs induces hypoglycemia and infusion of glucagon reverses the effects of somatostatin (Sperling et al., 1977). The rate of glucose output by fetal, neonatal, and adult ovine liver was increased similarly during glucagon infusions (Apatu and Barnes, 1991b); however, the effective dose of glucagon necessary to stimulate gluconeogenesis is greater in fetal liver (Girard and Sperling, 1983). Postnatal increases in glucagon receptor numbers and full development of intracellular signal transduction pathways along with a decrease in insulin receptor numbers favor regulation of gluconeogenesis in the neonate that is more sensitive to changes in glucagon concentrations (Girard and Sperling, 1983). Hepatic glucagon receptor numbers are lower in fetal and newborn ruminants than in adults. The effective dose of glucagon required to stimulate gluconeogenesis in adult sheep liver is not effective in fetal sheep liver (Girard and Sperling, 1983). In 21-day-old rats the number of liver glucagon receptors was only 40% of the receptor number for adult liver (Ganguli et al., 1983). Insulin receptor number and affinity are higher in fetal than in adult liver in rats and humans (Neufeld et al., 1980). High insulin and low glucagon receptor activity in utero favors glucose oxidation, whereas the coupling of glucagon receptor to cAMP synthesis combined with an increase in glucagon receptor numbers in early postnatal life favors gluconeogenesis (Girard and Sperling, 1983).

6. GLUCONEOGENESIS IN NEONATAL AND DEVELOPING RUMINANTS Gluconeogenesis from lactate is similar between fetal and maternal liver in the bovine (Prior and Scott, 1977). The rates of [2-14C]propionate and [2-14C]lactate incorporation to glucose

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and glycogen obtained from liver slices from adult sheep and newborn lambs are similar (Ballard and Oliver, 1963). In contrast, the rate of gluconeogenesis from [2-14C]pyruvate is greater for liver slices from neonatal lambs compared with adult tissue. The rate of pyruvate metabolism to glucose appears to peak at about 2–4 weeks of age in lambs (Ballard and Oliver, 1963). Likewise the rates of metabolism of lactate are markedly reduced in weaned lambs (Savan et al., 1986) and calves (Donkin and Armentano, 1995). In developing ruminants there is a marked decline in the capacity to metabolize lactate to glucose coupled with a reduced sensitivity to the effects of insulin (Donkin and Armentano, 1995). Radioisotope tracer data indicate that there is almost exclusive flux of lactate through pyruvate carboxylase (PC) to glucose in neonatal calf liver and very little isotope exchange with carbon of the TCA cycle (Donkin and Armentano, 1994). The substantial loss in lactate metabolism to glucose during the preruminant to ruminant transition (Donkin and Armentano, 1995), and similar use of propionate for gluconeogenesis between the two groups (fig. 2), suggests a loss in capacity to draw lactate into the gluconeogenic pathway. These results are perplexing in light of the extensive use of lactate for gluconeogenesis in nonruminants, but agree with the 10-fold lower rate of glucose recycling in vivo in adult versus neonatal (5- or 21-day old) sheep (Muramatsu et al., 1974). These changes suggest developmentally regulated differences in gluconeogenesis that are unique to lactate. Lactate is equilibrated rapidly with pyruvate in liver. The rates of [1-14C]lactate and [1-14C]pyruvate metabolism to glucose are not different for hepatocytes obtained from preruminating calves (Donkin and Armentano, 1994). This measurement has not been made directly in hepatocytes from ruminating calves, but is not likely the limiting step in gluconeogenesis from lactate based on a lack of control of gluconeogenesis in response to alterations in cytosolic redox state (Aiello and Armentano, 1987). As indicated above, pyruvate formed from lactate is carboxylated to oxaloacetate by pyruvate carboxylase (PC) and oxaloacetate is either metabolized to phosphoenolpyruvate (PEP) or metabolized in the TCA cycle. The similar ratios of [14C]glucose:14CO2 from [2-14C]propionate and carbons 2 and 3 of lactate support a common oxaloacetate pool for the metabolism of propionate and lactate in bovine hepatocytes (Donkin and Armentano, 1994). Therefore it is likely that decreased

Fig. 2. Effect of developmental state on gluconeogenesis from propionate and lactate in calves. Hepatocytes were isolated from preruminating (n = 4) and ruminating calves (n = 3) and cultured for 48 h. The rate of gluconeogenesis from [2-14C]propionate or [U-14C]lactate was determined during the last 3 h of incubation. Adapted from Donkin and Armentano (1995).

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flux through PC is the cause of decreased gluconeogenesis from lactate during postnatal development in calves. Chronic exposure of neonatal bovine hepatocytes to insulin results in decreased gluconeogenesis from lactate (Donkin et al., 1997), which is consistent with data suggesting that a portion of the reduction in gluconeogenesis from lactate in milk-fed calves may be due to chronically elevated insulin concentrations (Breier et al., 1988). However, the direct actions of insulin do not fully explain the reduction in gluconeogenesis from lactate that is observed in calves during the transition from the preruminant to the ruminant state. Comparing the rate of gluconeogenesis from lactate relative to propionate metabolism suggests additional changes in hepatic lactate metabolism. Gluconeogenesis from lactate is reduced to 32% of the rate of propionate conversion to glucose following chronic exposure to insulin (Donkin et al., 1997), but the developmental change in lactate metabolism reduces gluconeogenesis from lactate to only 10% of the rate of gluconeogenesis from propionate (Donkin and Armentano, 1995). The data described above point to PC as a primary control point for gluconeogenesis in developing ruminants and is supported by data from adult ruminants suggesting that PC may be a control point for gluconeogenesis. In cattle and sheep the activity of PC is responsive to nutritional and physiological states that impose the greatest demands for endogenous glucose production such as lactation and feed deprivation (Greenfield et al., 2000; Velez and Donkin, 2000). In contrast, the activity of PEPCK is relatively invariant between different nutritional and physiological states in ruminants, diabetes being the exception (Filsell et al., 1969; Taylor et al., 1971). When both PEPCK and PC activity are examined in response to physiological state or nutrient supply, the ratio of their activities suggests that an increase in capacity for lactate metabolism is primarily responsible for increases in hepatic gluconeogenesis. The dramatic reduction in basal rate of gluconeogenesis from lactate appears to be a due to a reduction in PC activity and gene expression. Data, from sheep, examining the relationship between prenatal development of gluconeogenic enzymes and activities found in maternal liver fail to reveal any striking differences in activity of PC, PEPCK, or PK (Edwards et al., 1975; Stevenson et al., 1976). Analysis of PC mRNA in liver biopsy samples from 7 through 84 days of age indicates a decline in expression of PC mRNA (Donkin et al., 1998) and suggests a decrease in capacity for lactate metabolism. A decline in PC mRNA expression was observed in both milk-fed calves and ruminanting calves by 84 days of age that mirrors a reduction in gluconeogenesis from lactate (Donkin et al., 1998). Taken together, these data suggest a developmental decrease in PC expression that is likely reflected as a decrease in lactate recycling (Muramatsu et al., 1974) and reduced lactate metabolism to glucose in the weaned calf (Donkin and Armentano, 1995). Data from lactating cows indicate that PC activity and mRNA expression are induced when demands for gluconeogenesis are elevated at calving (Greenfield et al., 2000) and during restricted feed intake (Velez and Donkin, 2000). The onset of rumen development is marked by the production and absorption of volatile fatty acids (VFA). Acetate and propionate form the bulk of VFA produced by rumen fermentation. Acute exposure to propionate decreases gluconeogenesis from lactate equally in hepatocytes from preruminating and ruminating calves (Donkin and Armentano, 1995). Prolonged exposure of hepatocytes from preruminating calves to valerate (which can be metabolized to acetate and propionate) had no effect on subsequent capacity for gluconeogenesis from propionate (Donkin and Armentano, 1993). However, an intermediate of propionate metabolism, methyl malonyl-CoA, can directly inhibit lactate metabolism (Blair et al., 1973) and is thought to be responsible for the acute effects of propionate in limiting gluconeogenesis from lactate in bovine hepatocytes (Donkin and Armentano, 1994). At present the nature of the developmental suppression of PC activity and gene expression is unknown.

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Long-term regulation of gluconeogenesis in nonruminants has been characterized by changes in the expression of genes encoding glucoregulatory enzymes (Pilkis and Claus, 1991). It is well established that insulin represses and glucagon (or cAMP) and glucocorticoids induce the activity of the PEPCK enzyme by directly regulating expression of the PEPCK gene (reviewed in O’Brien and Granner, 1990). From control strength studies in rats, gluconeogenesis from lactate is distributed between pyruvate kinase and the reactions involving PC and PEPCK (Sistare and Haynes, 1985). Glucocorticoids have little effect on flux through the PK-catalyzed reaction; therefore an increase in gluconeogenesis from lactate in glucocorticoidtreated rats or hepatocytes is mainly due to the combined increases in flux through reactions catalyzed by PC and PEPCK (Jones et al., 1993).

7. GLUCOCORTICOIDS AND POSTNATAL DEVELOPMENT Uncomplicated neonatal growth depends on maturation of vital organs and critical metabolic pathways including lung and cardiac development, thyroid axis, somatotropic axis, initiation of thermogenesis, and control of glucose homoeostasis. In altricial species, such as rodents, a rise in cortisol at birth is necessary to initiate neonatal maturation of many of the critical metabolic pathways including gluconeogenesis (Dalle et al., 1985; Gluckman et al., 1999). In ruminants, the concentration of fetal cortisol usually exceeds maternal cortisol concentrations; therefore caution should be exercised when extending data on the effects of glucocorticoids from rodent studies to the biology of liver metabolism in neonatal ruminants. The central role of glucocorticoids in regulation of expression of PEPCK, G-6-P, PC, and CPT-I (Jitrapakdee and Wallace, 1999; Van Schaftingen and Gerin, 2002) is established and there are indications in nonruminants that these effects are mediated through peroxisome proliferator-activated receptor γ coactivator-1 (Louet et al., 2002). Cortisol injected into developing sheep fetuses induced activity of hepatic G-6-Pase, fructose-6-phosphatase, PC, and PEPCK by 2- to 3-fold (Fowden et al., 1993). Glucocorticoids may also play a more general role in switching the fetal physiological state to a postnatal state (Liggins, 1977; Fowden, 1995). For example, gastrointestinal tract developmental, gastrin secretion, and intestinal absorption of immunoglobulins are stimulated by cortisol in neonatal piglets and play a role in maturation of the fetal exocrine pancreas of pigs and lambs (Sangild, 2001). Glucocorticoids are important regulators of the glucose status after birth in the immature neonatal calf. Cortisol concentrations decreased after birth in neonates (Baumrucker and Blum, 1994; Hadorn et al., 1997; Hammon and Blum, 1998). Importantly, plasma cortisol concentrations depend on the level and source of nourishment (milk or colostrum) after birth (Hammon and Blum, 1998). Calves fed milk replacer from birth were characterized by higher plasma cortisol concentrations and lower plasma glucagon concentrations than calves fed colostrum (Hammon and Blum, 1998). The prepartum cortisol surge may play an important role in initiating the perinatal switch of the somatotropic axis from the fetal to the postnatal status and function (Breier et al., 2000). Glucocorticoids stimulate gluconeogenesis in vivo by increasing plasma glucagon concentrations as well as augmenting the effects of glucagon to stimulate gluconeogenesis (Marco et al., 1973; Wise et al., 1973; Lecavalier et al., 1990). Furthermore, glucocorticoid treatment induces insulin resistance in late gestation in sheep (Challis et al., 2001) and postnatally in humans (Weinstein et al., 1995; Dirlewanger et al., 2000) and in dairy cows (Maciel et al., 2001). The interaction of glucocorticoids and growth hormone has not been fully characterized for neonatal ruminants, but postnatal growth is characterized by changes from a substrate-limited prenatal growth to enteral feeding with the somatotropic axis becoming the dominant endocrine regulatory system.

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Glucocorticoids enhance the maturation of the somatotropic axis and the prepartum cortisol surge may play an important role in initiating the perinatal switch of the somatotropic axis from the fetal to the postnatal status and function (Breier et al., 2000; Carroll et al., 2000). Cortisol acts to stimulates hepatic growth hormone receptor (GHR) numbers and IGF-I mRNA levels in the sheep fetus (Li et al., 1996). In vivo studies using porcine hepatocytes indicate that IGF-I mRNA expression is more responsive to GH in the presence of dexamethasone and thyroxine (Brameld et al., 1999). Therefore, it might be speculated that in precocious species such as ruminants, elevated cortisol levels at birth serve to enhance postnatal maturation of the somatotropic axis. The somatotropic axis in neonatal calves is functional, but immature at birth (Hammon and Blum, 1997) owing partly to reduced GH-binding capacity of the liver in neonatal calves (Breier et al., 1994). Little is known about the ontogeny of the growth hormone receptor in the neonatal bovine or its coordination with other hepatic functions including glucose metabolism. Growth hormone as well as the GH receptor are present in the bovine fetus but growth hormone does not affect IGF-I production in the liver (Gluckman et al., 1999), perhaps owing to GHR numbers, activity of receptors, or both (Fowden, 1995; Freemark, 1999).

8. GLUCONEOGENESIS AND REGULATION OF GENE EXPRESSION Most of the enzymes for gluconeogenesis, including PC, PEPCK-M, fructose 1,6-bisphosphatase, and G-6-Pase, have substantial activity in near-term fetuses of ruminants (Edwards et al., 1975; Stevenson et al., 1976; Narkewicz et al., 1993). The classic work of Ballard and Hanson (1967) established PEPCK-C as the limiting step in development of gluconeogenesis in rats. These data have been substantiated for rabbit and other species (Girard et al., 1992), but there is no limitation in development of PEPCK-C activity in ruminant liver (Edwards et al., 1975; Stevenson et al., 1976; Narkewicz et al., 1993). In rodents the rapid increase in PEPCK-C is linked to the process of birth rather than fetal age (Girard et al., 1992) and is related to the late fetal appearance of developmentally regulated transcription factors such as CCAAT/ enhancer-binding protein (Cassuto et al., 1999). Therefore it would follow that these transcription factors or their functional homologs are likely to be present in utero in liver of the developing ruminants. The expression of PC is tissue-specific with the highest catalytic activity of the enzyme found in liver, kidney, adipose tissue, brain, adrenal gland, and lactating mammary tissue. Changes in PC abundance, through alteration in rate of synthesis, constitute long-term regulation of pyruvate metabolism for gluconeogenesis and lipogenesis (Barritt, 1985). Short-term allosteric regulation of PC activity by acetyl-CoA is well noted; however, sustained changes in the activity of the PC enzyme require parallel increases in PC mRNA (Zhang et al., 1993). Northern analysis of total RNA indicates the presence of a single 4.2 kb mRNA for rat and human PC that is the product of a single copy gene (Jitrapakdee et al., 1996). However, selective amplification of the 5′ untranslated region (UTR) of PC cDNA indicates the presence of five alternative forms of PC cDNA that are generated through differential splicing of RNA transcripts and use of two tissue-specific promoters (Jitrapakdee et al., 1996). Transcripts generated from the proximal promoter are restricted to gluconeogenic and lipogenic tissue whereas those generated from the distal promoter are expressed in several tissues. These 5′ UTR isomers of the PC primary transcript share the same open reading frame and result in one PC protein. The liver expresses the C, D, and E forms of PC transcript, although the C and D forms predominate. During the suckling to weaning transition the abundance of the C isoform decreases as does PC mRNA and enzyme activity (Jitrapakdee et al., 1998). The fact that

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the C transcript is more functionally potent (2× greater than D) in translation reactions suggests that a small increase in the C transcript may result in proportionately greater increase in PC activity than changes in the D form. Furthermore an increase in C transcript and an offsetting decrease in the D transcript would result in no net change in PC mRNA abundance by Northern analysis but would increase PC translation and maximal PC activity. The entire coding sequence of bovine PC has been cloned (Agca et al., 2000) and the coding sequence contains 3075 bases with 85% identity to human PC. Furthermore, bovine PC is expressed as six 5′ UTR variants of different lengths (Agca and Donkin, 2001). Experiments are ongoing to test the functional significance of bovine PC variants relative to gluconeogenesis and neonatal development in cattle. Regulation of PEPCK expression has been extensively studied in liver of rodents as well as rat and human hepatoma cell lines. Glucagon acting in the presence of dexamethasone is one of the primary stimulators of PEPCK gene expression. The activity of PEPCK-C is determined by the rate of transcription of the PEPCK-C gene and the rate of turnover of its mRNA whereas the activity of PEPCK-M appears to be constitutive (Hanson and Reshef, 1997). The coding sequence for bovine PEPCK-C and a fragment of bovine PEPCK-M have been cloned recently (Agca et al., 2002). Unlike PEPCK-C the expression of PEPCK-M mRNA is not responsive to changes in physiological state (Greenfield et al., 2000; Agca et al., 2002). Control of PEPCK-C activity is largely exerted through transcription of the gene through activation of basal, tissue-specific, and hormone-dependent promoter elements within the 5′ region of the PEPCK-C gene (Hanson and Reshef, 1997). Crucial liver control elements are located within −460 to +73 of the promoter and six primary protein-binding sites have been characterized by DNAse I footprinting; these six sites contain docking sites for at least 15 separate transcription factors (Hanson and Reshef, 1997). The cAMP response element I (CRE-I) acting synergistically with protein-binding sites 3 and 4 is primarily responsible for the cAMP-mediated increase in PEPCK-C transcription (Hanson and Reshef, 1997). Insulin counteracts the effects of cAMP by repressing the promoter, perhaps by blocking the ability of glucocorticoids to promote activity of accessory factor-2 (O’Brien and Granner, 1990). Although the PEPCK-C gene is generally thought to be transcriptionally controlled, there is regulation through stability of the PEPCK-C mRNA which is mediated through cAMP action on a 3′ noncoding sequence (Lemaigre and Rousseau, 1994). There is some indication that PEPCK-C expression may be inhibited directly by glucose as is the case with other insulinresponsive genes. The lack of appreciable glucokinase activity in ruminant liver leads to questions regarding a similar control in ruminants.

9. FUTURE PERSPECTIVES There is no question that gluconeogenesis is critical to the survival and normal development of fetal, neonatal, and postnatal ruminants. The precocious development of gluconeogenic machinery in the liver of the ruminant fetus provides a number of advantages for survival at birth. There are many developmental aspects of gluconeogenesis that have been described in detail for nonruminants that are applicable to the developing ruminant, but several processes are species-specific. Information is lacking on the basic biology that accompanies the onset of metabolic competence in the developing ruminant, including processes that may modulate gluconeogenesis, and in many cases parallels must be drawn from rodent models. Issues associated with the initiation of expression of key genes for gluconeogenesis including PC and PEPCK in ruminants and the molecular cues that initiate development of gluconeogenesis remain to be clarified. Several aspects of gluconeogenesis in developing ruminants have been

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identified, but a more complete characterization of fetal and neonatal gluconeogenesis is needed to identify unique regulatory controls including the molecular and biochemical events that accompany the postnatal reduction in gluconeogenesis from lactate. Conversely the biochemical anomalies identified for ruminants, such as the inherent lack of hepatic G-6-Pase activity, could provide unique opportunities to study glucose trafficking in liver in order to better understand metabolic diseases of humans.

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Ganguli, S., Sinha, M.K., Sterman, B., Harris, P., Sperling, M.A., 1983. Ontogeny of hepatic insulin and glucagon receptors and adenylate cyclase in rabbit. Amer. J. Physiol. 244, E624–E631. Girard, J., 1986. Gluconeogenesis in late fetal and early neonatal life. Biol. Neonate 50, 237–258. Girard, J., 1990. Metabolic adaptations to change of nutrition at birth Biol. Neonate 58, Suppl. 1, 3–15. Girard, J., Sperling, M., 1983. Glucagon in the fetus and the newborn. In: Lefebre, P.J. (Ed.), Glucagon: Handbook of Experimental Pharmacology. Springer-Verlag, New York, p. 66. Girard, J., Ferré, P., Pégorier, J.P., Duée, P.H., 1992. Adaptions of glucose and fatty acid metabolism during perinatal period and suckling-weaning transition. Physiol. Rev. 72, 507–562. Gluckman, P.D., Sizonenko, S.V., Bassett, N.S., 1999. The transition from fetus to neonate – an endocrine perspective. Acta Pædiat. 428, 7–11. Greenfield, R.B., Cecava, M.J., Donkin, S.S., 2000. Changes in mRNA expression for gluconeogenic enzymes in liver of dairy cattle during the transition to lactation. J. Dairy Sci. 83, 1228–1236. Gresham, E.L., James, E.J., Raye, J.R., Battaglia, F.C., Makowski, E.L., Meschia, G., 1972. Production and excretion of urea by the fetal lamb. Pediatrics 50, 372–379. Hadorn, U., Hammon, H., Bruckmaier, R.M., Blum, J.W., 1997. Delaying colostrum intake by one day has important effects on metabolic traits and on gastrointestinal and metabolic hormones in neonatal calves. J. Nutr. 127, 2011–2023. Hamada, T., Matsumoto, M., 1984. Effects of nutrition and ontogeny on liver cytosolic and mitochondrial phosphoenolpyruvate carboxykinase activity of the rat, hamster, guinea-pig, pig, kid, calf and chick. Comp. Biochem. Physiol. B. 77, 547–550. Hammon, H., Blum, J.W., 1997. The somatotropic axis in neonatal calves can be modulated by nutrition, growth hormone, and long-R3-IGF-I. Amer. J. Physiol. 273, E130–E138. Hammon, H.M., Blum, J.W., 1998. Metabolic and endocrine traits of neonatal calves are influenced by feeding colostrum for different durations or only milk replacer. J. Nutr. 128, 624–632. Hanson, R.W., Reshef, L., 1997. Regulation of phosphoenolpyruvate carboxykinase (GTP) gene expression. Annu. Rev. Biochem. 66, 581–611. Hay, W.W. Jr., Sparks, J.W., Wilkening, R.B., Battaglia, F.C., Meschia, G., 1984. Partition of maternal glucose production between conceptus and maternal tissues in sheep. Amer. J. Physiol. 245, E347–E350. Hod, Y., Cook, J.S., Weldon, S.L., Short, J.M., Wynshaw-Boris, A., Hanson, R.W., 1987. Differential expression of the genes for the mitochondrial and cytosolic forms of phosphoenolpyruvate carboxykinase. Ann. N.Y. Acad. Sci. 478, 31–35. Hodgson, J.C., Mellor, D.J., Field, A.C., 1982. Foetal and maternal rates of urea production and disposal in well-nourished and undernourished sheep. Brit. J. Nutr. 48, 49–58. Holcomb, T., Curthoys, N.P., Gstraunthaler, G., 1995. Subcellular localization of PEPCK and metabolism of gluconeogenic substrains of renal cell lines. Amer. J. Physiol. 268, C449–C457. Hosokawa, M., Thorens, B., 2002. Glucose release from GLUT2-null hepatocytes: characterization of a major and a minor pathway. Amer. J. Physiol. Endocrinol. Metab. 282, E794–E801. Howarth, R.E., Baldwin, R.L., Ronning, M., 1968. Enzyme activities in liver, muscle, and adipose tissue of calves and steers. J. Dairy Sci. 51, 1270–1274. Huntington, G.B., 1990. Energy metabolism in the digestive tract and liver of cattle: influence of physiological state and nutrition. Reprod. Nutr. Dev. 30, 35–47. Jitrapakdee, S., Booker, G.W., Cassady, A.I., Wallace, J.C., 1996. Cloning, sequencing and expression of rat liver pyruvate carboxylase. Biochem. J. 316, 631–637. Jitrapakdee, S., Wallace, J.W., 1999. Structure and regulation of pyruvate carboxylase. Biochem. J. 340, 1–16. Jitrapakdee, S., Gong, Q., MacDonald, M.J., Wallace, J.C., 1998. The rat pyruvate carboxylase gene structure: alternate promoters generate multiple transcripts with the 5’-end heterogeneity. J. Biol. Chem. 272, 20522–20530. Jones, C.G., Hothi, S.K., Titheradge, M.A., 1993. Effect of dexamethasone on gluconeogenesis, pyruvate kinase, pyruvate carboxylase and pyruvate dehydrogenase flux in isolated hepatocytes. Biochem. J. 289, 821–830. Kalhan, S., Parimi, P., 2000. Gluconeogenesis in the fetus and neonate. Semin. Perinatol. 24, 94–106. Lecavalier, L., Bolli, G., Gerich, G., 1990. Glucagon-cortisol interactions on glucose turnover and lactate gluconeogenesis in normal humans. Amer. J. Physiol. 258, E569–E575. Lemaigre, F.P., Rousseau, G.G., 1994. Transcriptional control of genes that regulate glycolysis and gluconeogenesis in adult liver. Biochem. J. 303, 1–14.

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Leury, B.J., Bird, A.R., Chandler, K.D., Bell A.W., 1990. Glucose partitioning in the pregnant ewe: effects of undernutrition and exercise. Brit. J. Nutr. 64, 449–462. Li, J., Owens, J.A., Owens, P.C., Saunders, J.C., Fowden, A.L., Gilmour, R.S., 1996. The ontogeny of hepatic growth hormone receptor and insulin-like growth factor I gene expression in the sheep fetus during late gestation: developmental regulation by cortisol. Endocrinology 137, 1650–1657. Liggins, G.C., 1977. The role of cortisol in preparing the fetus for birth. Reprod. Fertil. Dev. 6, 141–150. Louet, J.F., Hayhurst, G., Gonzalez, F.J., Girard, J., Decaux, J.F., 2002. The coactivator PGC-1 is involved in the regulation of the liver carnitine palmitoyltransferase I gene expression by cAMP in combination with HNF4 alpha and cAMP-response element-binding protein (CREB). J. Biol. Chem. 277, 37991–38000. Maciel, S.M., Chamberlain, C.S., Wettemann, R.P., Spicer, L.J., 2001. Dexamethasone influences endocrine and ovarian function in dairy cattle. J. Dairy Sci. 84, 1998–2009. Marco, J., Calle, C., Román, D., Díaz-Fierros, M., Villanueva, M.L., Valverde, I., 1973. Hyperglucagonism induced by glucocorticoid treatment in man. N. Engl. J. Med. 288, 128–131. Mashek, D.G., Bertics, S.J., Grummer, R.R., 2002. Metabolic fate of long-chain unsaturated fatty acids and their effects on palmitic acid metabolism and gluconeogenesis in bovine hepatocytes. J. Dairy Sci. 85, 2283–2289. Muramatsu, M., Sugawara, M., Tsuda, T., 1974. Changes in rates of recycling and turnover of glucose during development of sheep. Agr. Biol. Chem. 38, 259–267. Narkewicz, M.R., Carver, T.D., Hay, W.W. Jr., 1993. Induction of cytosolic phosphoenolpyruvate carboxykinase in the ovine fetal liver by chronic fetal hypoglycemia and hypoinsulinemia. Pediat. Res. 33, 493–496. Neufeld, N.D., Scott, M., Kaplan, S.A., 1980. Ontogeny of the mammalian insulin receptor: studies of human and rat fetal liver plasma membranes. Dev. Biol. 78, 151–160. O’Brien, R.M., Granner, D.K., 1990. PEPCK gene as model of inhibitory effects of insulin on gene transcription. Diabetes Care 13, 327–339. Park, E.A., Song, S., Olive, M., Roesler, W.J., 1997. CCAAT-enhancer-binding protein alpha (C/EBP alpha) is required for the thyroid hormone but not the retinoic acid induction of phosphoenolpyruvate carboxykinase (PEPCK) gene transcription. Biochem. J. 322, 343–349. Pearce, J., Unsworth, E.F., 1980. The effects of diet on some hepatic enzyme activities in the pre-ruminant and ruminating calf. J. Nutr. 110, 255–261. Pilkis, S.J., Claus, T.H., 1991. Hepatic gluconeogenesis/glycolysis: regulation and structure/function relationships of substrate cycle enzymes. Annu. Rev. Nutr. 11, 465–515. Prior, R.L., 1980. Glucose and lactate metabolism in vivo in ovine fetus. Amer. J. Physiol. 239, E208–E214. Prior, R.L., 1982. Gluconeogenesis in the ruminant fetus: evaluation of conflicting evidence from radiotracer and other experimental techniques. Fed. Proc. 41, 117–122. Prior, R.L., Scott, R.A., 1977. Ontogeny of gluconeogenesis in the bovine fetus: influence of maternal dietary energy. Dev. Biol. 58, 384–393. Sangild, P.T., 2001. Transitions in the life of the gut at birth, In: Lindberg, J.E., Ogle, B. (Eds.), Digestive Physiology of Pigs. CABI Publishing, New York, pp. 3–17. Savan, P.M., Jeacock, M.K., Shepherd, D.A., 1986. Gluconeogenesis in foetal, suckling and weaned lambs. J. Agr. Sci. 106, 259–268. Seoane, J., Trinh, K., O’Doherty, R.M., Gomez-Foix, A.M., Lange, A.J., Newgard, C.B., Guinovart, J.J., 1997. Metabolic impact of adenovirus-mediated overexpression of the glucose-6-phosphatase catalytic subunit in hepatocytes. J. Biol. Chem. 272, 26972–26977. Sistare, F.D., Haynes, R.C., 1985. The interaction between the cytosolic pyridine nucleotide redox potential and gluconeogenesis from lactate/pyruvate in isolated rat hepatocytes: implications for investigations of hormone action. J. Biol. Chem. 260, 12748–12753. Sperling, M.A., Grajwer, L., Leake, R.D., Fisher, D.A., 1977. Effects of somatostatin (SRIF) infusion on glucose homeostasis in newborn lambs: evidence for a significant role of glucagon. Pediat. Res. 11, 962–967. Stevenson, R.E., Morris, F.H., Jr., Adcock, E.W. III, Howell, R.R., 1976. Development of gluconeogenic enzymes in fetal sheep liver and kidney. Dev. Biol. 52, 167–172. Swiatek, K.R., 1971. Development of gluconeogenesis in pig liver slices. Biochim. Biophys. Acta 252, 274–279. Taylor, P.H., Wallace, J.C., Keech, D.B., 1971. Gluconeogenic enzymes in sheep liver: intracellular localization of pyruvate carboxylase and phosphoenolpyruvate carboxykinase in normal, fasted and diabetic sheep. Biochim. Biophys. Acta 237, 179–191.

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Tsoulos, N.G., Colwill, J.R., Battaglia, F.C., Makowski, E.L., Meschia, G., 1971. Comparison of glucose, fructose, and O2 uptakes by fetuses of fed and starved ewes. Amer. J. Physiol. 221, 234–237. Van Schaftingen, E., Gerin, I., 2002. The glucose-6-phosphatase system. Biochem. J. 362, 513–532. Velez, J.C., Donkin, S.S., 2000. Administration of bST elevates phosphoenolpyruvate carboxykinase mRNA in lactating dairy cows. J. Anim. Sci. 78, Suppl. 1, 144. Warnes, D.M., Seamark, R.F., Ballard, F.J., 1977. Metabolism of glucose, fructose and lactate in vivo in chronically cannulated foetuses and in suckling lambs. Biochem. J. 162, 617–626. Watford, M., Hod, Y., Chiao, Y.B., Utter, M.F., Hanson, R.W., 1981. The unique role of the kidney in gluconeogenesis in the chicken: the significance of a cytosolic form of phosphoenolpyruvate carboxykinase. J. Biol. Chem. 256, 10023–10027. Weinstein, S.P., Paquin, T., Pritsker, A., Haber, R.S., 1995. Glucocorticoid-induced insulin resistance: dexamethasone inhibits the activation of glucose transport in rat skeletal muscle by both insulin- and non-insulin-related stimuli. Diabetes 44, 441–445. Williamson, J.R., Kreisberg, R.A., Felts, P.W., 1966. Mechanism for the stimulation of gluconeogenesis by fatty acids in perfused rat liver. Proc. Natl. Acad. Sci. USA 56, 247–254. Wise, J.K., Hendler, R., Felig, P., 1973. Influence of glucocorticoids on glucagon secretion and plasma amino acid concentrations in man. J. Clin. Invest. 52, 2774–2782. Zhang, J., Xia, W., Brew, K., Ahmad, F., 1993. Adipose pyruvate carboxylase: amino acid sequence and domain structure deduced from cDNA sequencing. Proc. Natl. Acad. Sci. USA 90, 1766–1770.

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Energy metabolism in the developing rumen epithelium B. W. Jesse Department of Animal Science, Rutgers, The State University of New Jersey, 84 Lipman Drive, New Brunswick, NJ 08901-8525, USA

The physical changes occurring during rumen epithelial development have been extensively characterized. However, relatively little information is available concerning development of energy metabolism in rumen epithelium. Available data indicate that both ontogenic and physiological/dietary factors are necessary for complete rumen epithelial metabolic development. Changes in the expression of specific genes, e.g. those for ketone body production, in response to ontogenic and physiological/dietary factors appear to be responsible for the changes in energy metabolism in developing rumen epithelium. Future research efforts will need to identify the mechanisms regulating gene expression within the developing rumen epithelium to obtain a better understanding of this process.

1. INTRODUCTION Energy metabolism in the rumen epithelium of mature sheep and cattle has been extensively characterized over the years. The specific oxidizable substrates required for energy production in the rumen epithelium in neonates of these species have received some attention, while the establishment of the rumen fermentation, and the physical changes occurring to the rumen epithelium during development, have been extensively researched. However, the changes in energy metabolism that occur during neonatal rumen epithelial metabolic development, and most importantly the timing and control mechanisms regulating those changes, have received relatively little attention. This review will provide a historical overview of the state of our knowledge in this area, and will discuss in more depth recent evidence that examines metabolic development in the neonatal rumen epithelium. It will become apparent that relatively little is known concerning the mechanisms driving rumen metabolic development, and that this is the result of relatively little research having been conducted in this area. The vast bulk of the research literature examining rumen development has focused on the rumen fermentation itself, or on physical changes manifested by changes in rumen epithelial morphology and blood chemistry in the growing ruminant. While the focus of the review will be on rumen

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epithelium, it should be noted that developmental changes will also be occurring in the reticular and omasal epithelia. This review will conclude with a brief discussion of key areas for future research into the topic of rumen epithelial metabolic development. 2. Historical Perspective To understand the changes in energy metabolism occurring in developing rumen epithelium, it is necessary to first define the points that mark the limits of these changes, that is, the energy metabolism present at the start (in the neonatal rumen) and at the endpoint (in the mature rumen) of rumen development. For the purpose of this review this time period will generally coincide with the 8-week period following birth, the time period in which the bulk of the physical and metabolic changes occur in the developing rumen epithelium.

2.1. Energy metabolism in mature rumen epithelium 2.1.1. Substrates and their metabolism The primary compounds that have been investigated as potential energy-yielding substrates within the mature rumen epithelium are fatty acids (both short- and long-chain), glucose, and glutamine (Weigand et al., 1975; White and Leng, 1980; Harmon, 1986; Harmon et al., 1991; Jesse et al., 1992; Britton and Krehbiel, 1993; Remond et al., 1995; Baldwin and McLeod, 2000). Of these, the short-chain or volatile fatty acids (VFA) are quantitatively the most important energy sources for the ruminal epithelium under most circumstances. The VFA, predominantly acetate, propionate, and butyrate, are the products of the rumen fermentation, and are absorbed by the rumen epithelium for release into the portal circulation. Prior to release into the portal circulation, the VFA may undergo metabolism within the rumen epithelium. Depending upon the specific VFA considered, a variable amount of metabolism occurs (Remond et al., 1995). VFA metabolism may include either oxidation, or conversion into other intermediates (e.g. lactate, β-hydroxybutyrate [BHBA], acetoacetate [AcAc]), for release into the portal circulation. The activities of numerous enzymes in the glycolytic pathway, the citric acid cycle, the ketogenic pathway, the acyl-CoA synthetases for activation of VFA, and the various enzymes involved in VFA uptake have been determined (Young et al., 1969; Bush and Milligan, 1971; Ash and Baird, 1973; Nocek et al., 1980; Scaife and Tichivangana, 1980; Bush, 1982; Leighton et al., 1983; Harmon et al., 1991). These assays have generally been conducted under saturating substrate concentrations to yield maximal activities of the assayed enzymes. Consequently, relatively little information is available concerning the kinetic properties of rumen epithelial enzymes. Activation of VFA to the coenzyme A thioester has been proposed as the key regulatory point for rumen epithelial VFA metabolism (Ash and Baird, 1973). However, others have indicated that knowledge of both the kinetic properties of these enzymes as well as the tissue substrate and inhibitor concentrations within the rumen epithelium is needed to fully justify that statement (Britton and Krehbiel, 1993). While various researchers have reported on the effects of dietary changes (composition, level of intake) on VFA metabolism and activities of specific enzymes within the rumen epithelium, no consensus has yet emerged from these studies. Harmon et al. (1991) noted an overall increase in ruminal epithelial metabolism in cattle fed at twice maintenance requirements than in cattle fed at maintenance. These authors also noted some increase in acyl-CoA synthetase activities of rumen epithelium from cattle fed a high-forage diet. Some researchers report no change in activity for a number of enzymes in rumen epithelium from cattle fed

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a high-grain versus hay diet (e.g. Young et al., 1969). Others have noted differences in VFA transport across the rumen epithelium as well as changes in some enzyme activities (propionyl-CoA synthetase, glutamate dehydrogenase, and aspartate aminotransferase) as a result of changes in ration physical form and level of rumen-degradable nitrogen (Nocek et al., 1980). A recent paper suggests that changes in rumen epithelial energy metabolism in response to dietary energy intake and composition is due in part to changes in tissue mass rather than to changes in metabolism per unit epithelial mass (McLeod and Baldwin, 2000). This is in agreement with other studies noting increases in rumen epithelial mass and papillae length in response to increased dietary energy intake (Liebich et al., 1987). Of the three major VFA absorbed by rumen epithelium, the proportion of absorbed acetate that is metabolized is lower than any of the other VFA (18–30%; Remond et al., 1995). However, since significantly more acetate is absorbed than propionate and butyrate, the absolute amount of acetate metabolized can be relatively large. The literature indicates that, compared to butyrate, relatively little acetate is converted to ketone bodies (BHBA and AcAc; Remond et al., 1995). Acetate undergoes primarily oxidation to carbon dioxide by the rumen epithelium, thereby contributing to the energy needs of the rumen epithelium (Britton and Krehbiel, 1993). The situation with propionate metabolism in the ruminal epithelium is the least clear of the VFA. Propionate is not used for the synthesis of ketone bodies, but is converted primarily to lactate, with some complete oxidation to carbon dioxide, some pyruvate formation, and some transamination of pyruvate to alanine (Remond et al., 1995). Some studies have estimated that as much as 70% of absorbed propionate is converted to lactate prior to release into the portal circulation, although more recent data suggest that the proportion is much less than that (30%; Remond et al., 1995). Propionate oxidation to carbon dioxide by ruminal epithelium is minimal at physiological concentrations of propionate, presumably to spare propionate and its metabolites for hepatic gluconeogenesis (Remond et al., 1995), although at high propionate concentrations rumen epithelium in vitro can oxidize propionate at relatively high rates (Harmon et al., 1991). Butyrate has been noted to inhibit propionate activation to propionyl-CoA, thereby minimizing propionate metabolism and further sparing propionate for release into the portal circulation (Harmon et al., 1991). Butyrate has long been known to be the VFA most extensively metabolized by the rumen epithelium, undergoing both oxidation to carbon dioxide and conversion to BHBA and AcAc (Bergman, 1990; Remond et al., 1995). Various researchers have estimated that as much as 90% of the absorbed butyrate undergoes metabolism by the ruminal epithelium (Bergman, 1990; Remond et al., 1995). Generally, a higher proportion of butyrate is converted to ketone bodies, and a lower proportion to carbon dioxide, than occurs with acetate, although the absolute rates of carbon dioxide production from acetate and butyrate are comparable (Harmon et al., 1991). This may simply be a reflection of the relative rate of activation of these two VFA by their respective acyl-CoA synthetases (Harmon et al., 1991). Acetyl-CoA synthetase activity is significantly lower than either propionyl- or butyryl-CoA synthetase activities (Harmon et al., 1991). A lower rate of acetate activation may provide sufficient Ac-CoA for use in the TCA cycle, but not a sufficiently high concentration for use of Ac-CoA as a ketogenic substrate, at least when acetate is the sole substrate in vitro. The kinetic parameters of the ruminal enzymes responsible for use of acetyl-CoA in these pathways (i.e. citrate synthase and AcAc-CoA thiolase) are not known, however. Butyrate can inhibit acetate and propionate activation to their respective coenzyme A thioesters, while acetate and propionate have relatively little effect on butyrate activation (Harmon et al., 1991). These data are consistent with observations of Scaife and Tichivangana (1980), who isolated a partially purified short-chain acyl-CoA synthetase from sheep rumen epithelium. The kinetic properties of this

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fraction suggested the existence of two distinct enzyme activities, one specific for butyrate activation and the other capable of activating acetate, propionate, or butyrate (Scaife and Tichivangana, 1980). The long-chain fatty acid palmitate may be oxidized or used in the synthesis of ketone bodies by rumen epithelium in much the same manner as in liver (Jesse et al., 1992). Isolated rumen epithelial cells oxidized palmitate at one-quarter the rate of butyrate, and converted palmitate to ketone bodies at one-half the rate of butyrate (Jesse et al., 1992). Propionate, butyrate, and ammonia inhibited ketogenesis from palmitate, but only butyrate and ammonia inhibited palmitate oxidation (Jesse et al., 1992). These data suggest that during feed restriction, or possibly when consuming a high-fat diet, mature rumen epithelium would be capable of using long-chain fatty acids as a major energy source. Both glucose and glutamine can undergo oxidation to carbon dioxide, and conversion to lactate in the case of glucose, or to glutamate and alanine in the case of glutamine, within the ruminal epithelium (Remond et al., 1995). Glucose may be a major source of the lactate produced by rumen epithelium (Remond et al., 1995). Rates of glucose oxidation to carbon dioxide are comparable to lactate production rates from glucose, but glucose oxidation occurs at a lower rate than either acetate or butyrate oxidation (Harmon et al., 1991). Glutamine oxidation rates by ruminal epithelium reportedly were 7 times lower than glucose oxidation (Harmon et al., 1991), suggesting that glutamine is not a major energy source for ruminal epithelium, in contrast to the importance of glutamine as an energy source to other tissues of the digestive tract (Britton and Krehbiel, 1993). More recent data indicate that glutamine in vitro can be oxidized by rumen epithelial cells at rates faster than butyrate, if present at sufficiently high concentrations (50 mM; Baldwin and McLeod, 2000). However, the glutamine concentration required for half-maximal oxidation rates by rumen epithelial cells (6 mM; Baldwin and McLeod, 2000) is about 30 times greater than the glutamine concentration found in vivo (0.20 mM; Alio et al., 2000; Noziere et al., 2000; Hanigan et al., 2001). This suggests that little glutamine oxidation would be expected to occur in vivo (Baldwin and McLeod, 2000), as was noted by Harmon et al. (1991). 2.1.2. Substrate uptake Prior to activation and metabolism within the rumen epithelium, energy substrates must be transported into the epithelium. For the rumen VFA this presents a unique challenge, as at the pH typical of rumen fluid (5.6–6.2), VFA exist predominantly in the ionized form. Ionized VFA would be unable to diffuse through the plasma membranes of the rumen epithelial cells. Consequently, some mechanism must exist for the movement of VFA across the epithelium. While early data supported a transcellular rather than a paracellular mechanism, the exact mechanism was not known (Remond et al., 1995). Early research suggested the importance of carbonic anhydrase in the absorption of VFA by rumen epithelium (Aafjes, 1967; Bergman, 1990). The proposed mechanism involved production of HCO 3− and H+ within the rumen epithelium, movement of the protons and bicarbonate across the rumen mucosa and into the rumen fluid, and neutralization of the VFA followed by passive diffusion into the rumen mucosa down a concentration gradient (Bergman, 1990). Recent data suggest the existence of both a carrier-mediated transport mechanism and a passive diffusion mechanism (Sehested et al., 1999a), with the mediated transport mechanism coupled with sodium, chloride, and bicarbonate (Sehested et al., 1999b). These results are summarized in fig. 1. While the situation for acetate and propionate is unknown, butyrate transport appears to be energy-dependent, as inhibition of ATP synthesis in rumen epithelium blocks butyrate uptake (Gabel et al., 2001).

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Fig. 1. Diagram of volatile fatty acid (VFA) transport into rumen epithelial cells. Protons and bicarbonate are generated within the rumen epithelial cells by the action of carbonic anhydrase. Circles represent the presence of specific transport molecules. Based on Sehested et al. (1999a).

This suggests that ATP is either directly involved in butyrate transport, or that energydependent metabolism of butyrate is necessary for butyrate uptake by rumen epithelium. Until recently, the mechanism of glucose transport into rumen epithelium had not been examined. The general concept was that glucose utilized by the rumen epithelium was derived from the blood and absorbed at the serosal side of the epithelium. A recent report indicated the presence of GLUT5 (the basolateral facilitative glucose transporter) mRNA in sheep rumen epithelium (Zhao et al., 1998), which would perform the uptake of blood glucose by rumen epithelium. Surprisingly, mRNA for the Na+-dependent glucose transporter (SGLT1) was also detected. Functional analysis of 3-O-methylglucose transport by sheep rumen epithelium in vitro demonstrated the presence of SGLT1, which was subsequently confirmed by cloning a cDNA from rumen epithelium with 100% identity to the sheep intestinal SGLT1 (Aschenbach et al., 2000b). In vivo experiments also demonstrated the sodium-dependent absorption of physiological concentrations of glucose by sheep rumen epithelium (Aschenbach et al., 2000a). The authors suggested that this could be an important route of glucose absorption in ruminants consuming high-concentrate diets, especially as a mechanism to minimize the effects of rumen acidosis, as previously suggested by Ganter et al. (1993). This hypothesis was supported by the observation that sheep rumen epithelial uptake of glucose by SGLT-1 can be stimulated by β2-adrenoceptors, since increased sympathetic activity has been noted in acidotic ruminants (Aschenbach et al., 2002). Glucose uptake by mature rumen epithelium is thus more complex than previously believed. No studies appear to have examined the mechanism for palmitate or glutamine specific uptake by mature rumen epithelium. Alternatively, glutamine could enter the rumen epithelium as

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a component of peptides rather than as the amino acid (Webb et al., 1992). A specific peptide transporter has been detected in ruminal epithelium that could move glutamine-containing peptides into the ruminal epithelium by an electrogenic mechanism (Chen et al., 1999). However, these results are not universally accepted (Martens et al., 2001), indicating that the role of peptide transport in moving glutamine into the ruminal epithelium has yet to be fully resolved. 2.2. Energy metabolism in neonatal rumen epithelium 2.2.1. Substrates and their metabolism Much of the early work on rumen epithelial development was concerned with factors that would promote anatomical development of the rumen. For example, the classical work of Warner et al. (1956) was the first detailed study to examine the effect of different dietary treatments on both ruminal size and papillary development. This was the first study to postulate the importance of VFA from the microbial fermentation as inducers of rumen epithelial development. Subsequent research (Tamate et al., 1962; Hamada et al., 1976; Klein et al., 1987) confirmed the importance of VFA for the stimulation of papillary growth, and noted that increased rumen volume and musculature were dependent on bulk fill of the rumen. Relatively little research, however, examined the energy metabolism of the developing rumen epithelium. The first study examining energy metabolism in undeveloped rumen epithelium noted that, prior to papillary development, metabolism of VFA by rumen epithelium was low (Sutton et al., 1963). Blood glucose was subsequently identified as the primary energy substrate of neonatal calf rumen epithelium (Juhasz et al., 1976). Giesecke et al. (1979) performed the first systematic analysis of the changes in rumen epithelial metabolism that occur during rumen development. Using slices of rumen epithelium isolated from weaned and unweaned lambs of various ages, these researchers measured oxygen consumption and ketone body production by the rumen epithelial slices in vitro in the presence of glucose, lactate, butyrate, or propionate. The importance of a number of observations that were made by these authors has repeatedly been demonstrated in the intervening years. Oxygen consumption by rumen epithelium decreased with age independently of dietary changes and stage of rumen epithelial development. Glucose, lactate, and butyrate stimulated oxygen consumption by rumen epithelial slices from both 2-week-old and 6-month-old lambs. However, the ability of glucose and lactate to stimulate oxygen consumption in rumen epithelium from 6-month-old lambs was significantly less than in that from 2-week-old lambs, whereas butyrate stimulated oxygen consumption equally well in ruminal epithelium from lambs of either age. This was interpreted as a shift in substrate preference from glucose to VFA by the developing rumen epithelium, and was supported by a decrease in glucose uptake by the rumen epithelium from the older lambs. These results also implied that the rate of butyrate oxidation was not dependent on the stage of rumen epithelial development. Ketogenesis at different developmental stages of the rumen epithelium was also examined. In rumen epithelial slices from 9–10-week-old lambs either maintained on milk (undeveloped epithelium) or weaned to solid feed (developed epithelium), total ketone body production (BHBA + AcAc) was nearly 1.60-fold greater in the developed than the undeveloped epithelium, and was comparable to ketogenic rates observed in older lambs. There was also a shift in the BHBA:AcAc ratio from about 2.7 to 6.2 in the undeveloped and developed lamb rumen epithelium, respectively, indicating a shift in the redox potential of the rumen epithelium with age. These data indicated the importance of solid feed intake in promoting rumen

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metabolic development. Glucose addition to these in vitro incubations stimulated ketogenesis in a synergistic manner. Perhaps the most interesting observation, however, was the change in ketogenic capacity with age of rumen epithelium from milk-fed lambs. Total ketone body production (μmoles/(g tissue dry weight × hour)) by rumen epithelial slices from milk-fed lambs increased from 19.6 at 1 week of age, to 25.5 at 3–4 weeks of age, to 71.9 at 9–10 weeks of age. This was the first indication that changes in energy metabolism within the rumen epithelium could occur in the absence of solid feed intake and the consequent microbial production of VFA. The observations of Giesecke et al. (1979) concerning ruminal ketogenesis were subsequently confirmed and extended to rumen epithelium from milk-fed and normally reared (milk-fed to 28 days of age; starter and hay available after 10 days of age) calves by Bush (1988). This author examined rumen epithelium from 3-, 12-, 19-, 30-, and 60-day-old calves, providing a more complete time-course of the changes in ketogenesis occurring in developing rumen epithelium. Total ketone body production from butyrate by rumen epithelium from normally reared calves was detectable at 3 days of age, and slowly increased through 19 days of age. By 30 days of age ketogenic rates had jumped to about 40% of that observed in mature rumen epithelium, and by 60 days of age were similar to ketogenic rates in mature rumen epithelium. Ketogenesis from butyrate also increased with age in rumen epithelium from milk-fed calves, although the difference in total ketone body production rate by rumen epithelium from milk-fed and conventionally reared calves at 60 days of age was about 4.8-fold (Bush, 1988), in contrast to the 1.6-fold difference observed by Giesecke et al. (1979). Acetate conversion rate to ketone bodies was nearly 12.5-fold less than was observed with butyrate as substrate in rumen epithelium from 60-day-old conventionally reared calves, again similar to that in rumen epithelium from older animals (Bush, 1988). Two important observations were made in both of these studies (Giesecke et al., 1979; Bush, 1988). First, some changes in rumen epithelial energy metabolism occur in an ontogenic manner even in the absence of solid feed intake and the associated rumen fermentation. Second, complete rumen epithelial metabolic development requires solid feed intake, and is mediated presumably by the VFA from the resultant feed fermentation. 2.2.2. Substrate uptake The underlying mechanisms(s) responsible for the changes in glucose, butyrate, and ketone body metabolism in the developing rumen epithelium were in general not identified by Bush (1988) or Giesecke et al. (1979). Altered substrate uptake or metabolism, or a combination of both, could be responsible for the observed changes in energy metabolism during rumen epithelial development. Giesecke et al. (1979) did note a nearly 10-fold decrease in glucose uptake in rumen epithelium from 6-month-old lambs compared to that observed in 2-week-old lambs. (In contrast, lactate uptake by rumen epithelium from 6-month-old lambs were more than doubled.) Whether this decrease in glucose uptake was due to a decline in glucose transporter activity, or to a decrease in glucose metabolic capacity, during rumen epithelial development is not known. Similarly, changes in butyrate metabolism by developing rumen epithelium could be the result of changes in the activity of the appropriate transporter activity, or in the activity of butyrate metabolizing enzymes within the ruminal epithelium. Prior to 1992, no reports had been made concerning activity changes of metabolic enzymes in developing rumen epithelium that would provide an explanation for the major metabolic changes occurring during rumen epithelial development. There apparently was also no research into VFA absorption by the developing rumen epithelium. Similarly, although glucose oxidation

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by mature rumen epithelium had been observed at that time, no research had been conducted into the mechanism of glucose absorption by either mature or developing rumen epithelium.

3. RECENT DATA ON THE ENERGY METABOLISM OF DEVELOPING RUMEN EPITHELIUM Beginning in 1992, a series of papers appeared that attempted to define more finely the timing of the energy metabolism changes occurring in developing rumen epithelium, as well as to attempt identification of the mechanisms responsible for those changes. 3.1. Substrate oxidation In 1992, Baldwin and Jesse reported on the developmental changes in glucose and butyrate metabolism by rumen epithelial cells isolated from conventionally reared lambs of different ages (0, 4, 7, 14, 28, 42, and 56 [weaned] days of age). In this study, glucose oxidation (based on 14CO2 production from [1-14C]glucose by isolated rumen epithelial cells) increased from birth to 14 days of age, remained elevated until 42 days of age, and decreased by weaning at 56 days to rates lower than those observed at birth, but comparable to those observed in mature sheep (Baldwin and Jesse, 1992). Maximum glucose oxidation rates coincided with the time period of allometric rumen growth, suggesting the importance of glucose oxidation for energy generation during this time of rapid rumen tissue accretion. Surprisingly in this study, butyrate oxidation rates (based on 14CO2 production from [1-14C]butyrate by isolated rumen epithelial cells) were maximal at 4 days of age (nearly 7-fold greater than those observed at birth), clearly indicating the ability of undeveloped rumen epithelium to absorb and metabolize VFA. Butyrate oxidation rates decreased gradually until weaning at 56 days, and were comparable to those observed in older lambs. In rumen epithelial cells isolated from 28-day-old and younger lambs, addition of unlabeled glucose or butyrate decreased 14CO2 production from the other labeled substrate. The data presented could not distinguish between actual inhibition of oxidation of the alternative labeled substrate by addition of unlabeled substrate, or simple dilution of the specific activity within the acetyl-CoA pool from the labeled substrate by the unlabeled substrate prior to complete oxidation in the citric acid cycle (Baldwin and Jesse, 1992). Either explanation, however, is consistent with the ability of neonatal rumen epithelium to absorb and oxidize butyrate. What is not clear is why neonatal rumen epithelium should possess that ability in such a magnitude at a time when little if any butyrate is present within the rumen. The changes in rumen epithelial metabolism are summarized in fig. 2. 3.2. Ketogenesis Baldwin and Jesse (1992) also found that ketogenesis from butyrate, as measured by BHBA production rate, was undetectable at birth, but increased to a low, relatively steady rate

Fig. 2. Summary of the metabolic changes occurring during the development of lamb rumen epithelium. Based on Baldwin and Jesse (1992).

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from 4 through 42 days of age. After 42 days of age ketogenic rates increased markedly (about 8-fold), so that at weaning at 56 days of age butyrate conversion to ketone bodies was occurring at nearly adult rates (Giesecke et al., 1979; Bush, 1988; Harmon et al., 1991; Baldwin and Jesse, 1992). Based on these findings, lamb rumen epithelial development was suggested to occur in stages, with the two most prominent stages being the period of rapid rumen growth and keratinization occurring between about 28 and 42 days of age, followed by the onset of metabolic maturity as indicated by the onset of high rates of ketogenesis from butyrate (between 42 and 56 days of age) at weaning (Baldwin and Jesse, 1992). The suggestion was made that VFA from the rumen fermentation may be acting to promote rumen epithelial metabolic development in the same way that VFA had been noted to promote rumen papillary development (Warner et al., 1956). However, two confounding variables existed in this study, namely the change in diet (leading to physiological adaptation) and the increase in age of the lambs that would be associated with ontogeny of rumen epithelial development (ShiraziBeechey et al., 1991a; Baldwin and Jesse, 1992). To distinguish between these two possibilities, a study was conducted to determine the ability of VFA to stimulate rumen metabolic development (Lane and Jesse, 1997). Milk-fed lambs received either continuous intraruminal infusions of a physiological mixture of VFA (acetate, propionate, butyrate) or saline, or no intraruminal infusions, for 7–10 weeks. No significant differences in rumen epithelial parameters were found in this study, but several trends were noted. Papillae length tended to be longer in the VFA-infused lambs, suggesting that the VFA were acting to stimulate papillae growth as expected (Warner et al., 1956). Glucose oxidation tended to be lower, and AcAc production from butyrate higher, in the VFA-infused lambs than in the saline-infused or uninfused controls. No other metabolic differences were observed among the three infusion treatments. Both glucose oxidation and BHBA production from butyrate were similar between the various infusion treatments and conventionally reared lambs (Baldwin and Jesse, 1992; Lane and Jesse, 1997). The results of VFA infusion on stimulating rumen epithelial development in this study were inconclusive, in view of the lack of significant treatment differences. The minimal effect of VFA infusion on papillae development suggests that insufficient amounts of VFA may have been administered during this experiment to stimulate maximal rumen metabolic development. The similarity in glucose oxidation and BHBA production from butyrate between the various infusion treatments and conventionally reared lambs, however, provided additional support to the concept that ontogenic factors play a prominent role in rumen epithelial metabolic development. 3.3. Ontogeny of rumen metabolic development A subsequent study then addressed the role of ontogenic development of rumen metabolism by separating out the effects of age and diet on rumen epithelial metabolic development (Lane et al., 2000). Lambs were either maintained on a milk diet before slaughter (0, 4, 7, 14, 28, 42, 49, 56, or 84 days of age), or at 49 days of age were weaned onto solid feed and slaughtered at 84 days of age. Glucose oxidation by rumen epithelial cells isolated from the milk-fed lambs followed the same general pattern as observed with conventionally reared lambs (Baldwin and Jesse, 1992; Lane et al., 2000). Glucose oxidation rates by isolated rumen epithelial cells were not different among the 84-day-old milk-fed lambs, 84-day-old lambs weaned at 49 days of age (Lane et al., 2000), or conventionally reared lambs weaned at 56 days of age (Baldwin and Jesse, 1992). These results are similar to those of Giesecke et al. (1979), who observed no difference in glucose oxidation by rumen epithelial pieces from 8–12-week-old milk-fed or conventionally reared lambs.

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In contrast to that observed with normally reared lambs (Baldwin and Jesse, 1992), butyrate oxidation by isolated rumen epithelial cells was undetectable in lambs 7 days old or less (Lane et al., 2000). No explanation was given for this difference, especially since conventionally reared lambs at this age would not yet have begun to consume solid feed and would not differ physiologically from milk-fed lambs in that study (Lane et al., 2000). Different breeds of sheep were used in the two studies, however, which may have had some effect on the results. Subsequent to that age (7 days), little difference in butyrate oxidation by isolated rumen epithelial cells was observed through 94 days. Similar to that for glucose oxidation, butyrate oxidation was not different between rumen epithelial cells isolated from 84-day-old milk-fed lambs and 84-day-old lambs weaned at 49 days of age (Lane et al., 2000). Ruminal butyrate oxidation by the 84-day-old milk-fed lambs and 84-day-old lambs weaned at 49 days of age was ~75% of the rate observed with conventionally reared lambs weaned at 56 days of age (Baldwin and Jesse, 1992; Lane et al., 2000). The study of Lane et al. (2000) confirms the findings of Giesecke et al. (1979), and demonstrates that changes in glucose and butyrate oxidation in developing rumen epithelium can occur independently of diet. Prior to 42 days of age, ketogenesis from butyrate by isolated rumen epithelial cells, as measured by BHBA production, was relatively low in rumen epithelial cells from the milk-fed lambs, but increased thereafter to rates comparable to conventionally reared lambs (Baldwin and Jesse, 1992; Lane et al., 2000). Similar ketogenic rates were observed in rumen epithelial cells from the 84-day-old lambs weaned at 49 days of age. These results are consistent with those obtained by Giesecke et al. (1979), who observed increasing ketogenic rates from butyrate by rumen epithelial pieces from 1-week, 3–4-week, and 9–10-week-old lambs. In contrast to Lane et al. (2000), ketogenesis by the rumen epithelial pieces from 9–10-week-old milk-fed lambs was about 75% of the rate found in lambs of the same age that had been reared and weaned conventionally. Bush (1988) also noted increased rates of ketogenesis by rumen epithelial tissue with age from milk-fed calves. However, in that study ketogenesis by rumen epithelial tissue from conventionally reared and weaned calves was nearly 8-fold greater than in milk-fed calves of the same age. Thus, all three of these studies indicate that ketogenic capacity of rumen epithelium increases with age regardless of the diet consumed, although the magnitude of the reported increase did differ, perhaps due to species differences (Giesecke et al., 1979; Bush, 1988; Lane et al., 2000). This again suggests that rumen epithelial metabolic development can occur in the absence of the rumen fermentation and VFA production, although dietary responses may modulate that ontogenic process. 3.4. Differential gene expression in rumen metabolic development The mechanism responsible for the observed increase in ketogenesis by the rumen epithelium from milk-fed lambs may be an increase in expression of the genes encoding ketogenic enzymes (Lane et al., 2002). Northern blots of total rumen epithelial RNA isolated from conventionally reared and milk-fed lambs of different ages were probed with cDNA probes against AcAc-CoA thiolase and HMG-CoA synthase, the two enzymes that are generally regarded as regulating ruminal ketogenesis (Leighton et al., 1983). In conventionally reared lambs the relative abundances of AcAc-CoA thiolase and HMG-CoA synthase mRNA in rumen epithelium increased gradually (Lane et al., 2002), and generally paralleled the reported changes in ketogenesis in rumen epithelium from conventionally reared lambs (Baldwin and Jesse, 1992). Relative abundance of HMG-CoA synthase mRNA followed a similar pattern to that of AcAc-CoA thiolase mRNA, but exhibited a sharper increase between 42 and 49 days of age. Because this was the time when ketogenesis markedly increased in

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rumen epithelium from conventionally reared lambs (Baldwin and Jesse, 1992), these data suggest that expression of the HMG-CoA synthase gene may be the factor controlling rate of ketogenesis in the developing rumen epithelium. In milk-fed lambs, changes with age of relative AcAc-CoA thiolase mRNA abundance were similar to those observed in conventionally reared lambs. However, relative abundance of HMG-CoA synthase mRNA in rumen epithelium from milk-fed lambs differed from that observed in conventionally reared lambs. The relative abundance of HMG-CoA synthase mRNA in rumen epithelium from milk-fed lambs remained relatively low through 42 days of age, then exhibited an almost quantum jump between 42 and 49 days of age, remaining relatively high thereafter (Lane et al., 2002). Again, this time corresponds generally with the onset of marked ketogenesis in rumen epithelium from the milk-fed lambs. These data support the concept that rumen epithelial metabolic development can occur in the absence of rumen fermentation (i.e. the dramatic jump in relative abundance of HMG-CoA synthase mRNA after 42 days of age in milk-fed lambs). Nevertheless, solid feed intake and the concomitant production of rumen fermentation products (i.e. VFA) can modulate that process, as shown by the difference in change in relative abundance of HMG-CoA synthase mRNA in rumen epithelium between the conventionally reared and the milk-fed lambs. These data are also consistent with the findings of Giesecke et al. (1979) and Bush (1988). These results in rumen epithelium are consistent with the findings of other researchers in the small intestine, where both ontogenic development and dietary induction of various enzymes have been reported. For example, lactase activity decreases and dipeptidylpeptidase IV activity increases in the lamb small intestine regardless of dietary treatment (conventional rearing or maintenance on a milk diet), indicating ontogenic control of these enzymes (Shirazi-Beechey et al., 1991b). On the other hand, activity of the sodium-dependent glucose cotransporter in lamb intestine does change in response to dietary treatment (Shirazi-Beechey et al., 1991a). Harmon et al. (1991) have reported increased acyl-CoA synthase activites for acetate, propionate, and butyrate in adult bovine rumen epithelium in response to increased dietary energy intake. The response of ketogenic gene expression to solid feed intake in conventionally reared lambs may be the result of a mechanism similar to that resulting in increased acyl-CoA synthetase activity in adult rumen epithelium, acting in conjunction with ontogenic factors. The unique aspect of epithelial metabolic development in the neonatal rumen, especially ketogenesis, is the association of both ontogenic and physiological factors that apparently affect metabolic development by altering expression of the genes encoding ketogenic, and perhaps other metabolic, enzymes within the rumen epithelium (Lane et al., 2002). A recent report found similar ontogenic and physiological effects on sodium and chloride transport in developing calf rumen epithelium (Breves et al., 2002). Sodium transport by rumen epithelium increased with age of the calves, independently of dietary treatment (milkfed or weaned onto solid feed). Chloride transport by rumen epithelium also increased with age of the calf, but exhibited a greater increase in those calves weaned onto solid feed than in those maintained on a milk diet. Increased sodium and chloride transport by the developing rumen epithelium could reflect an increase in the VFA absorptive capacity by the rumen epithelium (Sehested et al., 1999b). These data provide further evidence of the importance of ontogenic events in the metabolic development of the rumen epithelium.

4. FUTURE PERSPECTIVES From the above discussion it should be clear that there are many unanswered questions concerning metabolic development in the neonatal rumen epithelium. Certainly the area of

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substrate uptake in developing rumen epithelium needs to be addressed. To date, no information is available on specific transporters for glucose or VFA in developing rumen epithelium, and how the activity of those transporters changes during development. The recent discovery of the SGLT1 glucose transport protein in mature rumen epithelium leads to the question of when that transporter appears during rumen epithelial development (Zhao et al., 1998; Aschenbach et al., 2000a,b, 2002), and the role the transporter plays in glucose metabolism by the developing rumen epithelium. Similarly, given the importance of sodium and chloride in VFA transport by rumen epithelium (Sehested et al., 1999b), the recent discovery that sodium and chloride transport increase during rumen epithelial development (Breves et al., 2002) is suggestive that VFA transport capacity may also increase during development. These issues should be addressed in the future. Related to VFA uptake is the issue of VFA activation, which has been suggested to be the rate-limiting factor in VFA metabolism (Ash and Baird, 1973). No information is available to indicate when the acyl-CoA synthetases appear during rumen epithelial development. Given the ability of neonatal rumen epithelial cells to utilize butyrate (Baldwin and Jesse, 1992), the acyl-CoA synthetases are likely to be present soon after, if not at, birth, but that needs to be determined. The role of ontogeny in rumen epithelial metabolic development is only now being recognized for the importance it plays in this process. The question then arises as to how that process is controlled. Further research into this area will certainly require the isolation of genomic clones encoding proteins that respond to dietary changes, e.g. structural proteins such as the small proline-rich proteins (Wang et al., 1996), as well as those that exhibit ontogenic patterns of development, such as HMG-CoA synthase (Lane et al., 2002). A comparison of the regulatory regions of these genes should provide information about the transcription factors potentially involved in regulating the expression of these genes. That information in turn could lead to identification of the signal transduction pathways that ultimately lead to the activation of these genes. Various reports have noted the importance of agents such as butyrate, insulin, and epidermal growth factor in stimulating the proliferation of rumen epithelial cells (Sakata et al., 1980; Galfi et al., 1991; Baldwin, 1999; Galfi and Neogrady, 2001). Since the signal transduction pathways of some of these agents have been identified, this information should be helpful in establishing the mechanisms regulating gene expression within the developing ruminal epithelium, and the interplay between physiological/dietary factors and ontogenic factors that result in complete rumen epithelial metabolic development. Ultimately a more complete characterization of the processes involved in rumen epithelial metabolic development should lead to more effective management techniques in rearing young ruminants. REFERENCES Aafjes, J.H., 1967. Carbonic anhydrase in the wall of the forestomachs of cows. Brit. Vet. J. 123, 252–256. Alio, A., Theurer, C.B., Lozano, O., Huber, J.T., Swingle, R.S., Delgado-Elorduy, A., Cuneo, P., Deyoung, D., Webb, K.E. Jr., 2000. Splanchnic nitrogen metabolism by growing beef steers fed diets containing sorghum grain flaked at different densities. J. Anim. Sci. 78, 1355–1363. Aschenbach, J.R., Bhatia, S.K., Pfannkuche, H., Gabel, G., 2000a. Glucose is absorbed in a sodiumdependent manner from forestomach contents of sheep. J. Nutr. 130, 2797–2801. Aschenbach, J.R., Wehning, H., Kurze, M., Schaberg, E., Nieper, H., Burckhardt, G., Gabel, G., 2000b. Functional and molecular biological evidence of SGLT-1 in the ruminal epithelium of sheep. Amer. J. Physiol. Gastrointest. Liver Physiol. 279, G20–G27. Aschenbach, J.R., Borau, T., Gabel, G., 2002. Glucose uptake via SGLT-1 is stimulated by beta2-adrenoceptors in the ruminal epithelium of sheep. J. Nutr. 132, 1254–1257. Ash, R., Baird, G.D., 1973. Activation of volatile fatty acids in bovine liver and rumen epithelium: evidence for control by autoregulation. Biochem. J. 136, 311–319.

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Baldwin, R.L. VI, 1999. The proliferative actions of insulin, insulin-like growth factor-I, epidermal growth factor, butyrate and propionate on ruminal epithelial cells in vitro. Small Ruminant Res. 32, 261–268. Baldwin, R.L. VI, Jesse, B.W., 1992. Developmental changes in glucose and butyrate metabolism by isolated sheep ruminal cells. J. Nutr. 122, 1149–1153. Baldwin, R.L. VI, McLeod, K.R., 2000. Effects of diet forage:concentrate ratio and metabolizable energy intake on isolated rumen epithelial cell metabolism in vitro. J. Anim. Sci. 78, 771–783. Bergman, E.N., 1990. Energy contributions of volatile fatty acids from the gastrointestinal tract in various species. Physiol. Rev. 70, 567–590. Breves, G., Zitnan, R., Schroder, B., Winckler, C., Hagemeister, H., Failing, K., Voigt, J., 2002. Postnatal development of electrolyte transport in calf rumen as affected by weaning time. Arch. Tierernähr. 56, 371–377. Britton, R., Krehbiel, C., 1993. Nutrient metabolism by gut tissues. J. Dairy Sci. 76, 2125–2131. Bush, R.S., 1982. Extraction of enzymes and assessment of metabolism in bovine rumen epithelium. Can. J. Anim. Sci. 62, 429–438. Bush, R.S., 1988. Effect of age and diet on in vitro metabolism in rumen epithelium from holstein calves. Can. J. Anim. Sci. 68, 1245–1251. Bush, R.S., Milligan, L.P., 1971. Enzymes of ketogenesis in bovine rumen epithelium. Can. J. Anim. Sci. 51, 129–133. Chen, H., Wong, E.A., Webb, K.E. Jr., 1999. Tissue distribution of a peptide transporter mRNA in sheep, dairy cows, pigs, and chickens. J. Anim. Sci. 77, 1277–1283. Gabel, G., Muller, F., Pfannkuche, H., Aschenbach, J.R., 2001. Influence of isoform and DNP on butyrate transport across the sheep ruminal epithelium. J. Comp. Physiol. B 171, 215–221. Galfi, P., Neogrady, S., 2001. The pH-dependent inhibitory action of N-butyrate on gastrointestinal epithelial cell division. Food Res. Int. 34, 581–586. Galfi, P., Neogrady, S., Sakata, T., 1991. Effects of volatile fatty acids on the epithelial cell proliferation of the digestive tract and its hormonal mediation. In: Tsuda, T., Sasaki, Y., Kawashima, R. (Eds.), Physiological Aspects of Digestion And Metabolism In Ruminants: Proceedings of the Seventh International Symposium on Ruminant Physiology. Academic Press, New York, pp. 49–59. Ganter, M., Bickhardt, K., Winicker, M., Schwert, B., 1993. Experimental studies of the pathogenesis of rumen acidosis in sheep. Zbl. Veterinärmedizin A 40, 731–740. Giesecke, D., Beck, U., Wiesmayr, S., Stangassinger, M., 1979. The effect of rumen epithelial development on metabolic activities and ketogenesis by the tissue in vitro. Comp. Biochem. Physiol. B 62, 459–463. Hamada, T., Maeda, S., Kameoka, K., 1976. Factors influencing growth of rumen, liver, and other organs in kids weaned from milk replacers to solid foods. J. Dairy Sci. 59, 1110–1118. Hanigan, M.D., Crompton, L.A., Metcalf, J.A., France, J., 2001. Modelling mammary metabolism in the dairy cow to predict milk constituent yield, with emphasis on amino acid metabolism and milk protein production: model construction. J. Theor. Biol. 213, 223–239. Harmon, D.L., 1986. Influence of dietary energy intake and substrate addition on the in vitro metabolism of glucose and glutamine in rumen epithelial tissue. Comp. Biochem. Physiol. B 85, 643–647. Harmon, D.L., Gross, K.L., Krehbiel, C.R., Kreikemeier, K.K., Bauer, M.L., Britton, R.A., 1991. Influence of dietary forage and energy intake on metabolism and acyl-CoA synthetase activity in bovine ruminal epithelial tissue. J. Anim. Sci. 69, 4117–4127. Jesse, B.W., Solomon, R.K., Baldwin, R.L. VI, 1992. Palmitate metabolism by isolated sheep rumen epithelial cells. J. Anim. Sci. 70, 2235–2242. Juhasz, B., Szegedi, B., Keresztes, M., 1976. The abomasal digestion in calves during development of the forestomachs. Acta Vet. Acad. Sci. Hung. 26, 281–295. Klein, R.D., Kincaid, R.L., Hodgson, A.S., Harrison, J.H., Hillers, J.K., Cronrath, J.D., 1987. Dietary fiber and early weaning on growth and rumen development of calves. J. Dairy Sci. 70, 2095–2104. Lane, M.A., Jesse, B.W., 1997. Effect of volatile fatty acid infusion on development of the rumen epithelium in neonatal sheep. J. Dairy Sci. 80, 740–746. Lane, M.A., Baldwin, R.L. VI, Jesse, B.W., 2000. Sheep rumen metabolic development in response to age and dietary treatments. J. Anim. Sci. 78, 1990–1996. Lane, M.A., Baldwin, R.L. VI, Jesse, B.W., 2002. Developmental changes in ketogenic enzyme gene expression during sheep rumen development. J. Anim. Sci. 80, 1538–1544. Leighton, B., Nicholas, A.R., Pogson, C.I., 1983. The pathway of ketogenesis in rumen epithelium of the sheep. Biochem J. 216, 769–772.

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Liebich, H.G., Dirksen, G., Arbel, A., Dori, S., Mayer, E., 1987. Feed-dependent changes in the rumen mucosa of high-producing cows from the dry period to eight weeks post partum. Zbl. Veterinärmedizin A 34, 661–672. Martens, H., Kudritzki, J., Wolf, K., Schweigel, M., 2001. No Evidence for active peptide transport in forestomach epithelia of sheep. J. Anim. Physiol. Anim. Nutr. (Berlin) 85, 314–324. McLeod, K.R., Baldwin, R.L. VI, 2000. Effects of diet forage:concentrate ratio and metabolizable energy intake on visceral organ growth and in vitro oxidative capacity of gut tissues in sheep. J. Anim. Sci. 78, 760–770. Nocek, J.E., Herbein, J.H., Polan, C.E., 1980. Influence of ration physical form, ruminal degradable nitrogen and age on rumen epithelial propionate and acetate transport and some enzymatic activities. J. Nutr. 110, 2355–2364. Noziere, P., Remond, D., Bernard, L., Doreau, M., 2000. Effect of underfeeding on metabolism of portal-drained viscera in ewes. Brit. J. Nutr. 84, 821–828. Remond, D., Ortigues, I., Jouany, J.P., 1995. Energy substrates for the rumen epithelium. Proc. Nutr. Soc. 54, 95–105. Sakata, T., Hikosaka, K., Shiomura, Y., Tamate, H., 1980. Stimulatory effect of insulin on ruminal epithelium cell mitosis in adult sheep. Brit. J. Nutr. 44, 325–331. Scaife, J.R., Tichivangana, J.Z., 1980. Short chain acyl-CoA synthetases in ovine rumen epithelium. Biochim. Biophys. Acta 619, 445–450. Sehested, J., Diernaes, L., Moller, P.D., Skadhauge, E., 1999a. Ruminal transport and metabolism of short-chain fatty acids (SCFA) in vitro: effect of SCFA chain length and pH. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 123, 359–368. Sehested, J., Diernaes, L., Moller, P.D., Skadhauge, E., 1999b. Transport of butyrate across the isolated bovine rumen epithelium: interaction with sodium, chloride and bicarbonate. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 123, 399–408. Shirazi-Beechey, S.P., Hirayama, B.A., Wang, Y., Scott, D., Smith, M.W., Wright, E.M., 1991a. Ontogenic development of lamb intestinal sodium-glucose co-transporter is regulated by diet. J. Physiol. 437, 699–708. Shirazi-Beechey, S.P., Smith, M.W., Wang, Y., James, P.S., 1991b. Postnatal development of lamb intestinal digestive enzymes is not regulated by diet. J. Physiol. 437, 691–698. Sutton, J.D., McGilliard, A.D., Richard, M., Jacobson, N.L., 1963. Functional development of rumen mucosa. II. Metabolic activity. J. Dairy Sci. 46, 530–537. Tamate, H., McGilliard, A.D., Jacobson, N.L., Getty, R., 1962. Effect of various dietaries on the anatomical development of the stomach in the calf. J. Dairy Sci. 45, 408–420. Wang, L., Baldwin, R.L. VI, Jesse, B.W., 1996. Identification of two cDNA clones encoding small proline-rich proteins expressed in sheep ruminal epithelium. Biochem. J. 317, 225–233. Warner, R.G., Flatt, W.P., Loosli, J.K., 1956. Dietary factors influencing the development of the ruminant stomach. J. Agr. Food Chem. 4, 788–792. Webb, K.E. Jr., Matthews, J.C., Dirienzo, D.B., 1992. Peptide absorption: a review of current concepts and future perspectives. J. Anim. Sci. 70, 3248–3257. Weigand, E., Young, J.W., McGilliard, A.D., 1975. Volatile fatty acid metabolism by rumen mucosa from cattle fed hay or grain. J. Dairy Sci. 58, 1294–1300. White, R.G., Leng, R.A., 1980. Glucose metabolism in feeding and postabsorptive lambs and mature sheep. Comp. Biochem. Physiol. A67, 223–229. Young, J.W., Thorp, S.L., Delumen, H.Z., 1969. Activity of selected gluconeogenic and lipogenic enzymes in bovine rumen mucosa, liver and adipose tissue. Biochem. J. 114, 83–88. Zhao, F.Q., Okine, E.K., Cheeseman, C.I., Shirazi-Beechey, S.P., Kennelly, J.J., 1998. Glucose transporter gene expression in lactating bovine gastrointestinal tract. J. Anim. Sci. 76, 2921–2929.

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Splanchnic carbohydrate and energy metabolism in growing ruminants1 N. B. Kristensena, G. B. Huntingtonb, and D. L. Harmonc aDepartment

of Animal Nutrition and Physiology, Danish Institute of Agricultural Sciences, DK-8830 Tjele, Denmark bDepartment of Animal Science, North Carolina State University, Raleigh, NC 27695-7621, USA cDepartment of Animal Sciences, University of Kentucky, Lexington, KY 40546-0215, USA

Ruminal fermentation precludes a simple description of nutrient availability based on nutrient intake. Thus, we must strive to understand the nutrient needs of the microflora and gut and then evaluate nutrient availability after these needs have been met. Glucose is extensively metabolized by gut tissues such that the net supply to the liver is often zero or negative. Despite this extensive metabolism, small intestinal digestion can significantly increase glucose availability and metabolism. Lactate is derived from the diet, from ruminal bacterial metabolism and from endogenous metabolism. Because of its ubiquitous nature, lactate production from the gastrointestinal tract and viscera varies widely. However, lactate is a major glucose precursor in ruminants, supplying 9–35% of hepatic glucose carbon. Short-chain fatty acids are the major currency of ruminant energy metabolism, accounting for 45% of digestible energy intake. Significant quantities of short-chain fatty acids are metabolized by ruminal epithelium; however, it appears that in the fed ruminant this epithelial metabolism is limited to butyrate and longer short-chain fatty acids. Estimates indicate that 5% of ruminally supplied propionate is metabolized by the rumen epithelium and 30% of arterially supplied acetate is metabolized by the portal-drained viscera. These findings allow estimates of ruminal short-chain fatty acid production to be obtained from portal appearance of short-chain fatty acids corrected for portal-drained visceral metabolism of arterial short-chain fatty acids and ruminal epithelial metabolism of butyrate.

1. INTRODUCTION Compared with other mammals, ruminants could seem less efficient in capturing energy in the form of body tissue, fetus, or milk. For example, a young pig on a nutritionally adequate 1Approved

as publication No. 02-07-97 by the Kentucky Agricultural Experiment Station.

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Biology of Metabolism in Growing Animals D.G. Burrin and H. Mersmann (Eds.) © 2005 Elsevier Limited. All rights reserved.

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diet captures 76% of ME (metabolizable energy) as tissue gain (Dunkin and Campell, 1982) whereas a growing steer on a forage diet captures 46% or less of ME as tissue gain (Varga et al., 1990). Does this indicate that ruminants are energetically inefficient? The answer to this question is not as straightforward as might be indicated by its simplicity. First of all, the relative growth rates of the pig and the steer will affect the energetic efficiency, i.e. lower relative growth rate means that a relatively higher proportion of the total energy is used for maintenance. A quite different aspect is that interchanging the diets would have disastrous consequences for the performance of the young pig whereas the steer might do well though likely to suffer from overfeeding. The pig will not be able to obtain sufficient amounts of nutrients from a forage diet to obtain its potential growth rate. These findings and the fact that ruminants utilize intravenously infused glucose as efficiently as nonruminants (Reid et al., 1980) indicate that the key to understanding both possibilities and limitations in ruminant nutrition and efficiency is related to the digestive strategy of ruminants. The forestomach fermentation in ruminants implicates that there is only an indirect relationship between the molecular composition of the feed and the actual nutrients available for absorption. The fermentation has a major influence on digestion and metabolism of all organic dietary components, i.e. carbohydrate, protein, fat, and vitamins. Carbohydrates make up the largest fraction of almost any diet for functional ruminants and the utilization of carbohydrate will therefore be of importance to both efficiency and performance. However, a number of controversies still exist connected to the availability of carbohydrate (starch) for postruminal digestion and absorption as well as quantitative relationships between carbohydrate fermentation and end-product (short-chain fatty acid; SCFA) availability to the animal. The purpose of this chapter is to detail some of the unique aspects of ruminant energy metabolism. Primarily, we aim to focus on the supply of glucose, lactate, and SCFA as sources of energy and their availability to body tissues. Only through a thorough understanding of these interrelationships can we hope to predict and explain growth responses based on dietary inputs.

2. GLUCOSE Because of pregastric fermentation much of the dietary carbohydrate is fermented to SCFA. This fermentation leaves little dietary carbohydrate available for absorption in the small intestine. Only when high-concentrate diets are fed are significant quantities presented to the small intestine for absorption (Huntington, 1997). Thus, pregastric fermentation necessitates a continual need for very high rates of gluconeogenesis (Bergman, 1973) to meet the glucose needs of the ruminant. The fermentation of dietary carbohydrates necessitates unique adaptations in ruminant glucose metabolism and many of these adaptations have been detailed in some excellent reviews (Leng, 1970; Bergman, 1973; Young, 1976); gluconeogenesis is also discussed in a separate chapter within this book (see Chapter 15 by Donkin and Hammon). We shall focus on how dietary influences affect the glucose economy of growing ruminants and on current information on the interorgan metabolism of glucose in ruminants.

3. SOURCES OF GLUCOSE Blood glucose concentrations are typically 4–6 mM in most mammals; however; ruminant concentrations tend to be lower, at 2–5 mM (Bergman, 1973). Despite low blood glucose concentrations and continual gluconeogenesis, ruminant blood glucose concentrations are very responsive to intestinal carbohydrate digestion and absorption (Larson et al., 1956).

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The major carbohydrate that ruminants consume in early life is lactose from milk. However, by 1–3 weeks of age ruminal fermentation is active and only through suckling will animals achieve closure of the esophageal groove and bypass significant quantities of materials to the abomasum for gastric digestion (Orskov et al., 1970). With the increase in ruminal fermentation there is a decline in the ability to digest lactose in the small intestine. Intestinal lactase (St. Jean et al., 1989) and glucose transport (Shirazi-Beechey et al., 1989) activities in the small intestine decline after weaning. Postweaning dietary carbohydrates contributing directly to glucose supplies include the various forms of α-linked glucose available in plants. Russell and Gahr (2000) described the classification of food carbohydrates as occurring in four forms: (1) free (not associated with the cellular structure), such as lactose in milk or fructose in honey; (2) intracellular, which includes soluble sugars and storage polysaccharides such as starch and fructans; (3) cell wall components including cellulose, hemicellulose, pectins, and gums; and (4) chitin, a component of the exoskeleton. For the functioning ruminant, only the intracellular storage polysaccharide, starch, contributes significantly to absorbed glucose. The remaining forms of food carbohydrate are first fermented to SCFA. Huntington (1997) summarized numerous digestion experiments with starch intakes ranging from 1.5 to 10.6 kg/d. In these experiments, ruminal starch digestibility ranged from 94% to 50%. The net result is that starch flow to the small intestine ranged from 90 to over 5000 g/d. These data demonstrate that starch intake can make a sizable contribution to the glucose needs of growing ruminants. However, to determine the contribution of starch intake to glucose availability, the efficiency of small intestinal digestion must be known.

4. IMPACT OF INTESTINAL DIGESTION ON GLUCOSE SUPPLY Several experiments have used animals fitted with hepatic portal vein and hepatic vein catheters to measure the quantity of glucose exiting the portal-drained viscera (PDV) and entering the liver (Huntington et al., 1989). This measurement provides a means of determining the net glucose contributions to the liver or peripheral tissues and measures the sum of glucose absorption and metabolism. Across a wide range of experiments encompassing varied diets, intakes, and physiological states, net glucose absorption is almost always zero or negative (Reynolds et al., 1994). This is not to say that glucose is not being absorbed, but rather that very large amounts of glucose from the arterial supply are being metabolized such that the “net” result from absorption and metabolism is zero or negative. In a study designed to quantitate intestinal contributions to portal glucose supply, Huntington and Reynolds (1986) abomasally infused glucose and corn starch into heifers. Overall, they recovered an average of 65% of the glucose and 35% of the starch as glucose in portal blood. No differences were observed for the amounts of glucose recovered from animals fed alfalfa hay or a high-concentrate diet at two intakes, suggesting little effect of adaptation for carbohydrate assimilation. Kreikemeier et al. (1991) fed steers alfalfa hay to minimize intestinal carbohydrate supply and abomasally infused them with glucose, corn starch, or corn dextrins at 20, 40, and 60 g/h. Infusions all lasted 10 h, with samples taken during the final 6 h. Glucose infusion resulted in 90% recovery of intestinal glucose disappearance in portal blood whereas only 19% and 32% of the dextrin and starch intestinal disappearance were recovered in portal blood, respectively. Factors such as microbial fermentation and gut tissue metabolism must certainly make a large contribution to small intestinal carbohydrate disappearance and emphasize the need for measures of tissue metabolism and intestinal disappearance to more accurately describe

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processes of digestion and absorption. The very high metabolic activity of the PDV tissues has been shown to be a major factor in the apparently low net rates of glucose absorption (Reynolds and Huntington, 1988a). These authors (Reynolds and Huntington, 1988a,b) measured directly the contribution of stomach and intestinal tissues to nutrient absorption in beef steers. When steers were fed a concentrate diet, comparatively large amounts of glucose were absorbed from the intestines; however, the amounts utilized by ruminal and other stomach tissues were so great that the overall net PDV absorption was negative. Attempts were made in previous studies to account for this negative net glucose absorption and thereby obtain a better estimate of net glucose absorption by including control (water) infusions (Kreikemeier et al., 1991). However, more recent work has shown that increasing the peripheral supply of carbohydrate, either through intraduodenal or intrajugular infusion of glucose, increases the metabolism of arterially supplied glucose by the PDV (Balcells et al., 1995), making these corrections tenuous at best.

5. DIET EFFECTS ON GLUCOSE METABOLISM In previous sections we have attempted to define relationships of intestinal supply and glucose availability. However, it needs to be clearly pointed out that the major determinant of glucose supply is dietary energy intake (Herbein et al., 1978). Experiments assessing wholebody glucose metabolism have clearly shown that glucose irreversible loss, a measure of the flow of glucose through the body pool never to return, and thus, at steady state, an indicator of glucose production, is a function of digestible energy intake. Schmidt and Keith (1983) tested this hypothesis using steers fed 70% corn vs. 70% alfalfa diets fed at equal energy intakes. They demonstrated that when steers were fed at equal energy intakes, glucose irreversible loss was equal. When dry matter intakes were equalized, glucose irreversible loss was greater for the 70% corn diet because of the greater energy intake with the corn. In a related study (Russell et al., 1986) it was demonstrated that glucose irreversible loss was directly related to energy intake independent of body size in steers ranging in weight from 136 to 470 kg. These relationships depend on the tight control between digestible energy intake and gluconeogenesis. Organic matter fermented in the rumen will supply glucose precursors, primarily propionate, to meet the glucose needs of the host. These relations are borne out in the work of Van Maanen et al. (1978), who determined ruminal propionate production and glucose irreversible loss in steers fed forage and grain-based diets with the propionateenhancing antibiotic, monensin. Monensin increased ruminal propionate production by 49% on the forage diet and by 76% on the grain diet. Associated with these increases in propionate were increases in glucose irreversible loss of 7% and 16% for the forage and grain diets, respectively. This study shows that increasing propionate supply can increase glucose irreversible loss, but not in direct proportion. These results were similarly borne out by Seal and Parker (1994) using intraruminal infusion of propionate in calves. Only at their highest propionate infusion (1 mol/d) was glucose irreversible loss increased. Interestingly, ruminal propionate infusion decreased PDV glucose use from 28% to 11% of glucose irreversible loss. The relationships between dietary energy intake and glucose irreversible loss depend upon two related assumptions: (1) ruminants have a very tight control of hepatic glucose production, and (2) digestion and absorption of starch in the small intestine contributes little to glucose irreversible loss in these studies because they are dependent on glucose derived from the products of ruminal fermentation. Bauer et al. (1995) infused phlorizin, a potent inhibitor of SGLT1, into the abomasum of steers and sheep and demonstrated that when glucose active

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transport was inhibited, hepatic glucose production increased resulting in no change in total splanchnic glucose output. This demonstrates that glucose production is well coordinated between the PDV and the liver. A different approach was used in the study by Harmon et al. (2001). They infused a partially hydrolyzed starch solution either ruminally or abomasally in growing steers. Shifting the site of starch digestion from the rumen to the small intestine increased glucose utilization by PDV tissues (132%), PDV glucose flux (310%), and irreversible loss of glucose (59%). Abomasal infusion resulted in greater total energy availability (28%) from the total splanchnic tissues. Thus, shifting starch digestion to the small intestine increases PDV glucose uptake and utilization without a corresponding decrease in hepatic glucose production. This shift results in greater glucose supplies to the periphery. This would seem in contrast to the results of Herbein et al. (1978), who related glucose irreversible loss solely to energy intake. These relationships may not hold if significant quantities of starch are digested and absorbed in the small intestine. Balcells et al. (1995) infused sheep jugularly with glucose and found that glucose irreversible loss increased over 2-fold. Accompanying this increase in systemic glucose availability was an increased utilization of glucose by the PDV. However, in their experiment, the fraction of whole-body glucose used by the PDV remained constant (30% of whole-body glucose irreversible loss) despite the increase in glucose irreversible loss. These results are in agreement with their later work (Cappelli et al., 1997) where sheep received exogenous glucose either intrajugularly or intraduodenally. Supplying glucose by either route increased whole-body glucose irreversible loss and portal glucose utilization, and again, portal glucose utilization was approximately 30% of glucose irreversible loss. These results suggest that the fraction of whole-body glucose irreversible loss used by the PDV is relatively constant. However, both of these studies were relatively short-term, lasting 6 to 8 h. They do not answer whether or not long-term exposure causes tissues to adapt and use more or less of the available glucose. In the study by Harmon et al. (2001) they infused a partially hydrolyzed starch solution either ruminally or abomasally in growing steers for 7 days. In their study, portal glucose utilization was 23% of whole-body glucose irreversible loss with the ruminal infusion and this increased to 34% when the carbohydrate was infused abomasally. Thus, despite a 58% increase in glucose irreversible loss, there was a concomitant increase in the fraction of glucose metabolized by PDV tissues. It is not known if this increase in metabolism was the result of tissue adaptation or simply differences in cattle and sheep. With the ruminal infusion an increase in metabolism could reflect more energy available as SCFA resulting in less PDV glucose use, as was seen with the ruminal propionate infusions of Seal and Parker (1994) described above. A decrease in net PDV glucose use has also been reported for steers fed 450 g/d sodium propionate (Harmon and Avery, 1987). McLeod et al. (2001) used the ruminal/postruminal infusion of carbohydrate model described above (Harmon et al., 2001) to study energy balance in growing steers. They reported that abomasal infusion of carbohydrate increased retained energy; however, based on calorimetric data, the energy retained was retained solely as fat. When combined, these results suggest that an increased availability of glucose increases the energetic efficiency and PDV metabolism of glucose, but this may also result in greater fat deposition. One could speculate that increased circulating glucose results in increased insulin and increased fat deposition. Others have suggested that there are specific effects of glucose on lipogenesis in ruminants. Pearce and Piperova (1984) compared duodenal infusions of glucose and dextrins in sheep and found that glucose infusion increased in vitro lipogenesis from acetate nearly 7-fold in subcutaneous adipose tissue as compared with control (noninfused) sheep.

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6. DIETARY AND DIGESTIVE SOURCES OF LACTATE Lactate entering portal blood of the gastrointestinal tract of a ruminant can come from the diet, can be a product of rumen fermentation, or can be a product of tissue metabolism. Dietary sources generally include lactate from fermented feeds, e.g., a product of lactobacilli in silages. Lactate is produced in fermented feeds by homo- or hetero-fermenting lactobacilli that vary in substrate (sugar) preferences and isomer of lactic acid produced. Most bacteria can produce either D(−)- or L(+)-lactic acid by virtue of isomer-specific lactate dehydrogenase and lactate racemase enzyme activity (Counotte and Prins, 1981; McDonald et al., 1991). Lactic acid concentrations in most silages prepared by adequate or competent techniques range from 3 to 12 g/kg DM. Treatments that limit fermentative activity, e.g. treatment with mineral acids, formic acid, or formalin, or wilting before ensiling, can reduce lactic acid concentration by one-half or more. Treatments that induce or enhance fermentative activity in the silo, e.g. inoculation with bacteria, addition of sugars or propionic acid, decreased particle size by precision chopping of herbage before ensiling, in general increase lactic acid concentration 1.5–2.0-fold (McDonald et al., 1991; Sheperd et al., 1995; Kung et al., 2000; Kung and Ranjit, 2001). The isomeric proportions of lactic acid in these feedstuffs have not been studied extensively; available reports indicate that L(+):D(−) ratios range from 0.3:1 to 1:1 (Schaadt, 1968; Hull, 1996; Kung et al., 2000). McDonald et al. (1991) suggested that as time of ensiling increases, the L(+):D(−) ratio approaches 1:1 because of racemase activity of lactobacilli. Lactate is both produced and used by ruminal microbes. Numbers (and activity) of lactate producers and users respond rapidly to readily fermentable substrate (Counotte and Prins, 1981; Goad et al., 1998), which means that ruminal lactate concentrations usually are very low (1–3 mM) to nondetectable. Calculations of lactate production in the rumen are in a similar range, 1–3 mmol/h (Counotte and Prins, 1981). In cases of abrupt changes in intake of readily available carbohydrates there can be a rapid increase in ruminal lactate concentrations, indicating that production can exceed use or removal from the rumen. For example, Harmon et al. (1985) dosed beef steers intraruminally with 12 g of glucose per kg of body weight and measured peak concentrations of L(+)- lactate and D(−)-lactate of 77 and 40 mM, respectively, 30 h after the dosing. As a result of rapid fermentation of the carbohydrates, the proportion of L(+):D(−)-lactate may change from predominantly L(+) to predominantly D(−). The change in isomeric ratio is more a function of increased production than differences in use rates, because both isomers are used by ruminal microbes at similar rates. The rapid production and accumulation causes a ruminal acidosis that is lethal to many ruminal protozoa, and also causes a systemic acidosis in the host ruminant (Dunlop, 1972; Counotte and Prins, 1981; Goad et al., 1998). Ruminal concentrations and isomeric proportions of lactate are the product of the effects of ruminal production, use, absorption from the rumen, and passage with digesta to more distal portions of the gastrointestinal tract.

7. ABSORPTION OF LACTATE FROM THE GASTROINTESTINAL TRACT L(+)-lactate

(and presumably D(−)-lactate) are transported across cell membranes by a family of monocarboxylate transporters (Price et al., 1998). These transporters also transport ketones, pyruvate, and acetate. Because lactate can be a product of tissue metabolism, a substrate for tissue metabolism, and the subject of transport across the plasma membrane of epithelial cells, it is difficult to discern the relative importance of, or interactions among, these processes on the rate of lactate appearance in portal blood draining the gastrointestinal tract.

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Further, dietary and endogenous factors that alter blood flow can negate or amplify in vivo changes in concentration differences in blood supplying and draining the PDV. The few data available for absorption of D(−)-lactate suggest that factors that promote production of D(−)-lactate in the rumen also promote its absorption and appearance in hepatic portal blood (Huntington et al., 1980, 1981; Harmon et al., 1985). For the remainder of this discussion of lactate absorption and metabolism, “lactate” and “L(+)-lactate” will be used synonymously unless otherwise indicated. The studies summarized in table 1 are representative of published literature that quantifies net flux of lactate across splanchnic tissues. The studies show that lactate absorption in sheep and cattle ranges from approximately 2 to 200 mmol/h. Increased intake of a given diet increases net absorption (Reynolds et al., 1991; table 1), as does increased body mass (usually accompanied by increased intake), albeit at a nonlinear rate (Eisemann et al., 1996; table 1). The data from Taniguchi et al. (1995; table 1) exemplify the positive relationship between increased ruminal fermentation and lactate absorption (alfalfa vs. alfalfa and ruminal starch infusion in table 1), and also indicate that increased intestinal appearance of glucose results in increased portal appearance of lactate, ostensibly as a result of postruminal gut tissue metabolism (ruminal vs. abomasal infusion of starch in table 1). The postruminal digestive tract accounted for about one-third of lactate absorption in beef steers fed alfalfa hay or a high-concentrate diet (Reynolds and Huntington, 1988b). The lactating dairy cows in the studies in table 1 had similar daily dry matter intakes (data not shown), but the cows eating the grass diet absorbed less lactate than the cows eating corn silage and supplement (Reynolds et al., 1991; De Visser et al., 1997; table 1). McLeod et al., (1997) (table 1) found that infusion of somatostatin decreased blood flow through PDV of sheep, but increased venoarterial difference of lactate (data not shown), resulting in increased net absorption of lactate. The study of Bauer et al. (1995; table 1) included intragastric infusion of phlorizin, which decreased net absorption of glucose (data not shown) but had no statistically significant effect on lactate flux. Other examples of lack of effects of metabolic regulators include similar net absorption of lactate in control beef steers vs. steers fed a β-adrenergic agonist (Eisemann and Huntington, 1993) or control steers vs. hyperinsulemic, euglycemic beef steers receiving intravenous infusion of insulin and glucose (Eisemann and Huntington, 1994). Lactate makes a small but measurable contribution to the overall energy supply for ruminants. Lactate accounted for approximately 4.3% of the sum of energy absorbed as SCFA and lactate by lactating dairy cows consuming all-forage diets (De Visser et al., 1997; table 1), 8% by lactating dairy cows consuming a 60:40 corn silage:supplement diet (Reynolds et al., 1991; table 1), 9% by steers consuming all-forage diets (Huntington et al., 1988), and 16% by heifers consuming a diet containing 780 g corn grain/kg of DM (Huntington and Prior, 1983).

8. HEPATIC METABOLISM OF LACTATE The metabolic importance of lactate for ruminants centers on its role as a glucose precursor in the liver; net lactate removal by the liver often exceeds portal supply (table 1) and can theoretically account for 9–35% of net hepatic glucose release (data not shown) in studies with bovines listed in table 1. Studies with infusions of radiolabeled glucose and lactate into lambs and steers indicate that from 5% to 11% of glucose carbon comes from L(+)-lactate, and less than 1% comes from D(−)-lactate (Huntington et al., 1980, 1981; Harmon et al., 1983). Recycling of carbon through lactate and glucose would cause underestimations from isotope infusions, and calculations from net fluxes likely overestimated the true conversion of lactate to glucose. In the sheep studies of McLeod et al. (1997; table 1) net lactate removal could

aPositive

40 321 321 236 438 522 253 253 253 645 500

36 36

BW, kg

Alfalfa hay, duodenal starch and casein infusion Alfalfa hay, duodenal starch and casein infusion, somatostatin injection Alfalfa hay, starch infusion Alfalfa:concentrate, low intake Alfalfa:concentrate, high intake Bromegrass hay:concentrate 60:40 Bromegrass hay:concentrate 60:40 Bromegrass hay:concentrate 60:40 Alfalfa hay Alfalfa hay, ruminal starch infusion Alfalfa hay, abomasal starch infusion Corn silage:supplement 60:40 Fresh ryegrass

Diet description

numbers indicate net absorption or release, negative numbers indicate uptake or removal.

Sheep wethers Beef heifers Beef heifers Beef steers Beef steers Beef steers Beef steers Beef steers Beef steers Lactating dairy cows Lactating dairy cows

Sheep wethers Sheep wethers

Species

2.2 45 82 47 67 63 39 50 75 216 121

4.8 6.6

PDV

5.4 28 54 −34 −34 −22 −24 −27 −7 −33 −23

−5 −4

−9.8 −10.6 3.2 −17 −28 −81 −101 −85 −63 −77 −68 −249 −144

TSP

Liver

Net flux, mmol/h Reference

Bauer et al. (1995) Reynolds et al. (1991) Reynolds et al. (1991) Eisemann et al. (1996) Eisemann et al. (1996) Eisemann et al. (1996) Taniguchi et al. (1995) Taniguchi et al. (1995) Taniguchi et al. (1995) Reynolds and Huntington (1988c) De Visser et al. (1997)

McLeod et al. (1997) McLeod et al. (1997)

Selected studies of L(+)-lactate fluxa across portal-drained viscera (PDV), liver, and total splanchnic (TSP) tissues of sheep and cattle

Table 1

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maximally account for 41–62% of net hepatic glucose production. Lactate contribution is not calculated for the data of Bauer et al. (1995; table 1) because in some of their treatments they measured net hepatic output of lactate. The range of these potential hepatic fluxes and potential contribution to hepatic gluconeogenesis attest to the flexibility and versatility of lactate to participate in postabsorptive metabolism. The complete data of Reynolds et al. (1991; table 1) for beef heifers showed an interaction between intake level and percentage of dietary concentrate; net hepatic lactate removal and potential contribution of lactate to gluconeogenesis increased when the heifers’ intake of a high-forage diet increased. However, lactate removal and potential contribution to gluconeogenesis decreased when the heifers’ intake of a high-concentrate diet increased. A mesenteric vein infusion of alanine in the same heifers (Reynolds and Tyrrell, 1991) increased net alanine removal and reduced net lactate removal by the liver, but did not affect net hepatic glucose output. These results indicate a replacement of lactate by alanine as a glucose precursor. The complete data of Eisemann et al. (1996; table 1) predict decreased net hepatic removal or extraction of lactate, and increased net hepatic removal of amino acids to support increased hepatic glucose production in beef steers as they grow from 235 to 525 kg of body weight. The somatostatin injection that increased net portal absorption of lactate in sheep also increased net hepatic removal of lactate and increased glucose output by the liver (McLeod et al., 1997; table 1). Steers fed a β-adrenergic agonist had an acute surge in lactate removal by the liver that could account for up to 63% of liver glucose output on the first day of treatment. Hepatic removal and potential contribution to gluconeogensis subsided after 7 days of treatment (Eisemann and Huntington, 1993).

9. PERIPHERAL METABOLISM OF LACTATE Circulating concentrations of L(+)-lactate range from 0.2 to 1.0 mM, and concentrations of D(−)-lactate are 0.10 to 0.50 of concentrations of L(+)-lactate (Huntington et al., 1980, 1981; Harmon et al., 1983); these studies are cited in table 1. Whole-body lactate turnover in beef cattle and sheep ranges from approximately 5 to 10 times net portal absorption (Huntington et al., 1980, 1981), indicating the importance of the Cori cycle in movement of carbon through lactate and glucose between the liver and peripheral tissues, mostly muscle. Excitement or agitation of animals can cause a rapid rise in blood lactate levels as a result of heightened muscle activity. The major fate of D(−)-lactate is oxidation, which accounted for essentially all D(−)-lactate turnover in steers (Harmon et al., 1983). In vitro studies with bovine tissues show significant potential for oxidation of D(−)-lactate, with the greatest activity in kidney cortex followed by heart and liver, the lowest activity being detected in muscle tissue (Harmon et al., 1984). Net flux of L(+)-lactate across hindlimbs of cattle varies in response to physiological state of the animal and physiological interventions by researchers. As stated previously, lactate interacts with glucose through the Cori cycle, but lactate also is used as a substrate for lipid synthesis. Therefore, depending on the contribution of fat to tissue makeup, the hindlimbs may be net users or net releasers of lactate (Prior et al., 1984; Eisemann et al., 1996). The acute response of beef steers to an orally administered β-adrenergic agonist was a dramatic increase in lactate production by hindlimbs which was not evident after 7 days of treatment (Eisemann and Huntington, 1993). Establishment of hyperinsulemia with euglycemia in steers enhanced glucose uptake by hindquarters, but did not significantly change lactate flux across those tissues (Eisemann and Huntington, 1994).

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10. SHORT-CHAIN FATTY ACIDS OVERVIEW Short-chain fatty acids are simple aliphatic carboxylic acids with straight or methyl-branched hydrocarbon chains of 2 to 5 carbons. The SCFA anions with 2 (acetate), 3 (propionate), and 4 (butyrate) carbons are the most prevalent SCFA in the rumen and colon (Bergman, 1990) and their production is closely related to the energy metabolism of rumen microbes (Russell and Wallace, 1988). The existence of acetate in the rumen was observed more than a hundred years ago; however, not until the 1940s was it discovered that SCFA are absorbed from the forestomachs and make a significant contribution to ruminant metabolism (Barcroft et al., 1944). About 67% of ruminal SCFA are absorbed across the rumen epithelium or taken up by the rumen microbes and about 33% are carried out of the rumen by liquid passage (Peters et al., 1990). Short-chain fatty acids leaving the rumen with liquid outflow are absorbed mainly in the omasum and abomasum (Masson and Phillipson, 1952; Rupp et al., 1994). In ruminants, as in other animals, a mixture of undigested feed and organic matter of endogenous origin enters the hindgut and is fermented into gasses, SCFA, and microbial organic matter. Fermentation in the hindgut is of little quantitative nutritional importance to the animal compared to the forestomach, mostly because microbial protein and other nonSCFA products of fermentation are not readily absorbed. The SCFA production in the hindgut can be estimated as 6–13% of the total gut production based on the propionate appearance across mesenteric drained tissues compared to the total PDV net appearance (Reynolds and Huntington, 1988b). Studies based on isotopic dilution in the rumen and cecum have yielded similar relative production rates (12%) between forestomach and hindgut (Siciliano-Jones and Murphy, 1989). Therefore, forestomach fermentation is quantitatively the most important fermentation in ruminants, and most focus is given to forestomach physiology. However, it must be kept in mind that total gut production of SCFA does contain a hindgut component.

11. TRANSPORT BY NONIONIC DIFFUSION The rumen is lined with a keratinized stratified squamous epithelium. The epithelium is a heterogeneous structure with a physical barrier formed by keratinized cells facing the lumen. The chemical barrier of the epithelium is below the keratinized cells. The majority of metabolic activity is located in the basal cells as indicated by their high concentration of mitochondria (Steven and Marshall, 1970; Henrikson and Stacy, 1971). Weak electrolytes, a group to which SCFA belong, can pass biological membranes via nonionic diffusion; the resulting unidirectional flux is a function of concentration (activity) and solubility in the membrane (Rechkemmer, 1991). In accordance with this theory, it has been shown in vivo (Thorlacius and Lodge, 1973) as well as in vitro (Sehested et al., 1999b) that the unidirectional flux rate of butyrate across rumen epithelium increases with decreasing pH. However, the lack of proportionality between concentration of protonized acids and acetate and propionate fluxes as well as a relatively high permeability of these acids compared to longer-chain fatty acids has been seen as a challenge for the absorption theory based on nonionic diffusion. Nevertheless, a generally observed phenomenon is that SCFA have a relatively high permeability to biological membranes relative to longer-chain fatty acids (Dietschy, 1978). This means that the membranes behave as rather polar structures toward small solutes such as SCFA. The relative absorption rates of SCFA from experiments with washed reticulorumens show that absorption rates of fatty acids longer than butyrate increase with increased chain length (pH 7), and that methyl-branched SCFA (isobutyrate and isovalerate) have lower absorption rates than their corresponding straight-chain fatty acids (Oshio and Tahata, 1984;

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Kristensen et al., 2000a). Although the membranes of the rumen epithelium apparently have a relatively high permeability to acetate, propionate, and butyrate, it still makes sense to describe their absorption as regulated by mass action (as long as we consider unidirectional membrane fluxes of SCFA). Recent work has suggested that anion exchangers may contribute to apical SCFA fluxes in rumen epithelium even though the quantitative importance is unknown (Kramer et al., 1996). So far, the data available on SCFA absorption from the forestomach seem to indicate that absorption of SCFA by diffusion can account for the quantitatively most important SCFA absorption.

12. CARRIER-MEDIATED TRANSPORT IN RUMEN EPITHELIUM Rumen epithelium mounted in Ussing chambers has consistently shown a remarkable difference in the net transport of butyrate compared with the net transport of acetate and propionate (Stevens and Stettler, 1967; Sehested et al., 1999a). While the rumen epithelium shows a small net secretion (net transport from blood to lumen side of the isolated epithelium) of acetate and propionate when epithelia are incubated without an electrochemical gradient of SCFA, a relatively large net absorption of butyrate carbon usually occurs. The secretion of acetate and propionate by the epithelium at first seems to argue against the concept of nonionic diffusion. However, most estimates of SCFA flux in vitro have been based on 14C-labeled acids, implying that release of any substance carrying carbon from SCFA will be interpreted as SCFA flux. A small proportion of acetate and propionate transported across the epithelium will be oxidized under these conditions and the epithelium has been shown to primarily excrete the CO2 on the luminal side, explaining at least partly the net excretion of these acids (Sehested et al., 1999a). The rumen epithelium has long been known to be capable of metabolizing SCFA and, in particular, to have high affinity and capacity for metabolism of butyrate (Pennington, 1952). This in fact is the key to explaining the differences in the epithelial transport of butyrate compared with acetate and propionate. The metabolism of butyrate into acetoacetate and 3-hydroxybutyrate and the subsequent release of these compounds across the basolateral membrane would be in agreement with the apparent normal metabolic activity of the epithelium and would also explain why [14C]-butyrate was transported differently from acetate and propionate. It is likely that the products of butyrate metabolism are transported to the serosal (blood side) buffer carrying the label from butyrate. Keto- and hydroxyacids such as acetoacetate, 3-hydroxybutyrate, and lactate are more polar than SCFA because of their hydrophilic, secondary functional group, and consequently these acids have a lower permeability in biological membranes. In skeletal muscle a monocarboxylate transporter which co-transports lactate and protons solves an analogous transport problem for lactate across the cell membrane (Juel, 1997). The missing piece of the puzzle would therefore be to find monocarboxylate transporters in the epithelium that enable polarized transport of acetoacetate and 3-hydroxybutyrate. Recently, this transporter was shown to be present in rumen epithelium which agrees with this sequence of events (Müller et al., 2001). It has also been shown that blocking cellular metabolism abolishes the active component of butyrate absorption in vitro (Gäbel et al., 2001), confirming that it is the ketone bodies formed from butyrate that are selectively transported to the serosal side of the epithelium and not butyrate itself.

13. RUMEN EPITHELIAL METABOLISM One of the central observations on SCFA metabolism in ruminants has been the apparently extensive metabolism of ruminally produced SCFA by the rumen epithelium. However, this

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has been among the most difficult features of SCFA metabolism to understand. Numerous reviews are available discussing SCFA metabolism (Bergman, 1990; Britton and Krehbiel, 1993; Seal and Reynolds, 1993; Rémond et al., 1995; Kristensen et al., 1998; Seal and Parker, 2000). Recent studies have challenged the view that the rumen epithelium has a dominant role in the metabolism of acetate and propionate absorbed from the rumen. The classic attempt to determine the quantitative relationship between SCFA production in the gut and SCFA absorption was the work by Bergman and Wolff (1971). The production of SCFA in the rumen based on isotopic dilution was compared with portal appearance of SCFA corrected for PDV uptake of arterial acetate. It was concluded that large amounts not only of butyrate, but also of acetate and propionate, were metabolized by gut epithelia. In support of this conclusion, rumen epithelium also seemed to metabolize a large fraction of SCFA transported in vitro (Stevens, 1970). Nevertheless, these figures have long been doubted when considering the large amounts of SCFA apparently being absorbed in high producing ruminants (Sutton, 1985). These figures also lead to the paradoxical conclusion that the rumen epithelium of a lactating cow should have oxidative needs comparable to the entire fasting heat metabolism of the animal (Kristensen and Danfær, 2001). Studies on rumen epithelial metabolism of absorbed SCFA may have overestimated the metabolism by the epithelium because the actual estimation is the mixed effect of rumen microbial and rumen epithelial metabolism. Studies on SCFA absorption under washed reticuloruminal conditions that minimize bacterial activity have shown that the portal appearance of acetate, propionate, and isobutyrate could account for the entire disappearance of these acids from the rumen when the PDV uptake of arterial acetate is taken into account and 5% of the propionate is assumed metabolized into lactate by the rumen epithelium (Kristensen et al., 2000a). Butyrate was also extensively metabolized by the rumen epithelium under washed reticulo-rumen conditions and no more than 23% of the butyrate disappearance from the rumen could be accounted for by portal appearance of butyrate. It has previously been observed that there is increasing portal recovery of butyrate with increasing disappearance rates of butyrate from the rumen of sheep (Kristensen et al., 1996b, 2000b; Nozière et al., 2000). This effect is in agreement with a saturable metabolic capacity of the epithelium. To what extent there is interspecies differences in the metabolic capacity of butyrate in the rumen epithelium is not yet clear, but in a study with steers, the portal recovery of butyrate did not increase with increasing ruminal infusion rates of butyrate (Krehbiel et al., 1992). The recovery was relatively high at all infusion levels in the steers (25%), and was equivalent to the highest recovery level obtained in the sheep experiments. In sheep, increasing ruminal butyrate infusion not only leads to increasing portal recovery of butyrate, but also to increasing portal recovery of ruminal valerate (Kristensen et al., 2000b). These results point to a redefinition of the role of the rumen epithelium in SCFA metabolism and suggest that the rumen epithelium is not metabolizing large amounts of acetate and propionate as previously assumed.

14. IS BUTYRATE OXIDIZED TO CARBON DIOXIDE DURING ABSORPTION? In vitro studies have shown that rumen epithelium is able to oxidize all of the three quantitatively most important SCFA (Baldwin and McLeod, 2000); however, the epithelial production of 3-hydroxybutyrate and acetoacetate imply that butyrate oxidation is far lower than its disappearance across the epithelium. Studies comparing net portal appearance of butyrate and butyrate infusion into the rumen have indicated that major parts of the butyrate were

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oxidized (lost), because net portal appearance of butyrate, 3-hydroxybutyrate, and acetoacetate accounted only for 25–45% of ruminal butyrate infusion (Krehbiel et al., 1992; Kristensen et al., 1996b). However, the PDV has been shown to utilize 3-hydroxybutyrate from arterial blood equivalent to 32–42% of the whole-body flux in sheep and thereby mask the true production rate by the gut epithelia (Kristensen et al., 2000c). Intraruminal microbial pathways might also utilize part of the infused butyrate and thereby contribute to what could be interpreted as epithelial oxidation. This latter effect has been indicated by relatively high recoveries (as compared to expected recovery in the fed animal) of butyrate when infused into animals maintained under total intragastric nutrition (Gross et al., 1990a) or temporarily washed reticulo-rumen conditions (Kristensen et al., 2000a). In conclusion, the rumen epithelium has oxidative needs and butyrate is likely the most important carbon source. The majority of the butyrate absorbed is released as butyrate, acetoacetate, and most importantly, 3-hydroxybutyrate to the portal blood.

15. WHY DO EPITHELIA METABOLIZE BUTYRATE? Butyrate is generally considered a special metabolite for gut epithelial function (Topping and Clifton, 2001). One way to explain the special behavior of gut epithelia toward butyrate compared with acetate and propionate is that butyrate is important as an energy source for epithelial cells (Bugaut, 1987). However, the rumen epithelium has a range of other metabolites available, e.g. acetate and propionate absorbed from the rumen as well as arterially supplied glucose. One might speculate that butyrate’s role as an important substrate for epithelial energy metabolism might have evolved secondary to the basic need of having butyrate removed before it enters the blood stream. Butyrate metabolism by rumen and hindgut epithelia could therefore be seen as a protective mechanism that has two disposal pathways, oxidation and ketogenesis. It is obvious that butyrate is handled differently from acetate and propionate by the epithelia (Pennington, 1952), but another question remains to be answered: is butyrate a unique metabolite? Valerate, for example, is also efficiently metabolized by the rumen epithelium (Kristensen et al., 2000a,b) and it has been shown that the epithelium have the capacity to metabolize medium-chain (Hird et al., 1966) as well as longchain fatty acids (Jesse et al., 1992). Butyrate is an important substrate for gut epithelia compared with acetate and propionate, but it is apparently not a unique nutrient. Acetate, propionate, and isobutyrate are all metabolites of endogenous pathways in the organism. Acetate has the lowest membrane permeability, is utilized from peripheral arterial blood in major extrahepatic tissues (Pethick and Lindsay, 1982), and is a universal metabolite in the body in the form of acetyl-CoA. Propionate is the main donor of 3-carbon units for gluconeogenesis in the ruminant liver and is efficiently taken up by the liver (Leng and Annison, 1963). The endogenous sources of propionate include degradation of uneven chained fatty acids and some amino acids (methionine, threonine, isoleucine, and valine). Isobutyrate (an intermediate from catabolism of valine) appears in relatively low concentrations in the rumen, but is efficiently taken up by the liver for gluconeogenesis (Stangassinger and Giesecke, 1979). These SCFA are not only well tolerated in hepatic and peripheral tissues, but are key metabolites (especially acetate and propionate) in these tissues, and this agrees with a limited uptake of these SCFA in the gut epithelia. Butyrate, valerate, and probably longer, medium-chain fatty acids (MCFA) are less polar and will have a relatively high permeability in cell membranes. One way of controlling permeability is partial oxidation of these SCFA into acetoacetate and 3-hydroxybutyrate in the gut epithelia. When butyrate appears in the systemic circulation or is added to cell cultures,

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it has been shown to have a number of adverse effects: inhibition of growth and induction of morphological changes in cultured cells of different origins including ruminal epithelial cell lines (Prasad and Sinha, 1976; Gálfi et al., 1991); being an insulin secretagogue (Manns and Boda, 1967); inhibition of gastrointestinal motility by stimulation of epithelial receptors (Crichlow, 1988) and/or via systemic effects (Le Bars et al., 1954); stimulation of rumen epithelial development (Sander et al., 1959); or killing (2.5 mmol butyrate/kg BW in lambs) the animal (Manns and Boda, 1967). The epithelia of the gut have apparently evolved to perform gatekeeping functions by controlling the entry of butyrate and longer-chain fermentation acids into the peripheral circulation. It is tempting to speculate that the side effects of the gatekeeper function are that these metabolites also become quantitatively important oxidative substrates.

16. ACYL-CoA SYNTHETASES Activation of SCFA by an acyl-CoA synthetase (also named CoA ligase or thiokinase) is the first step in the metabolism of any SCFA in the cells of the gut epithelium, liver, or peripheral tissues (Groot et al., 1976). The acyl-CoA synthetases are therefore believed to be key enzymes in different tissues’ selectivity to metabolism of different SCFA. There exist a number of distinct acyl-CoA synthetases: acetyl-CoA, propionyl-CoA, butyryl-CoA, mediumchain fatty acid, and long-chain fatty acid-CoA synthetases. The acetyl-CoA synthetase (EC 6.2.1.1) has a high affinity for acetate, and some affinity for propionate (Campagnari and Webster, 1963; Groot et al., 1976; Ricks and Cook, 1981b). However, it is noteworthy that the activity of this enzyme has been found to be low in the rumen epithelium and liver of ruminants (Cook et al., 1969; Ash and Baird, 1973). These observations are in line with a limited role of the rumen epithelium and the liver in metabolism of absorbed acetate. The ruminant liver has a relatively high propionyl-CoA synthetase (EC 6.2.1.17) activity (Ash and Baird, 1973) and there exist a number of indications that propionyl-CoA synthetase is a distinct enzyme (Ricks and Cook, 1981a,c). Among the interesting features of this enzyme is that it is not present in rumen epithelium. This is not the same as denying any possible activation of propionate in rumen epithelium, which obviously can occur (Weekes, 1974), but it has been shown that the propionyl-CoA synthetase activity in the liver is almost insensitive to the presence of butyrate whereas the activity in the rumen epithelium is almost completely inhibited by the presence of butyrate (Ash and Baird, 1973; Harmon et al., 1991). As is the case with acetyl-CoA synthetase in rumen epithelium, the lack of propionyl-CoA synthetase activity is in agreement with in vivo observations showing a very limited uptake of propionate by the rumen epithelium. As described above, one of the most striking features of rumen epithelial metabolism is a high affinity and capacity for metabolism of butyrate. This feature is reflected in the butyrylCoA synthetase activity of the epithelium (Ash and Baird, 1973). The relative importance of the liver and the rumen epithelium in the metabolism of propionate and butyrate, respectively, is directly reflected in the acyl-CoA synthetase activities. Moreover, as butyrate was found to have an insignificant effect on propionate activation in the liver, propionate had no effect on butyrate activation in the rumen epithelium, but decreased the butyrate activation in the liver (Ash and Baird, 1973). A distinct butyryl-CoA synthetase (EC 6.2.1.2) was first purified from bovine heart mitochondria and this enzyme showed a high affinity for valerate and caproate (Webster et al., 1965). In ruminants, butyrate affinity is also found in xenobiotic/medium-chain fatty acid-CoA synthetases. These acyl-CoA synthetases activate a broad spectrum of

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straight-chain fatty acids: butyrate, longer SCFA, branched-chain fatty acids, and a number of xenobiotic (of foreign origin) carboxylic acids; others include benzoate and phenylacetate (Aas, 1971; Vessy et al., 1999). Indirect evidence from PDV flux studies indicates crossspecificity for valerate activation, which agrees with both types of butyrate activating systems. So far, no specific information seems to be available on the interaction of SCFA activation with longer-chain fatty acids or xenobiotic compounds absorbed from the rumen (Cremin et al., 1995); however, the fact that the isolated rumen epithelium or isolated rumen epithelial cells are able to use a wide range of fatty acids from SCFA to palmitate indicates the presence of some activity for activating medium- as well as long-chain fatty acids by the epithelium (Jesse et al., 1992; Hird et al., 1966).

17. HOW IS BUTYRATE METABOLIZED BY GUT EPITHELIA? Rumen epithelial ketogenesis is remarkable compared to hepatic ketogenesis by virtue of the fact that rumen epithelial ketogenesis is a main pathway in the fed state, and not a pathway turned on at fasting or when the organism is facing a high “metabolic drain”. This feature is obviously connected to the constant fueling of rumen epithelial ketogenesis via butyrate absorption in combination with an apparent need for removal of butyrate before entering the blood stream. The oxidation of butyryl-CoA to acetoacetyl-CoA in rumen epithelium (fig. 1) is, from a chemical point of view, identical to the initial steps of long-chain fatty acid β-oxidation. (For a review on this subject, see Eaton et al., 1996.) The first 3-hydroxybutyrate intermediate of this pathway is the L-(S)-isomer, which is not released to the peripheral circulation. Oxidation of L-3-hydroxybutyrate-CoA yields acetoacetyl-CoA. Acetoacetyl-CoA is a branching point between acetyl-CoA formation and ketone release because of the acetoacetyl-CoA thiolase (EC 2.3.1.9) catalyzed equilibrium between acetoacetyl-CoA and acetyl-CoA (fig. 2). The equilibrium constant of the reaction (6 × 10−6; Williamson et al., 1968) is strongly favoring acetyl-CoA and this means that the concentration of acetoacetyl-CoA probably will be relatively low in the mitochondrion. The production of ketone bodies from acetate (Harmon et al., 1991) or valerate (Weigand et al., 1975) in rumen epithelium confirms that acetoacetyl-CoA thiolase is present in the rumen epithelium, an observation also confirmed by assays on epithelial cell extracts (Baird et al., 1970). The main function of acetoacetyl-CoA thiolase is probably not to mediate ketogenesis from absorbed acetate, although this mediation is possible.

Fig. 1. Initial oxidation of butyryl-CoA to acetoacetyl-CoA in rumen epithelium proceeds via a pathway similar to the initial steps in β-oxidation. A number of isoenzymes are known for both acyl-CoA dehydrogenases (first dehydrogenase of the pathway) and 3-hydroxyacyl-CoA dehydrogenases (Eaton et al., 1996). However, the isoenzymes with specificity for short-chain acyl-CoA are likely to predominate in the rumen epithelium. The hydratase in the pathway is likely crotonase (EC 4.2.1.17), also an enzyme with the highest specificity toward short-chain acyl groups.

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Fig. 2. The acetoacetyl-CoA thiolase (EC 2.3.1.9) is catalyzing the reversible thiolytic cleavage of acetoacetyl-CoA into two acetyl-CoAs. The equilibrium between acetoacetyl-CoA and acetyl-CoA is favoring acetyl-CoA and as a result the acetoacetyl-CoA concentration in the mitochondrion will usually be low.

The fact that the rumen epithelium contains low amounts of acetyl-CoA synthetase, and the low affinity of the butyryl-CoA synthetase for acetate when butyrate is present, points to the conclusion that the main function of the acetoacetyl-CoA thiolase is feeding acetyl-CoA from the acetoacetyl-CoA pool to the TCA cycle. Therefore, even though butyrate metabolism in the epithelium cannot be explained from the point of the energy needs of the epithelium, butyrate metabolism is ensured to be the main oxidative substrate under in vivo conditions. Contrary to the consensus about the initial steps of butyrate metabolism, there has been more discussion of the subsequent metabolism of acetoacetyl-CoA. This compound can be deacylated directly (acetoacetyl-CoA deacylase; EC 3.1.2.11) or deacylated via the 3-hydroxy-3-methylglutaryl-CoA pathway (3-HMG pathway; 3-hydroxy-3-methylglutaryl-CoA synthetase and lyase; EC 4.1.3.5 and EC 4.1.3.4); however, other alternative pathways have been suggested and will be discussed briefly. The presence of the 3-HMG pathway (fig. 3) in rumen epithelium is supported by the fact that the enzymes of the pathway (3-hydroxy-3-methylglutaryl-CoA synthetase, and lyase) have been shown to be present in the epithelium in significant amounts (Baird et al., 1970; Leighton et al., 1983). However, isotopomer studies have had a dominant role in the arguments about ketogenic pathways in the epithelium. Hird and Symons (1961) investigated isolated ruminal and omasal epithelial metabolism of [1-14C]butyrate and [3-14C]butyrate into acetoacetate. The isotopomers of acetoacetate could be partly identified by measuring the label in position 1 (CO2 from decarboxylation of acetoacetate) and in the label in the acetone fraction after decarboxylation (interpreted as position 3). When the epithelium was incubated with [1-14C]butyrate, 80% of the label in acetoacetate was found in position 1 and 20% of the label was found in position 3. When the substrate was [3-14C]butyrate, 37% of the label in acetoacetate was found in position 1 and only 63% in position 3. The probable explanation for the 1 to 3 shifts in labeling is the thiolase-catalyzed equilibrium between acetoacetyl-CoA and acetyl-CoA (fig. 2). The labeling pattern also gives an indication of the relative importance of the pathway. The fact that 20% of the label in acetoacetate was found in position 3 could lead

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Fig. 3. The 3-hydroxy-3-methylglutaryl-CoA pathway (3-HMG pathway) ensures that acetoacetyl-CoA, despite its low concentration, can be “trapped” and deacylated. These steps of ruminal ketogenesis are similar to hepatic ketogenesis.

to the conclusion that 80% of acetoacetate was generated without degradation to acetyl-CoA. However, that is a false conclusion because if we assume that the labeling does not cause fractionation, then the acetoacetyl-CoA generated from acetyl-CoA (acetyl-CoA will be labeled in position 1) will be evenly distributed among carbon 1 and carbon 3 of acetoacetate. This means that at least 40% of the acetoacetate must have been equilibrating with acetyl-CoA to explain 20% of the total activity in position 3. The fact that the large majority of label from [1-14C]butyrate ends up in C1 of acetoacetate has been used as an argument against the function of the 3-HMG pathway in the epithelium. However, this argument might not be justified because this pathway will conserve C1 label in position C1, especially if the thiolase activity is relatively low compared to the flux through the 3-HMG pathway. The fact that Hird and Symons (1961) found a larger 3 to 1 shift in labeling of acetoacetate from [3-14C]butyrate is therefore in agreement with the 3-HMG pathway not only working on acetoacetyl-CoA derived from acetyl-CoA, but also on acetoacetyl-CoA from the initial-oxidation steps on butyrate. It seems puzzling that only 37% of the label in acetoacetate generated from [3-14C]butyrate was found in position 1, especially if the majority of the acetoacetate production is through the 3-HMG pathway. However, the relative enrichment of the acetyl-CoA pool and the acetoacetyl-CoA pool will have a major impact on the results. It is likely that the metabolism of [3-14C]butyrate will be accompanied by a lower specific activity of the acetyl-CoA pool compared with the [1-14C]butyrate because the [3-14C]butyrate will be less likely to deliver a labeled acetyl-CoA to the acetyl-CoA pool compared with [1-14C]butyrate as substrate. The only labeling of the acetyl-CoA pool from [3-14C]butyrate will be through the thiolase-catalyzed acetyl-CoA/acetoacetyl-CoA equilibrium. This implies that the 3 to 1 shift observed with the [3-14C] butyrate incubation indicates a far higher importance of the 3-HMG-CoA pathway than that apparently shown by the 37% of [3-14C]butyrate found in position 1 of acetoacetate simply because the specific activity of acetyl-CoA will be lower under these conditions. Though acetoacetate is the product of rumen epithelial ketogenesis, it is not the primary circulating ketone in plasma. A large proportion of acetoacetate is reduced to D-3-hydroxybutyrate (fig. 4) before leaving the epithelial cells catalyzed by 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30). Data on rumen epithelial enzyme activity and isotopomer distribution in acetoacetate suggest that the 3-HMG pathway is as quantitatively important in this tissue as it is in liver. Earlier denials (Annison et al., 1963) are partly correct in pointing out that butyrate is not completely degraded to acetyl-CoA before incorporation into ketone bodies.

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Fig. 4. D-3-Hydroxybutyrate is the dominating “ketone” in plasma due to the 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) catalyzed and NADH-dependent reduction of acetoacetate.

18. ALTERNATIVE KETOGENIC PATHWAYS Although the 3-HMG pathway is active in rumen epithelium, it might not be the only ketogenic pathway. One obvious alternative would be acetate release by the epithelium. Endogenous acetate production has been observed in vitro when the epithelium is incubated without substrate (Sehested et al., 1999a). The hydrolysis of acetyl-CoA into acetate and CoA is catalyzed by acetyl-CoA deacylase (EC 3.1.2.1; Grigat et al., 1979). We might wonder why the ruminant produces ketone bodies at all when it seems much simpler just to use acetate as a carrier of acetyl units. One of the reasons for the production of ketone bodies could be the cost of reactivation in recipient tissues because they would have to pay double the price for activation when acetate is the substrate compared with acetoacetate. Nevertheless, endogenous acetate production can be observed in vitro by rumen epithelium and acetate would be an obvious candidate for interorgan acetyl transfer. However, we have only limited and indirect evidence of endogenous acetate production by rumen epithelium in vivo (Kristensen et al., 2000a). It is unknown to what extent endogenous acetate from the PDV has a role in interorgan acetyl exchange (i.e. acetyl carbon originally absorbed in fatty acids other than acetate itself). Not only acetyl-CoA, but also acetoacetyl-CoA, might be directly deacylated (acetoacetylCoA deacylase; EC 3.1.2.11), and thereby lead to 3-HMG-CoA-independent acetoacetate synthesis. The acetoacetyl-CoA deacylase has been found in rumen epithelium though only at a low activity (Baird et al., 1970). One of reasons why direct deacylation of acetoacetyl-CoA might be of limited importance is the low acetoacetyl-CoA concentration in the mitochondrion. The low affinity of the acetoacetyl-CoA deacylase present in rat liver was quantitatively not important, although it was functional under in vitro conditions with high acetoacetyl-CoA concentrations (Williamson et al., 1968). A number of alternative pathways have been suggested to explain various parts of ketone body formation in rumen epithelium: succinyl-CoA:3-ketoacid CoA-transferase (Bush and Milligan, 1971); a L-3-hydroxybutyrate pathway not involving acetoacetate formation (Emmanuel et al., 1982); and a butyrate:acetoacetyl-CoA transferase pathway (Emmanuel and Milligan, 1983). These pathways all suggest metabolism of butyrate to 3-hydroxybutyrate as one unbroken C4 unit. The two latter pathways appear to be closely related to cytosolic pathways in tissues utilizing acetoacetate in de novo synthesis of fatty acids (Robinson and Williamson, 1980). However, it is difficult to determine the quantitative importance of non3-HMG-CoA pathways in the rumen epithelium from the limited data available.

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The situation for the succinyl-CoA:3-ketoacid CoA-transferase (EC 2.8.3.5; SCOT) is different because this enzyme is indeed anticipated to be a key enzyme in ketone body metabolism; however, its function is opposite to the function proposed in the rumen epithelium. This enzyme is a key enzyme in the activation of ketone bodies in peripheral tissues and the rare deficiency of this enzyme in human infants leads to severe ketoacidosis (Synderman et al., 1998). Succinyl-CoA:3-ketoacid transferase has been assumed to contribute to the acetoacetylCoA hydrolysis in rumen epithelium because addition of succinate was followed by increased disappearance of acetoacetyl-CoA (Bush and Milligan, 1971). If SCOT was important for the hydrolysis of acetoacetyl-CoA, it would suggest that the concentration of either acetoacetyl-CoA or succinate was higher in rumen epithelium compared with other tissues. However, owing to the true reversibility (Stern et al., 1956) of the reaction catalyzed by SCOT (acetoacetyl-CoA + succinate ↔ acetoacetate + succinyl-CoA), it might be suggested that this activity in the rumen epithelium was connected to the specific incubation conditions in vitro and not necessarily the pathway of acetoacetyl-CoA metabolism in vivo.

19. METABOLITE INTERACTIONS IN RUMEN EPITHELIAL KETOGENESIS If the rumen epithelium works in its usual position in a ruminant, or is maintained for a short period under in vitro conditions as epithelial slices or isolated cells, it will have an obligate requirement for chemical energy to maintain Na+, Ca2+, and K+ ion concentration gradients and other vital cell functions. Considering a situation with a relatively constant workload of the epithelium, it would then be expected that tissue supplied with small amounts of butyrate would oxidize a large fraction to CO2 simply to fulfill the basic needs of ATP and sustain basic cell functions. This relationship has been confirmed in vitro when different butyrate concentrations were compared. Increasing the supply of butyrate was followed by the oxidation of a decreasing fraction and an increasing fraction metabolized into ketone bodies (Beck et al., 1984). From a whole animal perspective, glucose is antiketogenic (Hamada et al., 1982) and initially it was surprising that glucose had the opposite effect on rumen epithelial ketogenesis, i.e. ketogenesis was stimulated by glucose (Stangassinger et al., 1979). A number of glucogenic substrates have been shown to impose a similar effect on epithelial metabolism. Some variability in the response concerning the uptake of butyrate and the proportion of butyrate oxidized has been observed, but generally a shift toward the more reduced “ketone body”, 3-hydroxybutyrate, compared with acetoacetate has been observed with the addition of a glucogenic substrate (Goosen, 1976; Beck et al., 1984; Giesecke et al., 1985; Baldwin and Jesse, 1996). Although the rumen epithelium is able to take up a broad range of metabolites including glucose, glutamine, and glutamate and oxidize them (Harmon, 1986; Baldwin and McLeod, 2000), this does not mean that glucose is the oxidative substrate that caused the shift in ketone body production. In fact, we would surmise from the discussion of butyrate metabolism (see above) that the epithelium had a source of acetyl-CoA from butyrate that would be able to fulfill any oxidative need. The reason might be that epithelium incubated without a glucogenic source will become depleted of TCA cycle intermediates and subsequently have difficulty maintaining ATP, NADH, and NADPH potentials. A very elegant example of this effect is the comparison between metabolite production from butyrate and valerate in rumen epithelium incubated in vitro (Weigand et al., 1975). When rumen epithelium was incubated with butyrate, 0.67 of the ketone bodies produced were acetoacetate; however, when incubated with valerate only 3-hydroxybutyrate was produced. This production was accompanied by

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lactate produced from the 3-carbon fraction of valerate. Therefore, there seems to be no reason to believe that glucose or any other glucogenic substrates play a particular role as regulators of rumen epithelial ketogenesis, but the results point to the general conclusion that rumen epithelium has a range of nutritional requirements for proper function.

20. THE IN VIVO/IN VITRO PROPIONATE ENIGMA In vitro, rumen epithelium metabolizes propionate into lactate (Weigand et al., 1975). In vivo, however, it has not been possible to demonstrate any major propionate metabolism into lactate using ruminal infusion of 14C or 13C labeled propionate (Weigand et al., 1972; Weekes and Webster, 1975; Kristensen et al., 2001). These matters have been even further confused by the fact that in vivo experiments on portal recovery of ruminal propionate indicate that a large proportion of propionate was metabolized by the epithelium (Bergman and Wolff, 1971). Because of the large capacity of the liver to metabolize propionate in vivo (Berthelot et al., 2002) it is difficult to explain why the rumen epithelium should limit the propionate supply to the liver. The reason for the large activation of propionate under in vitro conditions is probably the cross-specificity of the butyryl-CoA synthetase. In vivo, propionyl-CoA could be generated by thiolysis of 3-oxo-valeryl-CoA (from valerate). This latter source might be the explanation for the high capacity of propionyl-CoA-utilizing pathways in rumen epithelium. The usual metabolism of propionate via propionate carboxylation to methylmalonic acid followed by the TCA intermediate succinate will lead to the buildup of TCA intermediates. In the liver the main pathway to export surplus TCA intermediates is gluconeogenesis. Other tissues use nonessential amino acids (e.g. alanine and glutamine synthesis in muscles and other tissues) to control excess TCA intermediates. In rumen epithelium, it is apparently the malic enzyme (EC 1.1.1.40) catalyzed decarboxylation of malate into pyruvate (coupled to reduction of NADP) and the subsequent reduction of pyruvate to lactate that removes the surplus of propionyl-CoA from the rumen epithelial cells (Young et al., 1969). By these mechanisms we are able to explain the differing in vitro and in vivo observations on rumen epithelial metabolism.

21. FITTING THE CARBON BALANCE OF FERMENTATION IN THE GUT Although there is no doubt that SCFA are important in ruminant metabolism, no feed evaluation system has been able integrate knowledge of SCFA production, absorption, and metabolism in ruminants under production conditions. Simulation models constructed to describe fermentation and SCFA absorption, as well as other nutrients, need to improve in order to predict SCFA proportions in the rumen (Baker and Dijkstra, 1999). The problem has also been what to do with the apparently huge metabolic activity of the rumen epithelium. No model has been able to incorporate this metabolism, and this review attempts a possible explanation. Simulation models of ruminal fermentation and metabolism developed to date have been constructed and validated mainly against duodenal nutrient flows. The re-evaluation of the role of the gut epithelia in metabolism of SCFA has enabled an alternative method of model comparison. If the rumen and other gut epithelia do not metabolize significant amounts of acetate and utilize only a small percentage of the propionate flux, then the net rumen production of these acids would be predictable from PDV fluxes. Major corrections to be considered are, however, uptake of arterial acetate by PDV tissues and epithelial butyrate metabolism.

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The percentage of arterial acetate uptake by PDV tissues has been shown to be stable (about 32% of arterial flux) when evaluated across different rations and with relatively little postprandial variation in meal-fed sheep (Bergman and Wolff, 1971; Kristensen et al., 1996a). As discussed earlier, the portal recovery of butyrate has been shown to be a more complex function of its availability, and an increased portal recovery of butyrate with increasing ruminal production rates might be expected. In an attempt to compare data from studies on PDV fluxes with model predictions of gastrointestinal fermentation, Kristensen and Danfær (unpublished data) compared portal fluxes of SCFA from 36 studies in which a total of 58 different diets were fed to sheep and cattle under different physiological conditions (growing/maintenance, nonpregnant/pregnant, dry/lactating). The model used to predict fermentation and digestion of the diets was Karoline (Danfær, 1990) (version 8a, a dynamic simulation model of a lactating cow mainly validated against duodenal flow data; Danfær et al., unpublished). In the model, all diets were compared at a fixed dry matter intake of 20 kg/d and predicted carbon output in moles SCFA carbon per kg dry matter intake was compared to the observed/recalculated PDV fluxes in the studies. The experimentally observed PDV fluxes were corrected for acetate uptake in PDV tissues (assumed 32% of the arterial flux), propionate uptake by epithelial tissues (assuming that portal flux was equal to 95% of true absorption), and butyrate recovery [assuming portal recovery of gut butyrate production = 0.35 × P/(P + 0.05) where P = portal net appearance mmol × h−1 × (kg BW0.75)−1]. The portal recovery of butyrate is deliberately set to a higher level than those typically found following butyrate infusion into the normally functioning rumen. This recovery agrees with observations with sheep maintained on intragastric nutrition or short-term washed reticulo-rumen (Gross et al., 1990a,b; Kristensen et al., 2000a). The calculated SCFA production in the 36 experiments using the correction factors above was 11.9 ± 0.4 moles C in SCFA/kg dry matter intake. The simulated value was 12.3 ± 0.2 moles C in SCFA/kg dry matter intake and the mean bias was 0.4 moles C/kg DMI [Σ(predicted − observed)/number of observations; see Kohn et al. (1998)]. However, the root mean square prediction error (RMSPE) was 2.6 moles C in SCFA/kg DMI [(Σ(predicted − observed)2/ number of observations); see Kohn et al. (1998)]. On average, the model and the corrected experimental data are in good agreement. However, there is still a need for better models to predict net SCFA output. The corrected, experimentally determined SCFA production was, on average, 45 ± 2% of the simulated digestible energy. However, estimates based on intragastric tracer dilution, as discussed above, seem to overestimate the SCFA production and indirect evidence also supports these figures. In fact, the SCFA production accounting for 45% of digestible energy implies that 65% of total digested carbon is found in fermentation gases and SCFA. However, if the true relationship between portal absorption and gut production of SCFA is similar to the relationship described by Bergman and Wolff (1971), then the production of SCFA would need 116 ± 5% of the digested carbon to account for SCFA and fermentation gases. This would seem to be impossible. The good news is that values of portal absorption of SCFA actually make sense in terms of animal energy metabolism. It must be emphasized, however, that models of ruminal and hindgut fermentation still have a lot to gain in terms of precision of SCFA production, especially in the prediction of ruminal SCFA composition.

22. CONCLUSIONS Glucose is a major metabolic fuel for ruminant tissues, similar to most mammals. The pregastric fermentation dictates that gluconeogenesis serves to supply the glucose needs under

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most feeding situations. Lactate derived from the diet, ruminal bacterial metabolism and from endogenous metabolism is a major glucose precursor in ruminants, supplying 9–35% of hepatic glucose carbon, and is a key carbon intermediate in growing ruminants. Ruminants have been shown experimentally to be capable of contributing significant quantities of glucose through intestinal digestion and glucose absorption. This additional glucose does impact growth and retention of body tissues. The gut epithelia have a central function as gatekeepers for butyrate and longer-chain SCFA and MCFA. These acids have also become the main energy substrates of gut epithelia. There is no evidence suggesting that the rumen epithelium should have excessive requirements for energy metabolism, but rather intraruminal (luminal) isotopic dilution techniques overestimate net SCFA production because of microbial metabolism. Data on portal appearance of SCFA corrected for PDV metabolism of arterial metabolites is therefore the best direct measure of SCFA availability in ruminants. The average absorption of SCFA in ruminants is equivalent to about 45% of the digestible energy intake.

23. FUTURE PERSPECTIVES Current feeding systems often fail to meet today’s demand for accurate prediction of the nutrient needs of animals consuming a large menu of feedstuffs under a wide array of environmental conditions. To accomplish this task we need to understand all phases from digestion and nutrient assimilation to the subsequent use of nutrients by various tissues. Thus, successful models in the future will span concepts from commodity to animal product by incorporating the metabolic transformations in between. This chapter has reviewed recent findings on the impact of intestinal digestion on glucose availability, the contributions of lactate to meeting the glucose needs, and how our understanding of SCFA metabolism has been revised from long-accepted concepts. Developing models for predicting animal biological response are dependent on findings such as these to supply quantitative information to describe animal systems. Data are still greatly lacking for concepts as fundamental as SCFA production and glucose absorption, and their metabolism at various stages of growth and production. The near-global de-emphasis on agricultural production research may make these pieces of the puzzle long in coming. REFERENCES Aas, M., 1971. Organ and subcellular distribution of fatty acid activating enzymes in the rat. Biochim. Biophys. Acta 231, 32–47. Annison, E.F., Leng, R.A., Lindsay, D.B., White, B.A., 1963. The metabolism of acetic acid, propionic acid and butyric acid in sheep. Biochem. J. 88, 248–252. Ash, R., Baird, G.D., 1973. Activation of volatile fatty acids in bovine liver and rumen epithelium: evidence for control by autoregulation. Biochem. J. 136, 311–319. Baird, G.D., Hibbitt, K.G., Lee, J., 1970. Enzymes involved in acetoacetate formation in varous bovine tissues. Biochem. J. 117, 703–709. Baker, S.K., Dijkstra, J., 1999. Dynamic aspects of the microbial ecosystem of the reticulo-rumen. In: Jung, H.-J.G., Fahey, G.C. Jr. (Eds.), Nutritional Ecology of Herbivores. American Society of Animal Science, Savoy, IL, pp. 261–311. Balcells, J., Seal, C.J., Parker, D.S., 1995. Effect of intravenous glucose infusion on metabolism of portaldrained viscera in sheep fed a cereal/straw-based diet. J. Anim. Sci. 73, 2146−2155. Baldwin, R.L., Jesse, B.W., 1996. Propionate modulation of ruminal ketogenesis. J. Anim. Sci. 74, 1694–1700. Baldwin, R.L., McLeod, K.R., 2000. Effects of diet forage:concentrate ratio and metabolizable energy intake on isolated rumen epithelial cell metabolism in vitro. J. Anim. Sci. 78, 771–783.

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Reynolds, C.K., Harmon, D.L., Cecava, M.J., 1994. Absorption and delivery of nutrients for milk protein synthesis by portal-drained viscera. J. Dairy Sci. 77, 2787–2808. Reynolds, C.K., Tyrrell, H.F., 1991. Effects of mesenteric vein L-alanine infusion on liver metabolism in beef heifers fed on diets differing in forage:concentrate ratio. Brit. J. Nutr. 66, 437–450. Reynolds, C.K., Tyrrell, H.F., Reynolds, P.J., 1991. Effects of diet forage-to-concentrate ratio and intake on energy metabolism in growing beef heifers: net nutrient metabolism by visceral tissues. J. Nutr. 121, 1004–1015. Ricks, C.A., Cook, R.M., 1981a. Partial purification of enzymes of bovine kidney mitochondria activating volatile fatty acids. J. Dairy Sci. 64, 2344–2349. Ricks, C.A., Cook, R.M., 1981b. Regulation of volatile fatty acid uptake by mitochondrial acyl-CoA synthetases of bovine heart. J. Dairy Sci. 64, 2336–2343. Ricks, C.A., Cook, R.M., 1981c. Regulation of volatile fatty acid uptake by mitochondrial acyl-CoA synthetases of bovine liver. J. Dairy Sci. 64, 2324–2335. Robinson, A.M., Williamson, D.H., 1980. Physiological roles of ketone bodies as substrates and signals in mammalian tissues. Physiol. Rev. 60, 143–187. Rupp, G.P., Kreikemeier, K.K., Perino, L.J., Ross, G.S., 1994. Measurement of volatile fatty acid disappearance and fluid flux across the abomasum of cattle, using an improved omasal cannulation technique. Amer. J. Vet. Res. 55, 522–529. Russell, J.B., Wallace, R.J., 1988. Energy yielding and consuming reactions. In: Hobson, P.N. (Ed.), The Rumen Microbial Ecosystem. Elsevier Applied Science, London, pp. 185–215. Russell, R.W., Gahr, S.A., 2000. Glucose Availability and Associated Metabolism: Farm Animal Metabolism and Nutrition. CABI Publishing, Edinburgh, UK, pp. 121–147. Russell, R.W., Moss, L., Schmidt, S.P., Young, J.W., 1986. Effects of body size on kinetics of glucose metabolism and on nitrogen balance in growing cattle. J. Nutr. 116, 2229–2243. Sander, E.G., Warner, R.G., Harrison, H.N., Loosli, J.K., 1959. The stimulatory effect of sodium butyrate and sodium propionate on the development of rumen mucosa in the young calf. J. Dairy Sci. 42, 1600–1605. Schaadt, H., 1968. Effects of maturity, fermentation time and urea treatment on D(−) and L(+) lactate in corn silage. J. Dairy Sci. 51, 802–805. Schmidt, S.P., Keith, R.K., 1983. Effects of diet and energy intake on kinetics of glucose metabolism in steers. J. Nutr. 113, 2155–2163. Seal, C.J., Parker, D.S., 1994. Effect of intraruminal propionic acid infusion on metabolism of mesenteric- and portal-drained viscera in growing steers fed a forage diet. I. Volatile fatty acids, glucose, and lactate. J. Anim. Sci. 72, 1325–1334. Seal, C.J., Parker, D.S., 2000. Influence of gastrointestinal metabolism on substrate supply to the liver. In: Cronjé, P.B. (Ed.), Ruminant Physiology: Digestion, Metabolism, Growth and Reproduction. CABI Publishing, Wallingford, UK, pp. 131–148. Seal, C.J., Reynolds, C.K., 1993. Nutritional implication of gastrointestinal and liver metabolism in ruminants. Nutr. Res. Rev. 6, 185–208. Sehested, J., Diernæs, L., Møller, P.D., Skadhauge, E., 1999a. Ruminal transport and metabolism of short-chain fatty acids (SCFA) in vitro: effect of SCFA chain length and pH. Comp. Biochem. Physiol. A 123, 359–368. Sehested, J., Diernæs, L., Møller, P.D., Skadhauge, E., 1999b. Transport of butyrate across the isolated bovine rumen epithelium: interaction with sodium, chloride and bicarbonate. Comp. Biochem. Physiol. A 123, 399–408. Sheperd, A.C., Maslanka, M., Quinn, D., Kung, L. Jr., 1995. Additives containing bacteria and enzymes for alfalfa silage. J. Dairy Sci. 78, 565–572. Shirazi-Beechey, S.P., Kemp, R.B., Dyer, J., Beechey, R.B., 1989. Changes in the functions of the intestinal brush border membrane during the development of the ruminant habit in lambs. Comp. Biochem. Physiol. B 94, 801–806. Siciliano-Jones, J., Murphy, M.R., 1989. Production of volatile fatty acids in the rumen and cecum-colon of steers as affected by forage:concentrate and forage physical form. J. Dairy Sci. 72, 485–492. Stangassinger, M., Beck, U., Emmanuel, B., 1979. Is glucose ketogenic in rumen epithelium? Ann. Rech. Vét. 10, 413–416. Stangassinger, M., Giesecke, D., 1979. Quantitative measurement of gluconeogenesis from isobutyrate in sheep. Arch. Int. Physiol. Biochim. 87, 265–274. Stern, J.R., Coon, M.J., Del Campillo, A., Schneider, M.C., 1956. Enzymes of fatty acid metabolism. IV. Preparation and properties of coenzyme A transferase. J. Biol. Chem. 221, 15–31.

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Steven, D.H., Marshall, A.B., 1970. Organization of the rumen epithelium. In: Phillipson, A.T. (Ed.), Physiology of Digestion and Metabolism in the Ruminant. Oriel Press, Newcastle-upon-Tyne, UK, pp. 80–100. Stevens, C.E., 1970. Fatty acid tranport through the rumen epithelium. In: Phillipson, A.T. (Ed.), Physiology of Digestion and Metabolism in the Ruminant. Oriel Press, Newcastle upon Tyne, UK, pp. 101–112. Stevens, C.E., Stettler, B.K., 1967. Evidence for active transport of acetate across bovine rumen epithelium. Amer. J. Physiol. 213, 1335–1339. St. Jean, G.D., Rings, D.M., Schmall, L.M., Hoffsis, G.F., Hull, B.L., 1989. Jejunal mucosal lactase activity from birth to 3 weeks in conventionally raised calves. Amer. J. Vet. Res. 50, 1496–1498. Sutton, J.D., 1985. Digestion and absorption of energy substrates in the lactating cow. J. Dairy Sci. 68, 3376–3393. Synderman, S.E., Sansariq, C., Middleton, B., 1998. Succinyl-CoA:3-ketoacid CoA-transferase deficiency. Pediatrics 101, 709–711. Taniguchi, K., Huntington, G.B., Glenn, B.P., 1995. Net nutrient flux by visceral tissues of beef steers given abomasal and ruminal infusions of casein and starch. J. Anim. Sci. 73, 236–249. Thorlacius, S.O., Lodge, G.A., 1973. Absorption of steam-volatile fatty acids from the rumen of the cow as influenced by diet, buffers and pH. Can. J. Anim. Sci. 53, 279–288. Topping, D.L., Clifton, P.M., 2001. Short-chain fatty acids and human colonic function: roles of resistant starch and nonstarch polysaccharides. Physiol. Rev. 81, 1031–1064. Van Maanen, R.W., Herbein, J.H., McGilliard, A.D., Young, J.W., 1978. Effects of monensin on in vivo rumen propionate production and blood glucose kinetics in cattle. J. Nutr. 108, 1002–1007. Varga, G.A., Tyrrell, H.F., Huntington, G.B., Waldo, D.R., Glenn, B.P., 1990. Utilization of nitrogen and energy by Holstein steers fed formaldehyde- and formic acid-treated alfalfa or orchardgrass silage at two intakes. J. Anim. Sci. 68, 3780–3791. Vessy, D.A., Kelly, M., Warren, R.S., 1999. Characterization of the CoA ligases of human liver mitochondria catalyzing the activation of short- and medium-chain fatty acids and xenobiotic carboxylic acids. Biochim. Biophys. Acta 1428, 455–462. Webster, L.T. Jr., Gerowin, L.D., Rakita, L., 1965. Purification and characteristics of a butyryl coenzyme A synthetase from bovine heart mitochondria. J. Biol. Chem. 240, 29–33. Weekes, T.E.C., 1974. The in vitro metabolism of propionate and glucose by the rumen epithelium. Comp. Biochem. Physiol. B 49, 393–406. Weekes, T.E.C., Webster, A.J.F., 1975. Metabolism of propionate in the tissues of the sheep gut. Brit. J. Nutr. 33, 425–438. Weigand, E., Young, J.W., McGilliard, A.D., 1972. Extent of propionate metabolism during absorption from the bovine ruminoreticulum. Biochem. J. 126, 201–209. Weigand, E., Young, J.W., McGilliard, A.D., 1975. Volatile fatty acid metabolism by rumen mucosa from cattle fed hay or grain. J. Dairy Sci. 58, 1294–1300. Williamson, D.H., Bates, M.W., Krebs, H.A., 1968. Activity and intracellular distribution of enzymes of ketone-body metabolism in rat liver. Biochem. J. 108, 353–361. Young, J.W., 1976. Gluconeogenesis in cattle: significance and methodology. J. Dairy Sci. 60, 1–15. Young, J.W., Thorp, S.L., De Lumen, H.Z., 1969. Activity of selected gluconeogenic and lipogenic enzymes in bovine rumen mucosa, liver and adipose tissue. Biochem. J. 114, 83–88.

PART V Methodology

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Methodological approaches to metabolism research X. Guan and D. G. Burrin USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA

Advances in molecular biology and stable isotope techniques during the last decade have led to an explosion of research aimed at understanding the biological basis of metabolomics from the level of systemic physiology, to intermediary metabolism, to molecular regulation of critical proteins, and on down to genomic expression. We shall highlight principles, approaches, and applications of these cutting-edge molecular and metabolic techniques.

1. GENETICALLY ENGINEERED ANIMAL MODELS FOR METABOLISM RESEARCH With the development of transgenic technologies (gene overexpression, knockout, and conditional expression), one is able to explore physiological roles and metabolic functions of specific genes and to identify individual proteins involved in the control of specific aspects of metabolism. 1.1. Transgenic techniques Conventional transgenic technologies (gene overexpression and knockout) are invaluable for modeling genetic disorders and addressing developmental questions. However, this “all or nothing” mode is inflexible and cannot be used to fully answer subtle metabolic questions. In order to obtain precise information about the roles of a specific gene in a specific cell type at a critical stage of disease or development, conditional transgenic techniques that allow flexible spatial and temporal control of gene deletion or expression in transgenic animals must be used (Ryding et al., 2001). In these systems, the switching “on” or “off” of the expression of a particular gene is conditional upon exposure to a specific stimulus (Ryding et al., 2001). Three approaches have been used to inhibit specific gene expression in mammalian systems. First, the most common approach is specific gene ablation by homologous recombination in embryonic stem (ES) cells (Bronson and Smithies, 1994) and then reproduction of animals

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without expression of the specific gene. Targeted gene disruption by the homologous recombination technique takes advantage of the fact that pluripotent ES cells derived from mouse blastocysts can be cultured in vitro and remain viable for differentiation after injection into a different embryo. The most commonly used ES cells are those derived from mice that have an agouti coat color. These ES cells can be microinjected into embryos obtained from mice that have a black coat color. Offspring with a high degree of agouti coat color, indicating the transmission of ES cell-derived genes, can then be crossbred to obtain mice with a genetic background identical to that of the ES cells. Therefore, mice with specific gene modifications can be obtained by manipulation of the ES cell genome. Modification of specific genes in the ES cell genome depends on the ability of transfected DNA to recombine with the homologous gene in the chromosome. Isogenic DNA for the targeting construct is used to maximize hybridization of the targeting DNA to the proper gene locus in the chromosome. A selectable gene marker, such as the neomycin-resistant gene, is inserted into an exon to disrupt the coding sequence of the gene of interest. The chimeric targeting gene construct is used to transfect ES cells. Homologous recombination of the transfected DNA with chromosomal DNA at the target locus results in the replacement of a portion of the endogenous gene with the targeting construct, thus disrupting the coding sequence and inactivation of the endogenous gene. The use of the selectable gene marker allows the selection for cells that have taken up and expressed the transfected DNA. Growth of the ES cells in the presence of antibiotic indicates the integration of the transfected DNA into the ES cell genome. However, there are two limitations in this approach: the low rate of homologous recombination in mammalian cells and the high rate of random (nontargeted) integration of the vector DNA. Chimeric nucleases and triplexforming oligonucleotides may increase homologous recombination and decrease random integration in cells (Vasquez et al., 2001). Secondly, anti-sense DNA or RNA that inhibits gene expression by complementation to single-stranded mRNA (Izant and Weintraub, 1985), and trans-splicing ribozymes (Kohler et al., 1999) that catalyze RNA hydrolysis in a sequence-specific manner, have been used successfully to abolish gene expression in mammalian cells. Anti-sense RNA is useful for suppressing the expression of specific genes in vivo. The anti-sense plasmid construct can be introduced into eukaryotic cells by transfection or microinjection. Anti-sense transcripts complementary to 5′ untranslated target gene mRNA specifically suppress gene activity or direct against the protein-coding domain alone. Recently, trans-splicing ribozymes have been employed to repair mutant mRNAs in vivo. These trans-splicing ribozymes contain catalytic sequences derived from a self-splicing group I intron, which have been adapted to a chosen target mRNA by fusion of a region of extended complementarity to the target RNA and precise alteration of the guide sequences required for substrate recognition. The improved trans-splicing ribozymes may be tailored for virtually any target RNA, and provide a new tool for triggering gene expression in specific cell types. Thirdly, RNA interference (RNAi), an evolutionarily conserved pathway, uses these small interfering RNAs to degrade mRNAs before translation. Recently, RNAi has emerged as a specific and efficient method to silence gene expression in mammalian cells and to probe gene function on a whole-genome scale either by transfection of short interfering RNAs or by transcription of short hairpin RNAs (Hammond et al., 2001; Hannon, 2002; McCaffrey et al., 2002). Short interfering RNAs typically consist of two 21-nucleotide (nt) single-stranded RNAs that form a 19-bp duplex with 2-nt 3′ overhangs. Its antisense strand is used by an RNAi silencing complex to guide mRNA cleavage, so promoting mRNA degradation. It is certain that the ability of RNAi technology to silence specific genes will transform future studies of cellular systems and biology in mammalian cells (McManus and Sharp, 2002).

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1.2. Applications of genetically engineered animal models The use of nitric oxide synthase (NOS) gene knockout animals has helped elucidate the roles of different NOS isoforms in the synthesis and function of nitric oxide. Nitric oxide from neuronal NOS is a major inhibitory neurotransmitter; nitric oxide from endothelial NOS regulates blood flow under physiological conditions; and nitric oxide from inducible NOS causes hypotension during severe inflammatory conditions. Moreover, nitric oxide from each isoform has unique roles in tissue injury and inflammation. The neuronal NOS-deficient mice develop gastric dilatation and stasis; the endothelial NOS-deficient mice develop hypotension and lack vasodilatory responses to injury; and inducible NOS-deficient mice are more susceptible to inflammatory damage but more resistant to septic shock (Mashimo and Goyal, 1999). This example clearly demonstrates the enormous potential of genetically engineered mice lacking specific genes in elucidation of mechanisms specific in physiology and pathology. Another example is that gene silencing by siRNAs has provided insights into insulin regulation of glucose uptake and glycogen synthesis. The serine/threonine protein kinase Akt has been proposed to mediate insulin signaling in several processes. However, it is unclear if Akt is involved in insulin-stimulated glucose uptake, and which isoforms of Akt are responsible for each insulin action. Recently, experiments with isoform-specific siRNA have revealed that Akt2, and Akt1 to a lesser extent, has an essential role in insulin-stimulated glucose transporter-4 translocation and 2-deoxyglucose uptake in both Chinese hamster ovary cells and 3T3-L1 adipocytes, while Akt1 and Akt2 contribute equally to insulin-stimulated glycogen synthesis. These data suggest a prerequisite role of Akt in insulin-stimulated glucose uptake and distinct functions among Akt isoforms (Katome et al., 2003).

2. GENE EXPRESSION TECHNIQUES FOR METABOLISM RESEARCH The transcription of genomic DNA to produce mRNA is the first step in the process of protein synthesis, and differences in gene expression are indicative of cellular responses to environmental stimuli and perturbations and are responsible for both morphological and phenotypic differences between tissues and stages of development. Knowing when, where, and to what extent a gene is expressed is central to understanding the activity and biological roles of its encoded protein. In addition, changes in the multigene patterns of expression can provide clues about regulatory mechanisms and broader cellular functions and biochemical pathways (Lockhart and Winzeler, 2000). 2.1. Gene expression techniques There are many techniques for measuring gene expression. Both conventional methods (including Northern blots, RNase protection assay, in situ hybridization, and RT-PCR) and DNA microarrays have been employed to measure expression levels of specific genes, to characterize global expression profiles, and to screen for differences in mRNA abundance. These conventional methods are simply used in a more targeted fashion to follow up on the specific genes, pathways, and mechanisms implicated by the microarrays. 2.1.1. DNA microarray analysis for screening global gene expression profile DNA microarrays are a miniaturized, ordered arrangement of nucleic acid fragments from individual genes located at defined positions on a solid support, enabling the expression analysis of

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thousands of genes in parallel by specific hybridization (Arcellana-Panlilio and Robbins, 2002). This technology is a powerful tool for rapid, comprehensive, and quantitative analysis of global gene expression profiles of normal/disease statuses and developmental processes (Bednar, 2000; Lockhart and Winzeler, 2000). In general, there are two kinds of DNA microarrays: cDNA arrays and oligonucleotide arrays. For cDNA arrays, the nucleic acid fragments are spotted robotically onto a glass slide. The cDNA used for spotting are usually derived by PCR amplification of cDNA libraries. For oligonucleotide arrays, oligonucleotides are synthesized in situ by photolithography. Gene expression analysis using DNA microarrays is based on the competitive hybridization of differently labeled populations of cDNAs. Fluorescent dyes, usually Cy3 and Cy5, are used to label cDNA pools reverse transcribed from different mRNA samples (prepared from tissues or cells). The labeled cDNAs are applied to the microarray and allowed to simultaneously hybridize under conditions analogous to those established for Southern blotting. After washing off nonspecific hybridization, the slide is read in a confocal laser scanner that can differentiate between Cy3 and Cy5 signals. Because hybridization is governed by the recognition rules, the signal intensity at each position gives not only a measurement of the number of molecules bound, but also the likely identity of the molecules. Thus, the relative intensity of Cy5/Cy3 signal for each gene is used to assess the relative abundance of a specific mRNA. It should be noted that the extent of hybridization on a DNA microarray is influenced by time, concentration of solution-phase cDNA probes, and length of the arrayed DNA sequences (Stillman and Tonkinson, 2001). 2.1.2. mRNA quantitative techniques for measuring specific gene expression 2.1.2.1. Northern blotting analysis The Northern blotting analysis separates RNA species on the basis of size by denaturing gel electrophoresis followed by transfer of the RNA onto a nylon membrane by capillary, vacuum, pressure, or electrical-assisted blotting. The RNA is then irreversibly bound to the membrane by exposure to short-wave ultraviolet light or by heating at 80°C in a vacuum oven. The RNA sequences of interest are detected on the membrane by hybridization to a specific labeled probe. Probes for Northern blot detection generally contain full or partial cDNA sequences and may be labeled by enzymatic incorporation of radiolabeled (32P) nucleotides or with nucleotides conjugated to haptens such as biotin or digoxigenin. After washing off the unbound and nonspecifically bound probe, the hybridization signal is generally revealed by autoradiography or immunological detection after antibody incubation. Autoradiograph band intensities may be quantified by densitometry, by direct measurement of hybridized radiolabeled probe via storage phosphor imaging, or by scintillation counting of excised bands (for the technique in detail, see Rapley and Walker, 1998). The band identified by the probe indicates the size of the mRNA, and the intensity of the band corresponds to the relative abundance. The Northern blotting analysis can detect the steady-state level of a specific mRNA sequence in the sample. Association of the mRNA expression and the metabolic/physiological state provides important clues regarding gene regulation, developmental characterization, and responsiveness to stimuli. The abundance of mRNA is controlled by three major factors: gene transcription, mRNA processing and transport, and mRNA stability. More sensitive methods can be used for the analysis of rare transcripts including RT-PCR and RNase protection assay. However, the Northern blotting analysis is the only method that can determine mRNA size. In general, this method is semi-quantitative if a standard is used, and is suitable for determining relative abundances of mRNA species. To compare the relative abundances, each sample on a membrane must be hybridized with a probe for the specific mRNA of interest and

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a probe for an endogenous internal control. Constitutively expressed “housekeeping” genes, such as β-actin, cyclophilin, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and a constant level of 18S rRNA are used as the internal control. Variations of the Northern blotting technology, such as dot blots, slot blots, and fast blots, have been developed to simplify blot preparation and improve quantitative analysis. These techniques involve applying an RNA sample (dot and slot blots) or cell extract (fast blot) directly to the membrane without prior size fractionation on a gel. It is imperative that the probe used for dot/slot blot analyses is specific for the target mRNA without cross-hybridization or nonspecific hybridization to other sequences. 2.1.2.2. RNase protection assay Ribonuclease (RNase) protection assay is a technique used for detection and quantitative analysis of specific RNAs. In principle, cellular mRNA is hybridized with a gene-specific labeled single-stranded complementary RNA (labeled with 32P). After the hybridization, all unbound single-stranded RNA molecules are degraded by single-stranded-specific ribonuclease. The protected double-stranded (i.e. hybridized) fragments are separated by denaturing polyacrylamide gel electrophoresis, detected by exposure on x-ray film, and quantified by densitometry, or quantified by excising and scintillation counting the region of the gel that contain the protected fragments. The size of each protected fragment may be derived from the standard marker and the intensity of the bands directly corresponds with the absolute concentration of the specific mRNA. Unlike Northern blots, the size of product by the RNase protection assay does not depend on the size of the target mRNA, but on the size of the probe used in the assay. This assay can determine absolute abundance of mRNA at relatively high sensitivity (Reue, 1998). 2.1.2.3. In situ hybridization In situ hybridization (ISH) technique allows specific nucleic acid sequences to be detected in morphologically preserved chromosomes, cells, or tissue sections. In combination with immunocytochemistry, this technique can relate microscopic topological information to gene activity at the DNA, mRNA, and protein level. Localization of gene expression at the mRNA level is particular important to confirm the identity of cells expressing soluble or secreted proteins. Currently, nonradioactive labeled cRNA probes have become more feasible for detecting target mRNA in tissue sections. For example, digoxigenin (DIG)-labeled nucleotides may be incorporated at a defined density into nucleic acid probes by DNA polymerases, RNA polymerases, or terminal transferase. Usually, cRNA probes are generated by in vitro transcription from a linearized DNA template. Hybridized DIG-labeled probes may be detected with high-affinity anti-DIG antibodies that are conjugated to alkaline phosphatase (AP) or horseradish peroxidase. The antibodies conjugated to AP can be visualized with colorimetric or fluorescent AP substrates. The advent of the tyramide signal amplification (TSA) method has dramatically increased the sensitivity of nonradioactive ISH detection. Tyramide signal amplification is based on the horseradish peroxidase-catalyzed deposition of labeled tyramide molecules at sites of probe binding. In contrast, typical AP substrates precipitate diffusely at sites of AP activity. Dual fluorescent ISH and immunohistochemistry using TSA has provided a rapid and sensitive method to compare mRNA and protein localization (Zaidi et al., 2000), which offers the ability to distinguish between the cells responsible for production of the protein and its target cells. 2.1.2.4. Quantitative RT-PCR The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low abundance of steady-state mRNA (Wang and Brown, 1999). The RT-PCR is an in vitro method for enzymatically amplifying defined sequences of RNA and permits the analysis of different samples from as little

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as one cell. There are four types of RT-PCR: relative, competitive, comparative, and real-time RT-PCR. The first step in the RT-PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR. The reverse transcription step can be primed using specific primers, random hexamers, or oligo-dT primers. In theory, PCR should detect the cDNA derived from a single mRNA molecule; but in practice, ten or more mRNA copies are required because of the relative inefficiency of the reverse transcriptase reaction required to convert mRNA to cDNA for subsequent amplification. Typically, a small amount of total RNA (1 μg or less) is used for reverse transcription, and a fraction (1/20 to 1/50) of the resulting cDNA is used in the PCR. The cDNA is amplified exponentially via cycles of denaturation, annealing, and extension. Amplification products initially appear at undetectable levels, then accumulate at a nonlinear rate within an exponential phase, and eventually reach similar levels irrespective of initial template concentration. Thus, quantitative comparisons must be made during the exponential phase. One strategy to ensure that PCR products are analyzed within the exponential phase of the amplification reaction is to examine products at progressive cycles during the reaction. This may be accomplished by the use of real-time quantitative RT-PCR, wherein the whole reaction is monitored rather than just the end product. Real-time RT-PCR employs a fluorescent signal to report formation of PCR product as each cycle of the amplification proceeds, coupled with an automated PCR/fluorescent detection system (Heid et al., 1996). For absolute quantitative analysis of a target mRNA, an internal control template and corresponding control probe with a unique reporter fluorescent dye is included in each reaction tube (Gibson et al., 1996; for a review, see Bustin, 2000). It should be noted that real-time RT-PCR quantifies steady-state mRNA levels, which tells the researcher nothing about either transcription levels or mRNA stability (Bustin, 2002). 2.1.2.5. The method of choice The choice of mRNA quantitative analysis method is dependent on the study of interest. (1) The Northern blotting analysis is the first step in the characterization of mRNA expression as it allows visualization of intact mRNA. That is the only method providing information about the mRNA size, alternative splicing, and the integrity of the RNA. That also allows great flexibility, as the probe used for hybridization does not require preparation with specific cloning vectors or primers. (2) The RNase protection assay is the most useful for mapping transcript initiation and termination sites and intron/exon boundaries, and for discriminating between related mRNAs of similar size, which would migrate at similar positions on a Northern blot. (3) In situ hybridization is the most complex method of all, but is the only one that allows localization of transcripts to specific cells within a tissue. (4) In term of sensitive, specific, and reproducible quantification of mRNA, real-time RT-PCR is the method of choice (Bustin, 2000). The RNase protection assay and real-time RT-PCR are most readily applied to the analysis of mRNAs that have been previously characterized and sequenced, as they require production of specific vectors and primers for probe and control template preparation (Reue, 1998).

2.2. DNA binding assays for assessing DNA–protein interactions It has been known that, at the simplest level, transcription of genes into mRNAs is governed by transcription factors, which bind to cis-regulatory regions of the DNA in the vicinity of the target gene. The current challenges, however, include an understanding of (1) which specific cis-acting DNA sequence elements and which trans-acting factors (transcription factors) are required for the expression of a given gene; (2) how a given set of DNA–protein interactions

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regulates the expression of a tissue-specific gene; and (3) how these interactions are integrated into the overall regulation of gene expression during development (Yang, 1998). The highly specific interaction between a given transcription factor and its recognized binding sequence (DNA) forms the basis for the biochemical characterization, and provides insight into the overall molecular mechanisms controlling gene expression. 2.2.1. Electrophoretic mobility shift assay (EMSA) This assay consists of three steps. (1) A DNA-binding protein (present in a nuclear extract) is mixed with a 32P-labeled DNA fragment (probe). (2) The DNA–protein complexes migrate more slowly than free, unbound DNA during electrophoresis. (3) The two bands containing radiolabeled DNA are detected by autoradiography or a phosphor screen. In general, each additional protein binding to a DNA–protein complex alters its electrophoretic mobility and results in an additional retarded band. The EMSA is sufficiently sensitive for the binding of a monoclonal antibody to the protein–DNA complex to cause a supershift band, which can confirm the presence of a particular protein in the complex. Though it provides a quantitative measurement of the amount of a particular DNA binding activity, the EMSA does not give a direct readout of the DNA nucleotides that the protein recognizes. 2.2.2. DNase I protection (footprinting) assay A specific binding protein (in a nuclear extract) binds to a specific region within a singly end-labeled DNA fragment (probe). After digestion by DNase I, the DNA products are electrophoresed in a denaturing polyacrylamide gel. In the absence of any binding protein, the bands appear as a ladder without any interruption. However, in the presence of the specific binding protein, some bands disappear because DNase I cannot digest the region of DNA bound by the protein. This assay allows the determination of a short stretch of a protein-binding site within a relatively large DNA fragment. The exact nucleotide sequence in the protected region can readily be determined by concurrently running sequencing reactions of the same DNA fragment alongside the DNase I digestion products. 2.2.3. Chromatin immunoprecipitation (ChIP) assay An issue in gene transcription is the in vivo relevance of transcription factor binding sites that have been identified in vitro. The ChIP assay is being successfully exploited to confirm in vivo binding sites of specific transcription factors. In this assay, an antibody to a specific DNA-binding protein is used to immunoprecipitate cross-linked protein–DNA complexes. Then, the DNA is experimentally released from the complexes and detected by DNA footprinting (Lee Kang et al., 2002) or DNA microarray (Weinmann et al., 2002). In combination with the DNA microarray, the ChIP assay is used to probe the genome-wide pattern of DNA binding sites for specific transcription factors (Weinmann et al., 2002). Moreover, this technique can distinguish the direct targets of the transcription factors from indirect downstream effects (Shannon and Rao, 2002). 2.3. Applications: nutrient regulation of gene expression Effects of nutrition can be exerted at many stages between transcription of the genetic sequence and translation of a functional protein. Nutrients can influence gene expression

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through control of the regulatory signals in the untranslated regions of the gene (Hesketh et al., 1998). In the research of nutritional control of gene expression, it is important not only to focus on regulation through gene promoter regions but also to consider the possibility of post-transcriptional control (Hesketh et al., 1998). An example of nutrient regulation of gene expression is that the polyunsaturated fatty acid (PUFA) upregulates the expression of genes encoding proteins involved in fatty acid oxidation while simultaneously downregulating the expression of genes encoding proteins involved in lipid synthesis. The PUFAs appear to regulate gene transcription by modifying the DNA binding activity and/or the nuclear abundance of the transcription factors (Clarke, 2001). Furthermore, PUFAs govern the expression of enzymes in lipid oxidation and lipid synthesis by two independent mechanisms: activating peroxisome proliferator-activated receptor α (Clarke, 2001) and suppressing sterol regulatory element binding protein-1 (Xu et al., 1999, 2001). Duplus et al. (2000) have postulated multiple mechanisms for fatty acid control of gene transcription. One of them is that the fatty acid itself or its derivative acts as a ligand for a transcription factor, which then can bind to DNA at a fatty acid response element in the fatty acid-responsive gene and activate or repress transcription (Duplus et al., 2000). Another example is that amino acid availability regulates the expression of genes encoding proteins in the control of growth (Fafournoux et al., 2000). Limitation of several amino acids greatly increases the expression of a specific gene encoding the CHOP protein, a stressinducible nuclear protein that dimerizes with members of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors (Bruhat et al., 1997; Fafournoux et al., 2000). Elevated abundance of CHOP mRNA results from an increased rate of its transcription and an increased stability of its mRNA (Bruhat et al., 1997, 1999). The C/EBP family is involved in the regulation of processes relevant to gene expression, energy metabolism, cellular proliferation, and differentiation (Roesler, 2001). By forming heterodimers with members of the C/EBP family, the CHOP protein either as an inhibitor or an activator can influence expression of cell type-specific genes (Ubeda et al., 1996; Sok et al., 1999). In the promoter region of the CHOP gene, an amino acid response element (AARE) is found to bind in vitro the activating transcription factor 2, which is essential for leucine-induced transcriptional activation of the CHOP gene (Bruhat et al., 2000). Further work will be necessary to characterize the molecular steps by which the cellular amino acid availability can regulate gene expression, particularly to determine (1) the pattern of the AARE in the regulated genes; (2) the nature of the protein complexes bound to these elements; (3) the identity of the intracellular metabolites that mediate transcriptional activation by amino acid limitation; and (4) the signaling pathways involved in the control of translation by amino acids (Fafournoux et al., 2000). These studies will eventually provide insight into the role of amino acids in the regulation of cellular functions such as protein synthesis and proteolysis (Bruhat et al., 2002).

3. PROTEIN ABUNDANCE, ACTIVITY, AND LOCALIZATION Molecular mechanisms that govern cellular function and metabolism are controlled largely by the structure and function of genetically encoded products, the proteins. Post-transcriptional processing of mRNA and co-/post-translational processing of proteins lead to a fair degree of discordance between the open reading frames predicting protein structure and the actual functional product (Witzmann and Li, 2002). Consequently, it is necessary to quantitatively

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measure differential expression at the protein level. Moreover, many protein-mediated cellular functions are managed and regulated through mechanisms that do not even involve quantitative changes in protein expression. Instead, they are the consequences of protein interactions and chemical modifications of existing proteins (e.g. phosphorylation and glycosylation). It is essential to characterize these changes of the proteins using functional and structural proteomics. Finally, the localization of gene products, which is often difficult to predict from the DNA sequence, can be determined experimentally only at the protein level (Pandey and Mann, 2000).

3.1. Two-dimensional gel electrophoresis for screening protein expression profiles Two-dimensional (2D) gel electrophoresis/mass spectrometry can be used to visualize differential protein expression. In the 2D electrophoresis, proteins are subjected to orthogonal separation methods, the first based on protein charge via isoelectric focusing and then by mass in sodium dodecyl sulfate. The final product of the 2D electrophoresis separation is essentially an in-gel array of proteins, each assuming a coordinate position corresponding to the unique combination of isoelectric point and mass. Protein expression patterns are visualized by a number of staining methods such as fluorescent staining image analysis. Finally, the identity of the protein(s) in each spot is characterized by liquid chromatography–mass spectrometry (fig. 1).

Fig. 1. A schematic showing the two-dimensional gel approach. Cells (or tissue) derived from two different conditions, A and B, are harvested and the proteins solubilized. The crude protein mixture is then applied to a “first dimension” gel strip that separates the proteins based on their isoelectric points. After this step, the strip is subjected to reduction and alkylation and applied to a “second dimension” SDS–PAGE gel where proteins are denatured and separated on the basis of size. The gels are then fixed and the proteins visualized by staining methods. After staining, the resulting protein spots are recorded and quantified. The spots of interest are then excised and subjected to mass spectrometric analysis; Reproduced with permission from Pandey and Mann (2000).

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3.2. Protein microarrays for screening protein expression profiles Protein microarrays are being developed for high-throughput analysis of protein expression. There are two types of protein microarrays. One type of protein array is termed a protein function array, and consists of thousands of native proteins, recombinant proteins, or their domains immobilized in a defined pattern, which can be used to examine protein function (e.g. enzymatic activity or binding property). This type of array is incubated with a cell lysate containing putative interaction partners. After washing away unbound material, the bound proteins are eluted and then identified by mass spectrometry (Pandey and Mann, 2000). The other type of protein array is termed a protein-detecting array and consists of large numbers of protein-binding agents, which allows for screening protein expression profiles under various physiological stimuli (Kodadek, 2001). For example, based on antibody–antigen interactions, proteins isolated from cells in a particular physiological state are bound to an array containing specific antigens or antibodies. The extent of the specific binding is then detected by the fluorescence assay (Haab et al., 2001) or by the enhanced chemiluminescence assay (Huang, 2001).

3.3. Western blot analysis for measuring specific protein expression In Western blotting, a complex protein fraction is separated by electrophoresis and the proteins are transferred to a PVDF or nitrocellulose membrane, which is then hybridized with a primary antibody and visualized using a secondary antibody conjugated with horseradish peroxidase or alkaline phosphatase (Rapley and Walker, 1998).

3.4. Enzyme-linked immunosorbent assay (ELISA) for measuring specific protein activity In the ELISA, the antigen to be detected, being passively attached to the plastic surface of microplate wells, binds specifically to an antibody conjugated with an enzyme used for detection (e.g. horseradish peroxidase or alkaline phosphatase). The antigen–enzyme linked antibody complex is then reacted with a substrate/chromophore. The rate of color change, resulting from substrate metabolism by the enzyme, is proportional to the amount of enzyme in the complex. Many modified ELISAs have been developed to detect and quantify specific proteins (Rapley and Walker, 1998). 3.5. Confocal laser scanning microscopy (CLSM) for visualizing specific protein location The CLSM captures only the light coming immediately from the object point in focus and obstructs the light coming from out-of-focus areas of the sample. A laser beam is concentrated on a very small spot and then scans the sample in the X–Y direction. As a result, the part corresponding to the eliminated light is darkened in the image, making it possible to optically slice a thick tissue sample. It detects the fluorescence or transmits light from the sample, and displays an image on the monitor. The CLSM has high contrast and superior resolution in the light axis direction because of the use of confocal optics. In combination with immunohistochemistry, the CLSM provides specific information about protein expression patterns at the single-cell level and may indicate molecular changes relevant to metabolism.

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4. DNA AND RNA LABELING TECHNIQUES FOR MEASURING CELL PROLIFERATION AND APOPTOSIS IN VIVO 4.1. Cell proliferation 4.1.1. Bromodeoxyuridine (BrdU) labeling assay During cell proliferation, the DNA has to be replicated before the cell is divided into two daughter cells (Sawada et al., 1995). Because of the positive relation between fractional rate of DNA synthesis and proportion of new cells by counting (Macallan et al., 1998), the measurement of DNA synthesis is very attractive for assessing cell proliferation. Therefore, cell proliferation has been assayed by measuring incorporation of radiolabeled nucleosides (e.g. [3H]thymidine) into DNA. The amount of [3H]thymidine incorporated into the DNA is quantified by liquid scintillation counting. To avoid radioactivity hazards, a method of nonradioactive labeling of the DNA with 5-bromo-2-deoxyuridine (BrdU, a thymidine analogue) has been developed for measuring cell proliferation. It has been shown that the BrdU, like thymidine, is incorporated into cellular DNA. The incorporated BrdU during DNA synthesis could be detected by an enzyme immunoassay using monoclonal antibodies directed against BrdU, and used to quantify cell proliferation (Maghni et al., 1999). 4.1.2. Stable isotopic tracer incorporation methods DNA synthesis and breakdown have been measured by labeling DNA with pyrimidine deoxyribonucleosides (e.g. [3H]thymidine or BrdU); these techniques can be confounded by physiological factors other than the rates of cell proliferation and death per se (Hellerstein, 1999) and cannot be used safely in humans (Neese et al., 2002). Macallan et al. (1998) and Martini et al. (2002) have developed a stable isotopic tracer incorporation method for measuring DNA synthesis by labeling the deoxyribose moiety of purine deoxyribonucleotides through the de novo nucleotide synthesis pathway using [2H]glucose or [U-13C6]glucose or 2H O (Macallan et al., 1998; Martini et al., 2002; Neese et al., 2002). It allows measurement 2 of stable isotope incorporation into DNA and calculation of cell proliferation and death rates in vivo in humans and animals (Hellerstein et al., 1999; Neese et al., 2001, 2002). This method counts cell divisions by measuring the proportion of labeled DNA strands present assuming that each cell division in the presence of label generates two labeled DNA strands (one in each daughter cell) (Hellerstein et al., 1999). Compared to BrdU or [3H]thymidine labeling techniques, there are three differences (Macallan et al., 1998): (1) This method labels deoxyribonucleotides in DNA through the de novo nucleotide synthesis pathway instead of the nucleoside salvage pathway. The pathways for labeling of DNA are illustrated in fig. 2. The efficiency of de novo contribution to purine nucleosides is predictable and high in dividing cells. The activity of the de novo pathway for purine nucleosides is relatively unaffected by extracellular nucleoside concentrations and derives almost entirely from extracellular glucose, so that the precursor–product relationship can be used in a predictable way across cell types (Macallan et al., 1998; Hellerstein, 1999). (2) This method measures labeling in purine deoxyribonucleosides instead of pyrimidines (e.g. from [3H]thymidine or BrdU) (Macallan et al., 1998). (3) BrdU is a pyrimidine nucleoside that is used by the nucleoside salvage pathway and incorporated into DNA as a thymidine analogue. The efficiency of the pyrimidine nucleoside salvage pathway is variable and influenced by availability of extracellular nucleosides (Hellerstein, 1999). Moreover, BrdU does not truly quantify mitotic events, but rather labels descendants of dividing cells (Hellerstein, 1999).

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Fig. 2. Labeling pathways for measuring DNA synthesis and thus cell proliferation (adapted from Neese et al., 2002). GNG, gluconeogenesis; G6P, glucose-6-phosphate; R5P, ribose 5-phosphate; PRPP, phosphoribose pyrophosphate; NDP, nucleoside diphosphate; DNNS, de novo nucleotide synthesis pathway; DNPS, de novo purine/pyrimidine synthesis pathway; RR, ribonucleotide reductase; dNTP, deoxyribonucleoside triphosphate; dN, deoxyribonucleosides; dT, thymidine deoxyribonucleoside; BrdU, bromodeoxyuridine.

In particular, the incorporation of 2H2O into the deoxyribose moiety in newly synthesized DNA allows safe, convenient, reproducible, and inexpensive measurement of in vivo proliferation rates of slow-turnover cells in humans (Neese et al., 2002). The fractional production rate of dividing cells (k, per day) can be calculated based on the precursor–product relationship provided that blood glucose reproducibly provides about 65% of the deoxyribose present in purine nucleotides recovered from DNA of various dividing cells (Macallan et al., 1998; Hellerstein et al., 1999). Therefore, k = −In (1 − (IEd2-dA/IEd2-glucose × 0.65))/t where IEd2-dA and IEd2-glucose stand for isotopic enrichment of [2H2]deoxyadenosine and [2H2]glucose, respectively The absolute production rate of the specific-type cells can be derived by multiplying the k with their pool size (cells/μl). The half-life (survival time) is indicated by dividing 0.63 by k. 4.2. Cell apoptosis 4.2.1. TUNEL method The fragmentation of nuclear DNA is one of the endpoints in apoptotic pathways. DNA fragmentation can be determined by electrophoresis. However, an in situ labeling DNA method has been developed to quantify the DNA fragmentation on the basis of the terminal deoxynucleotidyl transferase enzyme reaction after adding deoxynucleotides labeled with biotin or digoxigenin to free 3′-ends of DNA fragments. Therefore, the formation of a DNA strand break early in apoptosis is detected by enzymatic labeling of the 3′-OH termini with modified nucleotides, which is visualized with streptavidin or anti-digoxigenin antibodies. This method is called terminal deoxynucleotidyl transferase nucleic acid end labeling (TUNEL). However, it must be noted that the TUNEL will also stain necrotic cells due to extensive DNA degradation (Walker and Quirke, 2001), and thus is a marker of the apoptotic process rather than a critical component of the death process itself. False positive staining in the TUNEL method to

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detect apoptosis in the liver and intestine is caused by endogenous endonucleases and is inhibited by diethyl pyrocarbonate (Stahelin et al., 1998). 4.2.2. Caspase method The cysteine–aspartic acid specific proteases (caspases) are activated in response to different inducers of apoptosis. The process of their activation is considered to be the key event of apoptosis (Shi, 2002). Caspases recognize a four-amino-acid sequence on their substrate proteins and target the carboxyl end of aspartic acid within the sequence. Several methods have been developed to detect the activation of caspases. After the pro-caspases are cleaved, their products can be revealed electrophoretically and identified on immunoblots using caspasespecific antibodies. For example, immunostaining of active caspase-3 is a reliable indicator of apoptotic rate (Marshman et al., 2001). Moreover, activation of caspases in situ can be measured by immunocytochemical detection of the epitope that is characteristic of the active form of caspases or by immunocytochemical identification of the specific cleavage products. In addition, fluorochrome-labeled inhibitors or substrates of caspases have also been used for measuring the activation of caspases with fluorescence microscopy and flow or laser scanning cytometry (Darzynkiewicz et al., 2002; Smolewski et al., 2002).

5. LASER CAPTURE MICRODISSECTION TO PROCURE PURE CELLS IN VIVO FOR MOLECULAR ANALYSIS Laser capture microdissection (LCM) is a powerful method to procure pure populations of targeted cells from specific microscopic regions of heterogeneous tissue sections (EmmertBuck et al., 1996; Bonner et al., 1997). In this technique, a transparent thermoplastic film (ethylene vinyl acetate polymer) is applied to the surface of a tissue section mounted on a glass slide. While the film is activated through pulsing a laser beam, it becomes focally adhesive and fuses to the cells of interest. When the film is removed from the tissue section, the selected cells remain adherent to the film. The film is then placed directly into the isolation buffer in a microfuge tube for the DNA, RNA, or protein analysis. For the LCM, individual cells can be identified based on histological morphology, immunophenotype, function-related antigen expression (Fend et al., 1999), or electronic images from serial sections (Wong et al., 2000). This technique allows in vivo analysis of tissue-, cell-, and function-specific molecular analysis. In combination with high-density oligonucleotide microarray, LCM-procured cells have been used to obtain gene expression profiles from a discrete cell population (Luzzi et al., 2001). This technique can be further coupled with real-time quantitative RT-PCR to quantify mRNA abundance (Betsuyaku et al., 2001), proteomic-based approaches (e.g. 2D-PAGE) to analyze protein expression (Craven and Banks, 2002; Craven et al., 2002), and biochemical assays to measure cellular metabolite concentrations and enzyme activities (Simone et al., 2000a,b; Stappenbeck et al., 2002).

6. STABLE ISOTOPIC TRACER TECHNIQUES FOR MEASURING PROTEIN SYNTHESIS AND BREAKDOWN IN VIVO Stable isotopically labeled tracer techniques have been used in the research of protein (amino acid, AA), lipid, and carbohydrate metabolism. The principles and practice of stable isotope tracer methodology have been introduced in detail (Wolfe, 1992). New developments and techniques will be highlighted in this section.

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6.1. Whole-body protein kinetics Assessment of whole-body protein turnover relies on the measurement of the dilution of tracer amino acids in plasma or whole blood, i.e. the rate of appearance (the flux) of the tracee amino acid assuming that the blood pool and tissue free amino acid pools are homogeneously mixed. Provided that a carbon-labeled indispensable amino acid is infused intravenously at a constant rate until isotopic equilibrium is attained in the plasma, the amino acid kinetic rates can be converted to whole-body protein kinetics rates using the average fractional contents of individual amino acids in body protein (Waterlow and Stephen, 1967). The equation can be expressed as follows: Whole-body flux of AA (Q) = Intake of dietary AA + Release of AA from protein breakdown = Utilization of AA for protein synthesis (nonoxidative disposal) + Oxidation of AA Whole-body flux of AA (Q) can be estimated by the isotopic enrichment (IE) of plasma AA at isotopic equilibrium, i.e. Q = I × [(IEi/IEp) – 1] where I is the infusion rate of the tracer AA, and IEi and IEp represent the isotopic enrichment of the labeled AA in infusate and that of plasma AA at plateau, respectively. Both protein synthesis and protein degradation can be solved from the equation. Because protein synthesis, breakdown, and amino acid oxidation are intracellular events, it is necessary to measure the isotopic enrichment of the intracellular free amino acid pool rather than its plasma isotopic enrichment to calculate these kinetics. There are three technique issues. The first one is how to assess the isotopic enrichment of the intracellular true precursor (i.e. amino acyl-tRNA) for protein synthesis (see section 6.2.1). The second one is how to assess the rate of oxidation. When a primed constant infusion of 13C-labeled tracer (e.g. 1-13C-leucine or 1-13C-phenylalanine) is employed to estimate rates of whole-body protein synthesis, one has to determine the rate of oxidation of the tracee by measuring the appearance rate of 13CO2. However, the labeling position of 13C tracer affects recovery of the 13CO2. For example, the recovery of the 2-13C label in breath CO2 is 58% relative to the 1-13C label, suggesting that a significant percentage (~42%) is retained in the body although a majority of the 2-13C label of leucine is recovered in the breath CO2, presumably by transferring to other compounds via the tricarboxylic acid cycle (Toth et al., 2001). Not all of the 1-13C liberated from oxidative disposal appears in the breath CO2. Ring2H -phenylalanine (Phe) and 1-13C-tyrosine (Tyr) are infused simultaneously to estimate 5 phenylalanine irreversible hydroxylation (Clarke and Bier, 1982). The hydroxylation rate of Phe into Tyr can be derived from the equation (Short et al., 1999): QPhe − Tyr = QTyr × IEd4−Tyr/IEd5− Phe where QTyr is whole-body flux of plasma Tyr that is estimated from 1-13C-Tyr infusion, IEd4-Tyr and IEd5−Phe are the isotopic enrichments of plasma L-ring-2H4-Tyr and L-ring-2H5-Phe, respectively. This approach has been employed to estimate the in vivo hydroxylation rate of Phe to Tyr in patients with phenlketonuria (van Spronsen et al., 1998). Other combinations (e.g. 15N-Phe and ring-2H4-Tyr or ring-2H5-Phe and ring-2H2-Tyr) have also been used to estimate whole-body hydroxylation (Meek et al., 1998; Short et al., 1999). The advantage of the Phe hydroxylation model is the rapid assessment of whole-body protein turnover from plasma samples alone without measurement of breath 13CO2 production (Clarke and Bier, 1982; Thompson et al., 1989).

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Fig. 3. A three-compartment model for deriving whole-body protein turnover using 15N,13C-Leu tracer (adapted from Gowrie et al., 1999). The model describes the kinetics events during an intravenous infusion of 15N,13C-Leu tracer. In brief, infused 15N,13C-Leu tracer enters the plasma free Leu pool (compartment 1) and further enters the intracellular AA free pool (compartment 2) where it may be irreversibly deaminated (indicated by irreversible loss of 15NH2, k02) or incorporated into the intracellular protein pool (compartment 3, indicated by the fractional transfer rate from compartment 2 to 3, i.e. k32). The isotopic enrichment of the intracellular free Leu pool can be diluted by unlabeled tracee Leu from release of protein breakdown (indicated by the fractional transfer rate from compartment 3 to 2, i.e. k23).

The third technique issue is how to model experimental data. Recently, a threecompartment model has been developed to assess whole-body protein synthesis and breakdown with a 15N,13C-Leu tracer (fig. 3) (Gowrie et al., 1999). This three-compartment model that represents 15N,13C-Leu tracer kinetics can be described by a set of differential equations. Fractional rate constants (fractional rates of protein synthesis and breakdown indicated by k32 and k23, respectively) can be solved using the SAAM II program. 6.2. Precursor method for measuring fractional synthesis rate of tissue protein 6.2.1. Constant infusion method The fractional synthesis rate (FSR) of protein has been evaluated by a direct precursor– product relationship. The constant infusion method has been used to measure both wholebody protein turnover and tissue protein synthesis. This method involves the infusion of a tracer amino acid at a constant rate until steady-state isotopic labeling of the precursor amino acyl-tRNA pool is reached. Specifically, when a precursor tracer (e.g. a labeled amino acid) is provided as a primed constant infusion into a system, the isotopic enrichment of a homogeneous product pool will increase as a monoexponential function of time (IEt), i.e. IEt = IEp (1 − e−kt), where IEp is the enrichment of the precursor pool. Therefore, FSR = k = [(IEt2 − IEt1)/(t2 − t1)]/IEp The FSR is determined by dividing the initial rate of change in the product isotopic enrichment by the precursor isotopic enrichment at the steady state (Patterson, 1997). For example, the FSR of human small intestinal mucosal protein is calculated by a primed constant infusion of 1-13C-leucine using this equation, in which IEt2 is the isotopic enrichment of mucosal protein-bound leucine (from the mucosal biopsy at time 2), IEt1 is the isotopic enrichment of mucosal protein-bound leucine (from the mucosal biopsy at time 1) or the isotopic enrichment of plasma protein-bound leucine at time 1, and IEp is the isotopic enrichment of the precursor pool (e.g. tissue-free fluid 13C-leucine or plasma 13C-ketoisocaproate (BouteloupDemange et al., 1998; Charlton et al., 2000). In order to avoid multiple tissue samples, an

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overlapping (i.e. staggered) infusion of multiple stable amino acid isotopomers has been developed to measure in vivo FSR when only a single tissue sample can be obtained (Dudley et al., 1998). Labeled Phe has been also used to assess the FSR of intestinal mucosal and muscle proteins (Stoll et al., 1997; Biolo et al., 1999). Furthermore, the isotopic enrichment of plasma VLDL ApoB-100-bound Phe has been used to represent that of the intracellular free Phe to calculate the FSR of hepatic protein synthesis (Stoll et al., 1997). The accuracy of this precursor method depends on the measurement of the isotopic enrichment of the intracellular true precursor pool. By definition, the tissue tRNA-bound amino acid is the immediate precursor used for protein synthesis (Watt et al., 1991). However, it is difficult to measure the isotopic enrichment of tRNA-bound amino acid, specifically when the precursor pool is not accessible. There are four alternative solutions to measuring the isotopic enrichment of the intracellular true precursor pool. The first is to measure isotopic enrichment of tissue free amino acids. During a constant infusion of labeled Leu, there is a quite close isotopic equilibrium between muscle-free and tRNA-bound leucine pools (Watt et al., 1991; Reeds and Davis, 1999). The isotopic enrichment of tissue fluid Leu in human skeletal muscle has been proved a valid surrogate measurement of the isotopic enrichment for intracellular leucyl-tRNA (Ljungqvist et al., 1997). The isotopic enrichment of tissue free Leu has also been used to estimate the FSR of intestinal mucosal protein (Stoll et al., 2000). The second approach is to measure the isotopic enrichment of a plasma metabolite that is exclusively derived from the intracellular metabolism of the precursor, e.g. measuring the isotopic enrichment of plasma α-ketoisocaproate (KIC) as an index of the isotopic enrichment of the intracellular free leucine to calculate the FSR of muscle and hepatic proteins (Mansoor et al., 1997). The KIC is formed intracellularly from leucine and is released, in part, into the systemic circulation. Thus, the isotopic enrichment of plasma KIC can be used to represent the isotopic enrichment of the intracellular free leucine pool (Matthews et al., 1982). However, isotopic enrichments of plasma KIC and leucine have been shown to be consistently higher than those of tissue leucyl-tRNA and tissue fluid leucine (Chinkes et al., 1996a). Therefore, using the isotopic enrichment of plasma KIC as a surrogate measurement of the isotopic enrichment for leucyl-tRNA will underestimate the FSR of muscle protein, whereas the isotopic enrichment of tissue fluid leucine is a valid surrogate measurement (Watt et al., 1991; Ljungqvist et al., 1997). In a reversal of this approach, constant infusion of α-[1-13C]KIC is more accurate than labeled leucine to determine the FSR of muscle protein (Chinkes et al., 1996a). When labeled KIC is infused, the isotopic enrichment of intramuscular free Leu is the same level as that of arterial Leu (Chinkes et al., 1996a). The third approach is to use the isotopic enrichment of newly synthesized protein-bound amino acid to represent the isotopic enrichment of the true precursor, e.g. using the isotopic enrichment of very-low-density lipoprotein apolioprotein B (VLDL ApoB)-100-bound amino acid as an index of the isotopic enrichment of the hepatic amino acid pool (Reeds et al., 1992). The VLDL ApoB-100 is made in the liver and has a very short half-life in the circulation. The isotopic enrichment of VLDL ApoB-100-bound amino acid rapidly rises to the same level as that of the precursor pool in the liver. Because of the heterogeneous composition of the hepatic intracellular precursor pool, the isotopic enrichment of VLDL ApoB-100-bound amino acid may provide a more valid measurement of the isotopic enrichment of the hepatic protein synthetic precursor than the hepatic free amino acid pool does (Stoll et al., 1997, 1999b). However, there are discrepancies in the literature. Isotopic enrichments of different precursors for liver protein synthesis have been compared with that of amino acyl-tRNA using 1-13C-Leu and 15N-Phe as tracers in miniature swine (Ahlman et al., 2001). It is shown in fig. 4

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Ratios of other precursors to amino acyl-tRNA in porcine liver (data from Ahlman et al., 2001).

that isotopic enrichment ratios of 13C-Leu and 15N-Phe in liver tissue fluid and 13C-KIC in plasma to those of respective amino acyl-tRNA are close to 1.0, indicating that isotopic enrichments of tissue fluid amino acid and plasma 13C-KIC are the best predictors of the isotopic enrichments of tissue amino acyl-tRNA in the liver and skeletal muscle under different physiological conditions (Barazzoni et al., 1999; Ahlman et al., 2001). In contrast, isotopic enrichments of plasma Leu and Phe are substantially higher, whereas that of plasma VLDL ApoB-100-bound amino acid is lower than that of the respective amino acyl-tRNA (Ahlman et al., 2001). Consequently, the FSR of the liver protein derived from isotopic enrichments of plasma 13C-Leu or plasma VLDL ApoB-100-bound amino acid would be underestimated or overestimated, respectively (Ahlman et al., 2001). Recently, the FSR of slow-turnover protein has been assessed by orally or intravenously administrating 2H2O to label nonessential amino acids (Hellerstein et al., 2002; Previs, 2002). This method takes advantage of the fact that through transamination reactions the α-hydrogen of nonessential amino acids (e.g. alanine and glutamine) equilibrates rapidly and completely with the 2H of body water. Thus, the FSR of tissue protein can be estimated by measuring the incorporation of 2H-alanine and/or 2H-glutamine into protein. However, at this time, there has been no demonstration of the equivalence of the isotopic enrichment of plasma alanine and glutamine and that of their tissue free pools. The fourth approach is to derive the isotopic enrichment of the intracellular true precursor pool from mass isotopomer distribution analysis (see section 9). In conclusion, using the constant infusion method for assessing FSR of tissue protein, the best estimate of the isotopic enrichment of intracellular true precursor (amino acyl-tRNA) pool seems to be the tissue free amino acid in muscle (Davis and Reeds, 2001). 6.2.2. Flooding dose method To avoid the problem in measuring isotopic enrichment of the intracellular true precursor (amino acyl-tRNA), the flooding dose method has been developed for measuring tissue protein synthesis. This approach involves giving a bolus injection of labeled amino acid with

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a large bolus dose of unlabeled amino acid (e.g. 5 to 10 times the endogenous flux of the tracee) to rapidly create a similar isotopic enrichment in the extra- and intracellular compartments. The FSR of liver protein is determined by flooding dose method with 2H5-Phe using the equation (Barle et al., 1999): FSR = IEp × 100/AUC where IEp is the isotopic enrichment of liver protein-bound Phe at the time of the biopsy and AUC is the area under the curve for the isotopic enrichment of plasma free Phe versus time. The flooding dose method assumes rapid equilibration of the isotopic labeling among the amino acyl-tRNA pool, the tissue free amino acid pool, and the blood free amino acid pool. This assumption has been recently validated by the fact that ratios of specific radioactivity are close to 1.0 for the tissue free Phe pool versus the phenylalanyl-tRNA pool in either skeletal muscle or liver (Davis et al., 1999). Under different nutritional and hormonal conditions, the isotopic enrichments of the tissue free Phe pool may be considered satisfactory for assessing the FSR of skeletal muscle and liver proteins when a flooding dose of Phe is administered (Davis et al., 1999). The short period of measurement with this method is especially valuable, as it allows the determination of acute changes in tissue protein synthesis within 30 min (Garlick et al., 1994). However, it has been observed that large doses of leucine might stimulate protein synthesis in muscle tissue (Ballmer et al., 1990). The FSR of muscle protein by the flooding dose method is higher than that measured by the constant infusion method when 13C-leucine is used (Garlick et al., 1994; Rennie et al., 1994). To study luminal versus basolateral modulation of protein metabolism in small intestinal mucosa, a local (luminal) flooding dose method has been used to determine the fractional rate of protein synthesis in intestinal mucosa (Adegoke et al., 1999a,b). 6.3. Tracee release method for measuring fractional breakdown rate of tissue protein To measure the fractional breakdown rate (FBR) of muscle protein, the tracee release method has been developed on the basis of the precursor–product principle (Zhang et al., 1996). This method involves infusing isotope tracer (e.g. ring-2H5-Phe or ring-13C6-Phe) until isotopic equilibrium is reached. The assessment of the rate of protein breakdown is achieved by measuring isotopic enrichment decay curves of the arterial and tissue free amino acid pools after the tracer infusion is stopped. Because there is no de novo synthesis of Phe, its appearance in the tissue free amino acid pool is solely attributed to transport from blood and release by proteolysis. At isotopic equilibrium, the isotopic enrichment in the tissue free amino acid pool is always lower than that in the arterial blood because the former is diluted by intracellular unlabeled amino acid released from protein breakdown. Once the isotopic infusion is stopped, the enrichment decay in the tissue free amino acid pool depends on the isotopic enrichment decay in the arterial blood, which provides tracer and a part of the tracee (i.e. Phe), and on the protein breakdown, which provides another part of the tracee. The calculation of FBR is based on the rate at which tracee is released from protein breakdown to dilute the isotopic enrichment of the tissue free amino acid pool using a modified precursor–product equation (Zhang et al., 1996), i.e.

FBR =

[ IEm (t2 ) −

IEm (t1 )] × (Qm / T )

t2 ⎡ ⎤ ⎢ P ∫ IEa (t )dt − (1 + P) ∫ IEm (t )dt ⎥ ⎢⎣ t1 ⎥⎦ t1 t2

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where P = IEm/(IEa − IEm) represents the ratio of fractional tracee from artery versus the fractional tracee from protein breakdown, IEa and IEm being the isotopic enrichments at plateau in the artery pool and muscle intracellular free pool, respectively. The IEm(t2) − IEm(t1) is the change of the isotopic enrichment in muscle intracellular free pool from time 1 (t1) to time 2 (t2) after stopping the isotopic infusion, t2

∫

t1

t2

IEa (t )dt and

∫ IEm (t )dt

t1

are areas under the decay curves of the isotopic enrichments in arterial and muscle intracellular free pools, respectively, from t1 to t2. Qm/T is the ratio of the intracellular free tracee mass versus protein-bound tracee mass in the muscle. The FBR assessed by the tracee release method is in agreement with that derived from the arterio-venous tracer balance method (see section 6.4). The tracee release method is the complement of the tracer incorporation method. These two methods can be combined to measure both muscle protein synthesis and breakdown in one infusion study and can be applied to other tissues (e.g. skin) if a few biopsies can be obtained (Zhang et al., 1996; Volpi et al., 2000). The tracee release method has been recently improved by using a pulse tracer injection (Zhang et al., 2002). This new approach does not require an isotopic steady state, and it can be completed within an hour and using one or two muscle biopsies. 6.4. Arterio-venous tracer amino acid balance method for measuring tissue amino acid transport, protein synthesis, and breakdown Since phenylalanine is neither synthesized nor degraded by muscle tissue, the measured removal of tracer and dilution of its isotopic enrichment across the hindlimb can be used to estimate rates of phenylalanine incorporation into and release from tissue protein. This measurement, coupled with an estimate of tissue blood flow, can provide a readily nondestructive method for estimation of protein turnover in specific muscle beds in vivo. Measurements can be made repeatedly over time in a single experiment, allowing the study of acute regulation of protein turnover (Barrett et al., 1987). This conventional arterio-venous tracer amino acid balance approach has been improved by measuring the isotopic enrichment of the intracellular free amino acid pool using muscle biopsy to calculate the relative proportions of intracellular amino acid derived directly from the blood (labeled) or from tissue protein breakdown (unlabeled) (Biolo et al., 1995a). Thus, tissue (e.g. muscle) protein synthesis and breakdown and transmembrane transport of the amino acid can be determined simultaneously. The arterio-venous tracer amino acid balance method (the A-V method) can be described in a three-compartment model. This model is based on an anatomic compartmentation of an indispensable amino acid (e.g. Leu or Phe) into three compartments: the arterial, the intracellular free, and the venous compartments (fig. 5). In this compartmental model, no interstitial free pool is assumed, i.e. isotopic tracers are assumed to enter their intracellular free compartments at the arterial values and leave from their intracellular free compartments at the venous values. It is also assumed that there is no recycling of isotopic tracers released from protein breakdown into the intracellular free compartment. Isotopic enrichments of the intracellular free compartment (in the tissue fluid) may also be represented by measurement of the isotopic enrichments of other compounds. For example, the isotopic enrichment of the local venous plasma 13C-KIC is used to represent that of the intracellular free 13C-Leu. The isotopic enrichments of liver-synthesized protein VLDL

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Fig. 5. A three-compartment model of amino acid kinetics across the porcine mammary gland during lactation (adapted from Guan et al., 2002). Free amino acid compartments in artery (A), main mammary vein (V), and mammary gland (MG) are connected by arrows indicating unidirectional fluxes of free amino acids between each compartment. Amino acids enter the MG via the mammary artery (Fa,o) and leave the MG via the main mammary vein (Fo,v). Other fluxes are designated as follows: Fv,a, direct flow of amino acids from artery to vein without entering the intracellular pool (by the arterial shunt); Fmg,a and Fv,mg, inward and outward transmembrane transport of amino acids from artery to the MG and from the MG to vein, respectively; Fmg,o, the rate of intracellular amino acid appearance from endogenous sources (i.e. release from protein breakdown (PB) and de novo synthesis (DS), if any); and Fo,mg, the rate of the intracellular amino’ acids disappearance (i.e. the rate of utilization of intracellular amino acids for protein synthesis (PS), oxidation (OX), and other metabolic fates (OM), if any).

ApoB-100-bound or mammary-synthesized casein-bound amino acids are also used to represent those of the intracellular precursor for hepatic or mammary tissue protein synthesis (Reeds et al., 1992; Bequette et al., 2000; Guan et al., 2002). Below, we use this three-compartment model to derive the rates of protein synthesis and breakdown and the transmembrane transport of amino acids across an organ of interest. 6.4.1. Protein breakdown Protein breakdown (PB) can be derived from appearance rate (Ra). Since Ra = PB + arterial influx, thus PB = Ra − arterial influx, i.e. PB = (Ca × IEa × BF)/IEi – Ca × BF, where IEa and IEi are isotopic enrichments of Phe in artery and tissue fluid (intracellular free pool), respectively, Ca is the arterial concentration of Phe, and BF is blood flow rate across the organ. Therefore, Protein breakdown (PB) = Ca × BF × [(IEa/IEi) − 1]

(1a)

Assuming that the isotopic enrichment of Phe in vein (IEv) can represent IEi, therefore, Protein breakdown (PB) = Ca × BF × [(IEa/IEv) − 1]

(1b)

In fact, eq. (1) can also be derived from irreversible loss of tracee (IL). Since IL − PB = Net mass balance (NB), thus, PB = IL − NB, i.e. PB = Tracer uptake/IEi − NB, therefore, PB = [(Ca × IEa – Cv × IEv) × BF]/IEi − (Ca − Cv) × BF; when simplified, its equation is identical to eq. (1a) . It is noted that IEv usually overestimates IEi, thus protein breakdown from eq. (1b) may be underestimated. Alternatively, PB can be derived from unidirectional influx (UI). Since NB = UI − PB, thus PB = UI − NB, where UI = Tracer fractional extraction

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rate × Arterial influx = [(Ca × IEa – Cv × IEv)/(Ca × IEa)] × (Ca × BF), and NB = (Ca − Cv) × BF, through rearrangement of the equation, therefore, Protein breakdown (PB) = Cv × BF × [1 − (IEv/IEa)]

(2)

6.4.2. Protein synthesis Protein synthesis (PS) can be derived from irreversible loss of tracee (IL). Since IL = PS + Oxidation (or hydroxylation of Phe to Tyr in liver or kidney), thus Protein synthesis (PS) = Irreversible loss − Oxidation (or Hydroxylation)

(3)

where irreversible loss (IL) = Tracer uptake/Ei, i.e. Irreversible loss (IL) = (Ca × IEa – Cv × IEv) × BF/IEi

(4)

In eq. (4), IEv may be used to replace IEi assuming that the isotopic enrichment of Phe in vein is proximate to that of its intracellular free pool. If 1-13C-Leu is infused, the isotopic enrichment of venous plasma 13C-KIC may represent IEi. In fact, IL can be derived from the difference between net mass balance and protein breakdown. Since NB = IL − PB, i.e. IL = NB + PB, thus Irreversible loss (IL) = (Ca − Cv) × BF + PB Oxidation rate across the organ can be determined by labeled CO2 production. If 1-13C-Leu is infused, oxidation of tracee = 13CO2 production/IEi, i.e. Oxidation = (CCO2v × IECO2v− CCO2a × IECO2a) × BF/IEi The isotopic enrichment of the intracellular free Leu may be indicated by the isotopic enrichment of venous plasma KIC. If different 13C labeling position or multiple 13C labeling of leucine is infused, oxidation estimated from eq. (5) should be adjusted by a correction factor. Instead of estimating oxidation, hydroxylation of phenylalanine to tyrosine across the organ Table 1 Applications of the arterio-venous stable isotopic tracer amino acid balance method Regional bed

Inflow

Outflow

Blood flow

Reference

Kidney

Femoral artery Hepatic artery Portal vein

Renal vein

The Fick method (paraaminohippurate) Doppler flow probe The Fick method (indocyanine green) The Fick method (internal Phe + Tyr) The Fick method (3H2O dilution) Transit-time ultrasound flow meter The Fick method (indocyanine green) The Fick method (indocyanine green)

Moller et al. (2000) Tessari et al. (1996) Tessari et al. (1996; Halseth et al. (1997)

Liver

Mammary gland Placenta

Carotid artery Maternal femoral artery Carotid artery

Portal-drained viscera Skeletal muscle Femoral artery Splanchnic bed Femoral artery

Hepatic vein

Mammary vein Umbilical vein Portal vein Femoral vein Hepatic vein

Guan et al. (2002) Paolini et al. (2001) Guan et al. (2003) Meek et al. (1998) Tessari et al. (1996) Meek et al. (1998) Moller et al. (2000) Tessari et al. (1996)

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can be determined (Moller et al., 2000). Note that if this hydroxylation is used to correct irreversible loss to obtain protein synthesis on the basis of eq. (3), oxidation should not be subtracted any more because oxidation is part of hydroxylation. This A-V method has been widely used to estimate amino acid kinetics across a particular organ (table 1). For example, in the fasting state, healthy human skeletal muscle is in a catabolic state to provide amino acids for protein synthesis required by the splanchnic bed. Net amino acid balance between splanchnic and skeletal muscle beds is achieved through differential regulations of protein metabolism in these tissues by insulin (Meek et al., 1998). In the fasting state of the dog, the splanchnic bed contributes about 40% to the whole-body protein breakdown and the gut and liver each contribute about 50% to the splanchnic bed (Halseth et al., 1997), indicating the equal significance of gut and hepatic proteolysis to whole-body proteolysis. It is important to accurately measure regional blood flow rate when using this A-V method. Three methods are used for measuring regional blood flow rate: fluorescent microsphere, external unmetabolized marker (e.g. indocyanine green and paraaminohippurate), and ultrasonic flow probe. If there are multiple entrance or exit vessels, blood flow rate may be appropriately measured by the Fick method based on the conservation of mass. For example, blood flow rates across the splanchnic bed and mammary gland have been estimated by the Fick method (Meek et al., 1998; Guan et al., 2002). It has been shown that mammary blood flow rates estimated by the ultrasonic method are comparable to those estimated by the Fick method (Trottier et al., 1997; Renaudeau et al., 2002). 6.4.3. Transmembrane transport of amino acids The same three-compartment model has been employed to assess amino acid inward and outward transport (Biolo et al., 1992, 1995a). Using the porcine mammary gland as an example, the calculations based on references are (Reeds et al., 1992; Bequette et al., 2000; Guan et al., 2002): Fa,o = Ca × BF Fo,v = Cv × BF Net mass balance = (Ca − Cv) ⋅ BF Based on the net mass balance of AA across the mammary gland (MG), Fa,o = Fmg,a + Fv,a Fo,v = Fv,mg + Fv,a Thus, (Ca − Cv) × BF = Fmg,a − Fv,mg Based on the tracer balance of AA across the MG, (Ca × IEa − Cv × IEv) × BF = Fmg,a × IEa − Fv,mg × IEi Therefore, Fmg,a ={[(IEi − IEv)/(IEa − IEi)] × Cv + Ca} × BF

(6)

Fv,mg ={[(IEi − IEv)/(IEa − IEi)] × Cv + Cv} × BF

(7)

The only source of tracer appearing in the mammary intracellular free AA compartment is transported inward from plasma. Thus, the isotopic enrichment of tracer AA in the intracellular

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free AA is diluted by endogenous sources (Fmg,o) (e.g. protein breakdown and de novo synthesis, if any). Therefore, Ra × IEi = Fmg,a × IEa , i.e.

Ra = (Fmg,a × IEa)/IEi

(8)

where Ra is the sum of inward transmembrane transport (Fmg,a) and the appearance rate of intracellular free AA from the endogenous sources (Fmg,o): Ra = Fmg,a + Fmg,o Thus, Fmg,o = Fmg,a × (IEa/IEi − 1)

(9)

At steady state, the total fluxes into the mammary intracellular free AA compartment are equal to the total fluxes out of this compartment, i.e. Fmg,a + Fmg,o = Fv,mg + Fo,mg Thus, Fo,mg = Fmg,o + NB

(10)

The disappearance rate (Fo,mg, i.e. utilization rate of the intracellular free AA) of intracellular free AA could also be directly calculated as the tracer balance divided by the precursor enrichment (IEi): Fo,mg = (Ca × IEa − Cv × IEv) × BF/IEi

(11)

It has been shown using this three-compartment model that increased net protein synthesis in human muscle (Volpi et al., 1998) and the porcine mammary gland (Guan et al., 2002) by intake of dietary indispensable amino acids is attributed to increased inward transmembrane transport of these amino acids into the respective organs, and that the net flow of amino acids from muscle to the gut in the fasting state is attributed to differences in their transmembrane transport rates (Biolo et al., 1995b). 6.4.4.

In vivo nitric oxide synthase activity

This arterio-venous tracer amino acid balance method can be used to assess in vivo nitric oxide synthase (NOS) activity across an organ. Guanidine-15N2-arginine is converted to ureido-15N-citrulline and 15NO through NOS reaction (Palmer et al., 1988), and used to quantify in vivo NOS activity across organs (Bruins et al., 2002). If guanidine-15N2-arginine and ureido-13C-5,5,2H2-citrulline are infused, unidirectional flux of guanidine-15N2-arginine to ureido-15N-citrulline (QArg→Cit) can be estimated on the basis of ureido-15N-citrulline tracer balance (see fig. 6), i.e. based on ureido-15N-citrulline tracer balance: QIN + QNOS = QOUT + QM where QIN = Ca × IE15N-Cit,a × BF QNOS = QArg→Cit × IE15N-Arg,i QOUT = Cv × IE15N-Cit,v × BF

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Fig. 6. A compartmental model of arginine and citrulline kinetics across the portal-drained viscera (PDV). QIN, QOUT, QM, and QArg→Cit represent unidirectional fluxes of ureido-15N-citrulline tracer entered from the artery, exited to the vein, metabolized in the organ, and de novo synthesized from arginine through the NOS reaction, respectively. Dotted lines and solid lines indicate fluxes of tracer and tracee, respectively.

QM = Irreversible loss × IE15N-Cit,i = [(Ca × IEd-Cit,a × BF − Cv × IEd-Cit,v × BF)/IEd-Cit,i] × IE15N-Cit,i where BF is the blood flow across the organ; Ca and Cv are concentrations of free citrulline in artery and vein, respectively; IE15N-Cit,a (IEd-Cit,a), IE15N-Cit,i (IEd-Cit,i), and IE15N-Cit,v (IEd-Cit,v) are isotopic enrichments of ureido-15N-citrulline (ureido-13C-5,5,2H2-citrulline) in artery, the intracellular compartment, and vein, respectively; and IE15N-Arg,i and IE15N-Arg,v are isotopic enrichments of guanidine-15N2-arginine in artery and vein, respectively. Assuming that isotopic enrichment of the tracer in vein is proximate to that in the intracellular compartment, i.e., IE15N-Arg,i = k1 × IE15N-Arg,v; IE 5N-Cit,i = k2 × IE15N-Cit,v; IE d-Cit,i = k3 × IE d-Cit,v; and k1 ≈ k2 ≈ k3 ≈ 1, therefore, QArg→Cit = [(IE d-Cit,a/IEd-Cit,v) × (IE15N-Cit,v/IE15N-Arg,v) − (IE15N-Cit,a /IE15N-Arg,v)] × Ca × BF (2) This unidirectional flux (QArg→Cit) indicates in vivo NOS activity across the organ (e.g. PDV). The principle of this method has been also applicable to assessment of the local conversion (e.g. hydroxylation of phenylalanine to tyrosine across the liver or kidney) (Moller et al., 2000). 6.4.5. First-pass utilization In amino acid tracer kinetic studies, ingested amino acid is taken up during its initial transit through the splanchnic bed and thus not all absorbed amino acids enter the systemic compartment. The amount of enterally delivered tracer (or tracee) sequestered by the splanchnic bed can be estimated by simultaneously administrating a labeled tracer intravenously (iv) and intraduodenally (id) (or intragastrically, ig) (Matthews et al., 1993a,b). To assess protein metabolism in the splanchnic bed, the infusion of tracer amino acid into the gastrointestinal tract should ideally avoid gastric emptying in the postabsorptive state. An intraduodenal administration of AA tracers is recommended to obtain plasma isotopic enrichments at steady state (Crenn et al., 2000). This method allows measurement of splanchnic extraction and

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indirect assessment of splanchnic protein metabolism under fasting and feeding conditions (Crenn et al., 2000). The splanchnic extraction coefficient of leucine is defined (Stoll et al., 1997, 1999b; Basile-Filho et al., 1998, 1999b) as: Splanchnic extraction = [Qid − Qiv]/Qid = 1 − Qiv/Qid in which Qid or Qiv (whole-body flux) = Rate of tracer infusion · [(IEi/IEp) − 1]. If whole-body flux is not corrected by the amount of tracer infused, then splanchnic extraction is simplified as (Crenn et al., 2000): Splanchnic extraction = 1 − [(IEp,id/Iid) / (IEp,iv/Iiv)] where IEp,id and IEp,iv are isotopic enrichments (tracer-to-tracee ratio, TTR) of plasma amino acids at plateau after a stable isotopic tracer (e.g. 2H3-Leu) is infused intraduodenally and intravenously, respectively. Iid and Iiv are infusion rates of the tracer via intraduodenal and intravenous routes, respectively. Fractional splanchnic oxidation (of whole-body oxidation) and fractional splanchnic hydroxylation of Phe to Tyr (of whole-body hydroxylation) have been estimated using this method (Basile-Filho et al., 1998). Because the rate of intragastrically infused tracer (13C-Phe) appearing in the nonsplanchnic pool is the product of the tracer infusion rate (Iig) and nonsplanchnic extraction (Fnsp,extraction = 1 − splanchnic extraction), which is handled in first pass in a manner similar to that for intravenously infused tracer, then the fractional nonsplanchnic oxidation (Fnsp,oxidation, of whole-body total oxidation) can be calculated as follows (Basile-Filho et al., 1998): Fnsp,oxidation = Fnsp,extraction × Riv,oxidation/Rig,oxidation and

Fsp, oxidation = 1 − Fnsp,oxidation

where Fsp,oxidation is the fractional splanchnic oxidation (of whole-body oxidation), and Riv,oxidation and Rig,oxidation are whole-body oxidation rates calculated by intravenously and intragastrically infused tracer, respectively. Similarly, fractional splanchnic hydroxylation (of whole-body hydroxylation) can be obtained with an intravenous infusion of ring-2H4-Tyr (see details in Basile-Filho et al., 1998). We have combined this method and the portal tracer amino acid balance to further assess amino acid metabolism in the gut and liver (Stoll et al., 1997; van Goudoever et al., 2000). Hepatic extraction is defined by the difference between splanchnic extraction (derived from above) and portal extraction (derived from the portal tracer amino acid balance). We have found that splanchnic extraction of Phe is attributed to fractional extraction of the gut and the liver by 75% and 25%, respectively (Stoll et al., 1997), and one-third of the dietary amino acids is metabolized in the gut (Stoll et al., 1998, 1999a; van Goudoever et al., 2000). It is important to assess amino acid metabolism and protein turnover in the splanchnic bed (including the gut and liver) in order to predict the post-splanchnic availability of absorbed dietary amino acids and to understand tissue protein metabolism under different nutritional, physiological, and pathological conditions.

7. STABLE ISOTOPIC TRACER TECHNIQUES FOR STUDYING LIPID METABOLISM Regulation of lipid metabolism is not only related to growth and fattening of animals, but also to the development of cardiovascular disease, insulin resistance, diabetes, and obesity in humans. The measurement of dynamic fluxes of lipids (biosynthesis, oxidation, and lipolysis) poses difficult challenges. Two fundamental advances have recently been made for

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measuring lipid biosynthesis, namely deuterium-labeled water incorporation method and mass isotopomer distribution analysis (MIDA). These techniques have resolved the central methodological problem in measuring the isotopic enrichment of the intracellular true precursor pool. In the 2H2O incorporation method, rates of deuterium incorporation into fatty acids and cholesterol are used to assess de novo lipogenesis and cholesterol synthesis, respectively (Diraison et al., 1997; Guo et al., 2000; McDevitt et al., 2001; Bassilian et al., 2002), in which 2H2O (tracer) equilibrates well among the intracellular precursors NADPH and water (Di Buono et al., 2000). Besides, the MIDA is also used to assess the FSR of the VLDL by measuring incorporation of repeating subunits of acetyl-CoA into the newly synthesized triglyceride (TG) after a constant infusion of [1-13C]acetate (Chinkes et al., 1996b). Labeled glycerol is also used in the study of lipid metabolism (Siler et al., 1998; Lemieux et al., 1999). 7.1. Whole-body lipolysis Whole-body lipolysis can be determined by the dilution method (e.g. at the infusion of [1,2,3,4-13C4]palmitate and [2H5]glycerol or [2-13C]glycerol) (Horowitz et al., 1999; Siler et al., 1999; Wang et al., 2000; Bergeron et al., 2001). With intravenous infusion of [2H5]glycerol, appearance rate (Ra) of plasma glycerol represents the rate of glycerol released into plasma from hormone-sensitive lipase hydrolysis of adipose tissue and intramuscular TG and the rate of glycerol released into plasma during lipoprotein lipase hydrolysis of VLDL-TG (Mittendorfer et al., 2001). However, it does not include the rate of glycerol released during lipolysis of intra-abdominal adipose tissue TG, which is cleared by the liver (Mittendorfer et al., 2001). Moreover, lipolysis is underestimated by the extent to which glycerol released by lipolysis does not enter the systemic circulation, as occurs when lipolysis takes places in the nonhepatic tissue of the splanchnic bed (Landau, 1999a). Thus, the glycerol Ra is used to calculate the lower limit for whole-body lipolysis (Aarsland et al., 1996). The rate of appearance of fatty acids (FA) in plasma (Ra) is determined by the equation: Ra = I × [(IEi/IEp) − 1] where I is the infusion rate of fatty acid tracer, and IEi and IEp are isotopic enrichments of the fatty acid in the infusate and in the plasma at plateau. However, FA may be re-esterified to TG in most tissues. 7.2. Fatty acid kinetics The constant infusion of U-13C-labeled fatty acids is used to determine the effects of hyperglycemia–hyperinsulinemia on whole-body, splanchnic, and leg fatty acid metabolism in humans (Sidossis et al., 1999). It has been demonstrated that an increase in glucose availability inhibits fatty acid oxidation across the leg and the splanchnic region under the constant availability of fatty acids (Sidossis et al., 1998, 1999). The fatty acid kinetic parameters for the leg and the splanchnic region are derived in the same manner as in section 6.4. In brief, Net rate of NEFA uptake or release = (Ca − Chv) × hepatic (or leg) plasma flow where Ca, Chv, and Cfv are arterial, hepatic venous, and femoral venous concentrations of nonesterified fatty acids (NEFA), respectively. Fractional extraction of labeled NEFA = [(IEa × Ca − IEhv × Chv)]/(IEa × Ca);

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= [(IEa × Ca − IEfv × Cfv)]/(IEa × Ca)

or

where IEa, IEhv, and IEfv are the isotopic enrichments of NEFA in the artery, hepatic vein, and femoral vein, respectively. Absolute rate of uptake of NEFA = Fractional extraction × Ca × Hepatic (or femoral) plasma flow. Uptake of NEFA that is released as CO2 (%) = Regional 13CO2 production/ regional uptake of labeled NEFA = (IECO2,hv × CCO2,hv − IECO2,a × CCO2,a)/ (IEa × Ca − IEhv × Chv) where IECO2,a and IECO2,hv are the isotopic enrichments of CO2 in the artery and hepatic vein, respectively, and CCO2,a and CCO2,hv are the concentrations of CO2 in the artery and hepatic vein, respectively. Absolute rate of oxidation of NEFA = Absolute rate of uptake of NEFA × % of NEFA uptake that is released as CO2/ the acetate correction factor. The acetate correction factor accounts for label fixation that might occur at any step between the entrance of labeled acetyl-CoA into the tricarboxylic acid cycle until the recovery of label CO2 in breath (Sidossis et al., 1995a). Because label fixation occurs not only via the bicarbonate pool, but also via isotopic exchange reactions in the tricarboxylic acid cycle (Sidossis et al., 1995b), bicarbonate cannot fully correct the label fixation.

7.3. Muscle triglyceride synthesis On the basis of the precursor–product relationship and the assumption that the intramuscular NEFA are the synthetic precursors during the infusion of [U-13C]palmitate (Guo and Jensen, 1998): Fractional synthesis rate (FSR) of intramuscular TG = (IEt2,TG-palmitate– IEt1,TG-palmitate)/ [Averaged IENEFA-palmitate × Time] where the numerator is the increment in 13C enrichment of muscle TG palmitate during a 2–4 h interval, and the denominator is the average 13C enrichment of intramuscular nonesterified palmitate over the same time interval. This measurement is across a particular muscle bed.

7.4. Hepatic de novo lipogenesis The rate at which de novo synthesized palmitate is secreted as VLDL-TG is assessed with a constant infusion of [1,2-13C]acetate using the MIDA. To calculate the fractional synthesis rate of VLDL-bound palmitate (FSR), the following formula is used (Aarsland et al., 1996): FSR = [(IE(t2) – IE(t1))/(t2 − t1)]/[8p(1−p)7] where t2 and t1 are the times when samples are taken, IE(t) is the doubly labeled enrichment at time t, and p is the MIDA-derived enrichment of the intrahepatic precursor pool (hepatic acetyl-CoA) for fatty acid synthesis. Here, the factor of 8 accounts for the fact that it requires

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eight acetate molecules to form one palmitate molecule (i.e. the principal product of mammalian de novo fatty acid synthesis). The factor of (1−p)7 accounts for the probability that seven unlabeled acetate molecules will be incorporated into a palmitate molecule (Chinkes et al., 1996b). The FSR is defined as the fraction of plasma VLDL-bound palmitate pool, per unit of time, which is newly synthesized. The absolute synthesis rate is then calculated by multiplying the FSR and the pool size of VLDL-bound palmitate.

8. MASS ISOTOPOMER DISTRIBUTION ANALYSIS Mass isotopomer distribution analysis (MIDA) is a new technique for quantifying synthesis rates of polymeric biomolecules from 15N-, 13C-, or 2H-labeled monomeric units in the presence of unlabeled polymer. Mass isotopomer distribution is analyzed according to a combinatorial probability model. The isotopomers of a given type of molecule are the various combinations of positions of labeled atoms. For example, when the 12C isotope can be replaced by 13C independently at each position in glucose, there are 64 (26 = 64) different isotopomers. The MIDA allows the isotopic enrichment of the monomeric precursor to be derived indirectly from the isotopic enrichment of the polymer (product). This derived precursor enrichment presumably represents the steady-state enrichment of the precursor. The MIDA has been used to measure fractional rates of cholesterol biosynthesis (Lindenthal et al., 2002), gluconeogenesis (Trimmer et al., 2002), lipogenesis, and protein synthesis. Monomer subunits are randomly selected from the precursor pool and incorporated into a polymer (the product). Theoretical distribution of newly formed product molecules can be predicted by binomial or multinomial expansion. The probabilities of incorporating a given number of labeled precursors into the product are determined by the isotopic enrichment of the precursor pool on the basis of a multinomial distribution (Hellerstein and Neese, 1999):

d ( z, σ , p ) =

z

z! (1 − p)( z −σ ) Pσ σ = 0 ( z − σ )!σ !

U

where σ is the number of labeled subunits present in the variable moiety of the polymer, z is the maximum number of monomer subunits that can be labeled in the variable moiety of the polymer, and p is the fraction of ΔA∞x /ΔA∞y isotopically labeled subunits in the subunit precursor pool. The value of p is calculated from the best-fit polynomial regression equation of p against the ratio of in an appropriate reference table (Hellerstein and Neese, 1999). Here, ΔA∞x and ΔA∞y are defined as the change in fractional abundance (i.e. excess mass isotopomer abundance) in the newly synthesized or isotopically perturbed polymers only (e.g. ratio of doubly to singly labeled product, or triply to doubly labeled product). The precursor enrichment (p) is determined from the measured ratio of ΔA∞x /ΔA∞y using this equation. Based on the precursor–product relationship, the fractional synthesis (f, the proportion of newly synthesized molecules present in the mixture) can be calculated using ΔA∞x at the value of p (derived from the ratio of ΔA∞x /ΔA∞y ), i.e. f = ΔAx (mixture)/ΔA∞x where ΔAx (mixture) is the change of the fractional abundance of a mass isotopomer Mx in the mixture (measured), and ΔA∞x is the enrichment at plateau, i.e. the precursor enrichment in a one-source biosynthetic system (calculated from the regression equation of ΔA∞x against p, representing the asymptotic value of ΔA∞x ). When all the ions in the mass isotopomer spectrum are not monitored, a correction equation is used for calculating f (Papageorgopoulos et al., 1999).

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Therefore, the fractional synthetic rate constant (ks) is calculated as follows (Papageorgopoulos et al., 1999): ks = −In(1 − f)/t 8.1. Protein synthesis It is difficult to assess protein synthesis using the conventional precursor–product method because it not easy to accurately measure the isotopic enrichment of the precursor pool (intracellular amino acyl-tRNA). This difficulty has been conquered with the MIDA. Using the MIDA to assess protein synthesis has become technically feasible and practical in vivo using proteolytically derived peptides (Papageorgopoulos et al., 1999). To obtain mass isotopomer distribution, a small peptide that contains repeats of a selected amino acid is generated from a whole molecule of protein. The kinetics of the peptide component presumably represents the kinetics of the intact protein. For example, [5,5,5-2H3]leucine is intravenously infused into rats, and then a leucine-rich peptide is isolated and purified from trypsin-digested rat serum albumin. Theoretic abundances and excess abundances of mass isotopomers are calculated and measured. Biosynthetic rates of rat serum albumin are estimated by the MIDA, which are similar to previously published values (Papageorgopoulos et al., 1999). Based on the exchange of 2H2O with α-hydrogen of non-essential amino acids (e.g. alanine and glutamine), the MIDA can be used for measurement of synthesis rates of slow-turnover proteins (Hellerstein et al., 2002). 8.2. Lipogenesis If [1-13C]acetate is infused in vivo, VLDL-bound palmitate enrichment can be measured by the tracer-to-tracee ratio (TTR). The precursor (acetyl-CoA) enrichment p is derived from the MIDA (Chinkes et al., 1996b): p = [2 × TTR(M + 2)/TTR (M + 1)]/[(n − 1) + 2 × TTR(M + 2)/TTR(M + 1)] This precursor enrichment is expressed in terms of acetate units, and is converted to units of single labeled palmitate (IEp) using the binomial equation: IEp = np(1−p)n−1 The FSR of VLDL-palmitate is calculated on the basis of the precursor-product relationship as follows: FSR = [(IE(t2) − IE(t1))/(t2 − t1)]/[np (1 − p)n−1] where IE(t) is the singly labeled product enrichment at time t, i.e. singly labeled VLDL-palmitate (M + 2) enrichment. If doubly labeled acetate ([1,2-13C]acetate) is infused rather than singly labeled acetate, palmitate will appear at the peaks M + 2 and M + 4 rather than M + 1 and M + 2. In the calculation of the precursor enrichment, TTR(M + 4)/TTR(M + 2) is used in place of TTR(M + 2)/TTR(M + 1). Recently, 2H2O has been used to label the glycerol moiety of triglyceride to simultaneously measure in vivo TG synthesis and de novo lipogenesis in adipose tissue (Antelo et al., 2002; Turner et al., 2002). 8.3. Gluconeogenesis (GNG) The rate of glucose production is the sum of rates of glycogenolysis and gluconeogenesis. The rate of glycogenolysis is the rate at which glucose is formed from glycogen, which can

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be determined by the decline in liver glycogen content measured by 13C nuclear magnetic resonance spectroscopy (Rothman et al., 1991). The rate of gluconeogenesis is the rate of glucose synthesis via glucose-6-phosphate from gluconeogenic precursors (e.g. lactate, alanine, pyruvate, and glycerol). Gluconeogenesis can be determined directly by two approaches. The first one is to assess the fractional contribution of gluconeogenesis by measuring the ratio of the 2H enrichment of the hydrogen bound to C-5 and to that of C-2 of blood glucose at steady state after oral intake of 2H2O (Landau et al., 1996; Chandramouli et al., 1997; Petersen et al., 1999). That is because a hydrogen atom from body water is bound to C-5 of every molecule of glucose formed via gluconeogenesis and none via glycogenolysis, while a hydrogen atom from body water is added at C-2 of glucose formed via both gluconeogenesis and glycogenolysis; the ratio of enrichment at C-5 to that at C-2 also provides a measure of that fraction (Chandramouli et al., 1997). The rate of gluconeogenesis is calculated by multiplying that ratio by the rate of glucose production, i.e. the rate of appearance of glucose. Gluconeogenesis determined by the ratio of the 2H enrichments will be overestimated by the degree of cycling between glucose-6-phosphate and triose phosphate, and/or loss of label via transaldolase exchange reactions that are part of the pentose cycle (Ackermans et al., 2001), for the contribution of the cycling between glucose-6-phosphate and triose phosphate results in an increase in the labeling of C-5 and, thus, in an overestimation of gluconeogenesis, i.e. the conversion of glycogen to triose phosphates (then used for glucose synthesis) is included in the estimate of the contribution of gluconeogenesis rather than glycogenolysis (Chandramouli et al., 1997). The second approach is to assess the fractional contribution of gluconeogenesis using the MIDA. Glucose can be considered as a dimer made of two triose subunits. The MIDA of glucose labeled from [2-13C]glycerol, [U-13C3]glycerol, [3-13C]lactate, or [U-13C3]lactate can be used for estimating the contribution of gluconeogenesis to glucose production (Neese et al., 1995). The MIDA of glucose is more precise with uniformly labeled than singly labeled 13C substrates (Previs et al., 1995). In the latter case, ratios of glucose molecules labeled with two 13C atoms (M ) versus with one 13C atom (M ) are very sensitive to a small error in the fairly 2 1 high background correction at M2. Moreover, the contribution of gluconeogenesis to glucose production is artifactually underestimated by loss of [2-13C]glycerol carbon via the pentose cycle when [2-13C]glycerol is infused (Previs et al., 1995; Kurland et al., 2000). It is also possible that a proportion of glucose is formed from glycerol and from amino acids not converted to glucose via pyruvate (Landau, 1999c). Thus, [U-13C3]lactate appears to be a suitable tracer for the MIDA of gluconeogenesis in vivo (Previs et al., 1995), especially for tracing low or moderate rates of gluconeogenesis (Previs et al., 1998). The MIDA of plasma glucose and lactate can be carried out during an infusion of [U-13C6]glucose. During an infusion of [U-13C6]glucose (M6 glucose), glycolysis leads to the production of labeled lactate (m3 lactate). When 13C carbon atoms are recycled in gluconeogenesis, glucose molecules with one, two, or three 13C substitutions (M1, M2, and M3 glucose) are produced. The appearance of mass isotopomers M1, M2, and M3 of glucose provides a measurement of the rate of gluconeogenesis. Because the chance of two labeled triose phosphates combining to form glucose is negligible, M6 glucose behaves as a nonrecyclable tracer, and the steady-state enrichment of M6 glucose in plasma allows the determination of the hepatic glucose production rate. Thus the infusion of [U-13C6]glucose has the advantage of being able to estimate simultaneously hepatic glucose output and fractional gluconeogenesis from the MIDA of plasma glucose and lactate and has been used to estimate gluconeogenesis by Tayek and Katz (Tayek and Katz, 1996, 1997; Katz and Tayek, 1999). However, different equations have been used to calculate the contribution of gluconeogenesis to glucose production.

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The dilution of the labeled lactate molecules by endogenous unlabeled lactate molecules (D) is calculated by the equation (Landau et al., 1998; Landau, 1999b; Radziuk and Lee, 1999): D = [0.5(M1 + M2 + M3) + M6]/(m1 + m2 + m3) where M1, M2, M3, and M6 are, respectively, the percentages of blood glucose molecules with one, two, three, and six 13C atoms, i.e. isotopomers M1, M2, M3, and M6. Correspondingly, m1, m2, and m3 are the percentages for blood lactate of isotopomers m1, m2, and m3, respectively. The fraction of glucose molecules in the blood that recycled (F), i.e. via the Cori cycle, is calculated by the equation (Landau et al., 1998; Landau, 1999b; Radziuk and Lee, 1999): F = 0.5(M1 + M2 + M3)/[0.5(M1 + M2 + M3) + M6] The product of the Cori cycle and the dilution of glycolysis by endogenous lactate represents the contribution of gluconeogenesis to the Ra glucose (Katz and Tayek, 1999). Thus, the fractional gluconeogenesis (% of glucose production) can be calculated by the following equation (Landau, 1999b; Radziuk and Lee, 1999; Mao et al., 2002), assuming that there is no loss of labeled molecules via the tricarboxylic acid cycle because when mi → mj, i ≥ j, labeled molecule is still counted (Kelleher, 1999; Radziuk and Lee, 1999): Fractional gluconeogenesis (% of glucose production) = (M1 + M2 + M3)/[2(m1 + m2 + m3)] Fractional gluconeogenesis can be derived directly by a binominal expansion approach (Kelleher, 1999; Radziuk and Lee, 1999): Gluconeogenesis (% of glucose production) = (M1 + M2 + M3)/[2 ⋅ m0 ⋅ (m1 + m2 + m3)] where m0 is approximate to 1. Fractional gluconeogenesis calculated from these equations is underestimated (Landau et al., 1998; Kelleher, 1999; Landau, 1999b; Radziuk and Lee, 1999; Mao et al., 2002), which results from the lack of isotope equilibrium in both the lactate (m3) and glucose (M3) compartments and the tracer dilution by other unlabeled gluconeogenic substrates (Mao et al., 2002). Finally, Rate of gluconeogenesis = Ra glucose × D × F Equations for D and F are applicable only when the rate of glucose infused is small relative to glucose production, which will result in relatively low enrichments and with negligible formation of M4 and M5 as well as M6 isotopomers.

9. NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY Nuclear magnetic resonance (NMR) spectroscopy now provides a noninvasive means to monitor metabolic flux and intracellular metabolite concentrations continuously. The basic principles of in vivo NMR spectroscopy have been described in detail (Roden and Shulman, 1999). In brief, some atomic nuclei (e.g. 1H, 13C, and 31P) possess magnetic properties, i.e. the magnetic moment or “spin”. Under experimental conditions, resonant waves (resonance) from various nuclei/compounds can be translated into a display of peak intensities vs. frequencies. The frequency of a peak is the characteristic of a certain nucleus/compound and the area under that peak corresponds to the concentration of that nucleus/compound. The ability to distinguish between different molecules containing the same nucleus relies on the “chemical shift”, given in parts per million (ppm). The nuclei of different molecules thereby experience

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an altered static magnetic field and in turn resonate at an altered frequency, i.e. chemical shift, which is typical for the respective molecule. Several measures are used to improve the signal-to-noise ratio of NMR spectroscopy. Increasing the field strength of the static magnetic field improves the signal-to-noise ratio and thereby the sensitivity of the technique. Studies in humans are routinely performed at 1.5–4.7 Tesla. To examine a defined small volume of tissue, surface coils are placed tightly over the region of interest to ensure homogeneous tissue filling in that region. The pulse angle and shape can be selected to suppress signals from other tissues such as the subcutaneous fat layer. In vivo NMR spectroscopy can measure the concentrations and synthesis rates of individual biological molecules such as glycogen and neurotransmitters within precisely defined areas of specific organs such as brain, liver, and muscle (Shulman and Rothman, 2001). Most studies to date have used 1H, 31P, and 13C to determine skeletal muscle glucose and glycogen metabolism. The suitability of a nucleus for NMR spectroscopy depends on its relative magnetic sensitivity, the tissue concentration range of the metabolite, and the chemical shift range. 9.1.

1H

NMR spectroscopy

Protons (1H) have a natural abundance close to 100% and overall offer the highest sensitivity for NMR spectroscopy. However, the relatively low concentration of metabolites (compared to the proton concentration in water) and the low chemical shift range (10 ppm) have limited the use of 1H for NMR spectroscopy. Measurement of intracellular triglyceride (TG) content in vivo at 1.5 Tesla by 1H NMR spectroscopy has been validated biochemically by liver biopsy (Szczepaniak et al., 1999). Furthermore, utilization of intramyocellular lipid in human muscle is measured by 1H NMR spectroscopy (Szczepaniak et al., 1999; Krssak et al., 2000). 9.2.

31P

NMR spectroscopy

Phosphors (31P) occur 100% in nature and allow quantification of intramuscular concentrations of adenosine triphosphate (ATP), adenosine diphosphate, inorganic phosphate, phosphocreatine, and glucose-6-phosphate (G6P) (Krebs et al., 2001). The concentrations of metabolites are determined by comparing the spectral areas to the area of the β-ATP resonance, which is used as an internal concentration standard (Bloch et al., 1993). Measurement of muscular G6P concentrations by 31P NMR spectroscopy has been validated by a chemical assay of its concentration in rat muscle frozen in situ (Bloch et al., 1993). Glucose-6-phosphate is an intermediate in the muscle glycogen synthesis pathway, and its concentration depends on the relative activities of muscle glycogen synthase enzyme and glucose transport into muscle. In addition, 31P NMR spectroscopy has been used to measure mitochondrial unidirectional ATP synthesis flux in vivo in rat skeletal muscle (Jucker et al., 2000a,b) and to measure G6P concentration in human muscle (Rothman et al., 1995). 9.3.

13C

NMR spectroscopy

In contrast to 1H and 31P, 13C has a natural abundance of 1.1% and therefore a relatively low sensitivity. Nevertheless, 13C NMR spectroscopy has been used to measure hepatic glycogen concentrations and thus estimate rates of net hepatic glycogen synthesis and glycogenolysis in vivo. Since the resonance of 13C in the C-1 position of glycogen is clearly resolved at 100.5 ppm and all 13C signals from glycogen are detected by 13C NMR spectroscopy, it can be used to measure 13C incorporation into glycogen during an infusion of [1-13C]glucose, which increases the

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sensitivity of the method by up to 100-fold. Measurement of tissue glycogen content by 13C NMR spectroscopy has been validated for skeletal muscle and liver by comparison with muscle (Gruetter et al., 1991) and liver (Gruetter et al., 1994) biopsies (Taylor et al., 1992; Krssak et al., 2000). Furthermore, using a 13C-glucose pulse–12C-glucose chase experiment, rates of hepatic glycogen synthesis and glycogenolysis can be assessed (Magnusson et al., 1994; Roden et al., 1996; Petersen et al., 1998). The peak intensity of the C-1 resonance of the glycosyl units of glycogen is monitored with 13C NMR spectroscopy during [1-13C]glucose infusion followed by unlabeled glucose infusion. Increment in the C-1 peak intensity during the [1-13C]glucose infusion represents glycogen synthesis, while decline in the C-1 peak intensity during unlabeled glucose infusion reflects glycogenolysis (Magnusson et al., 1994). Increments in muscle glycogen concentration can be calculated from the change in [1-13C]glycogen concentration and the isotopic enrichment of plasma [1-13C]glucose (Shulman et al., 1990). In human and rat brains 13C NMR measurements of the in vivo flux of 13C label from [1-13C] glucose into glutamate and glutamine simultaneously determine the rate of glucose oxidation (tricarboxylic acid cycle rate) and glutamate/glutamine neurotransmitter cycling between astroglia and neurons (Sibson et al., 1998, 2001; Shen et al., 1999; Shulman et al., 2001). The glutamate/glutamine neurotransmitter cycling, measured by 13C NMR spectroscopy, is the major pathway for neuronal glutamate repletion (Lebon et al., 2002), which accounts for 80% of glucose oxidation in the resting state (Shen et al., 1999). 1H-decoupled 13C NMR spectra yields sufficient signal-to-noise resonance at C-4 glutamate and C-4 glutamine in the rat brain in vivo at 7.0 Tesla (Sibson et al., 1998). It is possible to detect 13C labeling of glutamate and glutamine in liver by 13C NMR spectroscopy. Additionally, the in vivo 13C labeling kinetics of glutamate and glutamine in liver and glutamine in blood can be used to calculate the liver tricarboxylic acid cycle flux (Jucker et al., 1998). 13C NMR and 31P NMR can be combined to quantify glycogen synthesis rate and glucose-6-phosphate concentration in rat gastrocnemius muscle (Chase et al., 2001). The concentration of glycogen is calculated from the increment in the 13C spectra and the isotopic enrichment of [1-13C]glucose (Bloch et al., 1994).

10. FUTURE PERSPECTVES In this chapter, we have discussed some new approaches aimed at understanding the biological basis of metabolomics from systemic physiology, to intermediary metabolism, and to molecular regulation of critical gene and protein expression. Metabolomics has recently been developed as a platform for the quantitative measurement of the dynamic multiparametric metabolic response of living systems to genetic modification, developmental state, pathophysiological process, or environmental stimulus, which promises to identify gene function, evaluate drug efficacy and toxicity, and define in vivo metabolic profiling (of all the metabolites in an intact tissue, organ, or biofluid) (Raamsdonk et al., 2001; Brindle et al., 2002; Nicholson et al., 2002; Watkins et al., 2002). Metabolomics is becoming feasible directly in crude biological extracts with advances in nuclear magnetic resonance spectroscopy, mass spectrometry coupled with bioinformatics techniques, and multivariate statistical analyses. In fact, the metabolic status of an integrated biological system can be defined by its spectral metabolic profile. Because of metabolic dynamics caused by coordinated biochemical and molecular events, metabolic profiles are spatial-specific and temporal-dependent in response to developmental state and environmental stimuli, which may mirror tissue-specific and timerelated changes in transcriptomic and proteomic patterns, thus limiting any physiological relevance of single-time-point measurements of gene expression and protein abundance. Moreover, metabolomics may provide the most direct linkage between genetic function,

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metabolic pathway, and physiological process to decipher metabolic networks (e.g. control of glycolysis). In molecular regulation, identifying changes in gene expression using cDNA microarrays is just the start of a long journey from tissue to cell. At this step, the principal aim is to assemble microarray hits into groups for particular metabolic pathways and/or functional processes that provide an intelligible story of a cell’s state, or its metabolic responses to stimuli. Then, it is usual to select a subset of these genes to independently validate changes in their expression. Combination of laser capture microdissection with real-time quantitative RT-PCR is a helpful follow-up step that allows expression of selected genes to be quantified in a pure population of defined individual cells. The voyage from chip to single cell can be completed using sensitive new in situ hybridization and immunohistochemical methods based on tyramide signal amplification to identify cells that express mRNAs and proteins of interest (Mills et al., 2001). Finally, RNA interference can be used as a specific and efficient method to silence gene expression in mammalian cells and to confirm gene function on a wholegenome scale (McManus and Sharp, 2002). In intermediary metabolism, stable isotopic tracer methodology has become the most powerful tool to quantify metabolic fluxes both in the whole body and across an organ. For example, the arterio-venous tracer balance approach and mass isotopomer distribution analysis have been widely used to estimate in vivo enzyme activity (e.g. NOS activity) and nutrient metabolism (e.g. protein synthesis and breakdown, lipogenesis and lipolysis, and gluconeogenesis). In the future, it will be possible to integrate data from transcriptomics, proteomics, and metabolomics to provide an in vivo holistic picture of gene function and metabolic control (Nicholson et al., 2002; Fiehn and Weckwerth, 2003). REFERENCES Aarsland, A., Chinkes, D., Wolfe, R.R., 1996. Contributions of de novo synthesis of fatty acids to total VLDL-triglyceride secretion during prolonged hyperglycemia/hyperinsulinemia in normal man. J. Clin. Invest. 98, 2008–2017. Ackermans, M.T., Pereira Arias, A.M., Bisschop, P.H., Endert, E., Sauerwein, H.P., Romijn, J.A., 2001. The quantification of gluconeogenesis in healthy men by 2H2O and [2-13C]glycerol yields different results: rates of gluconeogenesis in healthy men measured with 2H2O are higher than those measured with [2-13C]glycerol. J. Clin. Endocrinol. Metab. 86, 2220–2226. Adegoke, O.A., McBurney, M.I., Baracos, V.E., 1999a. Jejunal mucosal protein synthesis: validation of luminal flooding dose method and effect of luminal osmolarity. Amer. J. Physiol. 276, G14–G20. Adegoke, O.A., McBurney, M.I., Samuels, S.E., Baracos, V.E., 1999b. Luminal amino acids acutely decrease intestinal mucosal protein synthesis and protease mRNA in piglets. J. Nutr. 129, 1871–1878. Ahlman, B., Charlton, M., Fu, A., Berg, C., O’Brien, P., Nair, K.S., 2001. Insulin’s effect on synthesis rates of liver proteins: a swine model comparing various precursors of protein synthesis. Diabetes 50, 947–954. Antelo, F., Strawford, A., Neese, R.A., Christiansen, M., Hellerstein, M., 2002. Adipose triglyceride (TG) turnover and de novo lipogenesis (DNL) in humans: measurement by long-term 2H2O labeling and mass isotopomer distribution analysis (MIDA). FASEB J. 16, A400. Arcellana-Panlilio, M., Robbins, S.M., 2002. Cutting-edge technology. I. Global gene expression profiling using DNA microarrays. Amer. J. Physiol. Gastrointest. Liver Physiol. 282, G397–G402. Ballmer, P.E., McNurlan, M.A., Milne, E., Heys, S.D., Buchan, V., Calder, A.G., Garlick, P.J., 1990. Measurement of albumin synthesis in humans: a new approach employing stable isotopes. Amer. J. Physiol. 259, E797–E803. Barazzoni, R., Meek, S.E., Ekberg, K., Wahren, J., Nair, K.S., 1999. Arterial KIC as marker of liver and muscle intracellular leucine pools in healthy and type 1 diabetic humans. Amer. J. Physiol. 277, E238–E244. Barle, H., Essen, P., Nyberg, B., Olivecrona, H., Tally, M., McNurlan, M.A., Wernerman, J., Garlick, P.J., 1999. Depression of liver protein synthesis during surgery is prevented by growth hormone. Amer. J. Physiol. 276, E620–E627.

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Index

3-methyl histidine, 72 5′deiodinase, 309, 311, 318 α-cardiac MHC, 360 β-adrenergic agonists, 77, 79, 289–291

A “Absorptive use”, 202 Acetyl CoA carboxylase (ACC), 224, 226 Acute-phase protein, 84, 85, 90–92 Acyl CoA synthetase (ACS), 224, 226 Adaptive immunity, 85 Adipose tissue, 337 Amino acid absorption, 198, 201, 207 Amino acid oxidation, 197, 207 Amino acid requirements, 128 Amino acids, 5, 6, 8–10, 24–27, 29, 35, 51, 53, 55–57, 59, 107–108, 110–113, 116–118, 120–122 Ammonia absorption and liver urea synthesis, 208 Animal growth, 21, 118 Anorexia, 86–88, 90 Apoptosis, 303, 313, 315 Arachidonic acid Functional roles, 13–15 Infant nutrition, 34–40 Metabolism, 8–12, 32–34 Milk composition, 26–29 Placental transfer, 23–26 Arginine, 161, 166, 168–169, 173, 177–180 Arterio-venous tracer balance method, 453 ATP, 356, 359, 361–362, 367

B Blood flow, 361 Brain development Docosahexaenoic acid, 18–23 N-3 fatty acid deficiency, 15–23 Polyunsaturated fatty acid accretion, 29–32 Polyunsaturated fatty acid metabolism, 30–34 Branched chain amino acids, 205 Bromodeoxyuridine (BrdU) labeling assay, 445 Brown adipose tissue (BAT)

β3-adrenergic receptors, 305 Adipocytes, 305 Brown unknown gene, 305 Fatty acid metabolism, 310–311 Fetal development, 305 Morphology, 304 Quantity, 304 Thermogenesis, 304 Uncoupling protein-1, 304, 308–309, 311–312 Brown adipose tissue, 276, 353, 357 Brown unknown gene (BUG), 6, 305

C Calcineurin, 13, 44 Calpain, 51, 84, 98–99 Cardiac output, 353, 356, 361 Carnitine palmitoyltransferase (CPT), 224–225, 229, 379, 383 Catecholamines, 14,18, 20–21, 368 Cathepsin, 84, 99 cDNA microarray, 437 Cell apoptosis, 446 Cell culture, 6, 279 Cell number, 277 Cell proliferation, 445 Cell signalling, 180 Cell-mediated immunity, 85 Chromatin immunoprecipitation assay, 441 Chylomicron, 329–330, 332, 340 Citrulline, 166, 169 Cold, 354–356, 358, 360, 362, 365, 367–368 Colostrum, 2, 4, 15, 17–19, 26, 57–58 Compartment modeling, 449 Composition, 281 Conceptus, 4, 6, 7, 11, 24 Confocal laser scanning microscopy, 444 Conjugated linoleic acid, 290 Constant infusion method, 449 Copper, 318 Corticotropin-releasing hormone, 87 Cortisol, 16, 18–20, 23, 368, 380, 383–384 Cost of urea synthesis, 208

480 CPT-1, 365–366 Cysteine, 169–170, 179, 181 Cytochrome c oxidase, 311–312 Cytokine, 83–100 Cytokines, 178, 182

D Development, 108, 119, 277, 282 Differentiation, 277–280 Distribution, 276 DNase I protection assay, 441 Docosahexaenoic acid Behaviour, 18–23 Brain accretion, 29–32 Brain function, 18–23, 37–40 Dopamine, 19–20 Functional roles, 241–243 Infant nutrition, 254 Metabolism, 237–241, 253–254 Milk composition, 248–249 Neurotransmitters, 19–21 Placental transfer, 23–26 Serotonin, 21 Visual function, 15–18, 36–40

E Electrophoretic mobility shift assay, 441 Endocrine functions, 282 Endocrine regulation–anabolism, 289–290t Endocrine regulation–catabolism, 18–22t Endogenous amino acid secretions, 198, 206 Energy metabolism, 353, 355, 359, 368–369 Energy stores, 356, 363, 369 Enteral, 161, 169–173, 176 Enterocytes, 108–118, 120–121 Essential fatty acids Requirements, 40–43 Deficiency, 7,12,13 Gene expression, 15 Infant nutrition, 34–40 Metabolism, 8–12, 32–34 Milk composition, 26–29 Placental transfer, 23–26 Essential fatty acids, 324, 334–335 Esterification, 329, 339, 342 Eukaryotic initiation factor, 94–96 Eukaryotic initiation factors, 29, 50–53, 55–57 Excess protein digestion, 208 Expression of enzyme data, 18, 287

Index Fatty acid synthesis, 282–290 Fatty acids Metabolism, 310–311 Polyunsaturated (PUFA), 316–317 Saturated, 316–317 Fatty acids, 5, 8–11, 23 Feed intake, 1–9, 83–88, 90, 94 Feeding, 51–57 Fetal programming, 4, 23 Fetus, 5–8, 11, 14–16, 19, 21 First pass metabolism, 141 First-pass utilization, 458 Flooding dose method, 451 Functions, 276

G Gastrocnemius muscle, 90–91, 95–97 Gene expression Polyunsaturated fatty acids, 15 Gene expression, 400 Glucagon, 15, 158, 164, 176–177, 379–380, 383–385 Glucocorticoids, 176, 379, 383–385 Gluconeogenesis, 6, 10, 12, 15, 213, 353, 367, 462–463 Glucose transport, 395 Glucose, 5–8, 10, 12, 14, 17–18, 21, 23, 406 Glucose-6-phosphatase, 377–378 Glutamate, 161, 166–171, 173, 179, 182 Glutamine, 92, 160–161, 166, 168–169, 173, 176–182 Glutathione, 24, 39, 164, 169–171, 179, 181 Glycogen synthesis, 466 Glycogen, 16, 17, 378–381 Glycogenolysis, 466 Glycolytic, 16, 22, 31, 38, 44–45, 48, 53 Growth hormone releasing hormone, 84, 93 Growth hormone, 15–17, 19–20, 22, 24, 29, 37, 41, 45, 56–58, 77, 84, 93, 383–384

H High-density lipoprotein, 333, 341 Humoral immunity, 85 Hydroxy-methylglutarylCoA synthase (HMGCS), 224 Hyperplasia, 277–278 Hypertrophy, 281–282

I F Fast–twitch muscle fibres, 22, 38, 44–45, 53, 55 Fast-twitch muscle, 91 Fatty acid binding protein, 328, 336 Fatty acid oxidation, 342–343, 353

IGF-1, 25, 30–31, 77, 175, 369 Immune system, 83–86, 90, 93, 99–100 Immunonutrients, 39, 157, 179, 183 in situ hybridization, 439 Indicator amino acid oxidation, 128 Innate immunity, 4–5 Insulin receptor substrate, 16, 93

Index Insulin, 12, 14–15, 18–19, 22–23, 28, 30, 46, 50, 53–59, 158, 164, 174, 176, 180–181, 288–289, 292, 379–383, 385 Insulin-like growth factor 1, 16, 20 Insulin-like growth factor 2, 15–16, 18, 20–22 Insulin-like growth factor binding protein, 19–21, 94–96 Insulin-like growth factor-I, 84, 92–96 Insulin-like growth factors, 40–41, 48, 56–58 Interferon, 85, 94 Interleukin-1, 83–91, 93, 97 Interleukin-6, 84–91, 93, 97–99 Interleukin-1 receptor antagonist, 88, 90, 95 Intestine, lipid metabolism, 328 Intrauterine growth retardation, 3, 11, 19, 21, 23 Involution, 310–311 Isotopic labelling, 201–202, 206

K Ketogenesis, 222–223, 225, 227–229, 394, 396–398, 400

L Lactate, 375, 377–383, 386, 410 Laser capture microdissection, 447 Leptin, 19, 22, 89, 337, 341 Leucine, 161, 164, 167–168, 172–173, 176, 180–181 Linoleic acid Placental transfer, 23–26 Deficiency, 7, 12–13 Dietary requirements, 40–43 Functional roles, 13–15 Infant nutrition, 34–40 Metabolism, 8–12, 32–34 Milk composition, 26–29 Linolenic acid (alpha) Deficiency, 7,12–14 Dietary requirements, 40–43 Functional roles, 13–23 Infant nutrition, 34–40 Metabolism, 8–12, 32–34 Milk, 26–29 Lipid degradation, 290f, 292 Lipid digestion, preruminants, 324, 327 Lipid digestion, ruminants, 326–327 Lipid metabolism, 337, 341 Lipid synthesis, 13f, 282 Lipogenesis, 337, 339, 353, 366, 461, 463 Lipolysis, 13, 24–26, 282f, 290–291, 340 Lipopolysaccharide, 6–8, 10–13, 16, 19, 23, 86 Lipoprotein lipase, 13, 20, 282, 288, 330 Lipoprotein metabolism, 329–330, 332, 341 Liver amino acid metabolism, 211 Liver, lipid metabolism, 325, 341 Long chain polyunsaturated fatty acid See also docosahexaenoic acid, arachidonic acid Brain, 243–246, 251–252 Dietary requirements, 253–258

481

Infant nutrition, 254 Metabolism, 237–241, 253–254 Milk, 248–249 Placental transfer, 246–248 Low-density lipoprotein, 341 Lysine, 165, 167, 173, 176

M Mammalian target of rapamycin, 50, 52–53 Mass isotopomer distribution analysis, 462 MCFA, 364–365 Melanocyte-stimulating hormone, 7 Membrane function By contractile activity, 75 By feeding and diet, 12–15, 76 By inflammation and injury, 75t By stress, 76–77 Endocrine and autocrine controls, 15–16, 75 Genetic makeup, 74 Metabolism, 408 Metabolomics, 467 Methionine, 23, 41–43, 169–170, 176–177, 179, 181–182 Methionyl-tRNA, 50 Microflora, 43, 163, 177–178, 182 Milk Polyunsaturated fatty acids, 241–246 Mitochondria, 306, 312, 353, 357–359, 362, 364–365 Molecular aspects–differentiation, 278–279 mRNA quantitative technique, 438 Mucin, 164, 169–170 Multi-catheterization, 202 Muscle, 1, 5, 7–12, 14, 16–18, 25 Muscle hyperplasia, 37, 39–41, 59 Muscle hypertrophy, 40, 47 Muscle, lipid metabolism, 335 Myoblasts, 41–42 MyoD, 39, 41 Myofibres, 37, 40–42, 58 Myofibril, 356, 359–360 Myofibrillar protein, 12, 21, 23–24, 26 Myofibrillar proteins, 38, 42, 46, 53, 57, 59 Myogenesis, 39–40 Myogenic regulatory factors, 24, 27–30 Myogenin, 39, 41 Myosin, 36, 38, 43–44 Myostatin, 40–41, 58, 60 Myotubes, 41–42

N Neuropeptide Y, 87 Newborn mammals, 275–276 Nonesterified fatty acid uptake and oxidation, 460–461 Norepinephrine (NE), 311, 316 Northern blotting analysis, 438 Nuclear magnet resonance spectroscopy, 465

482 Nucleotides, 3, 169, 179–180 Nutritional efficiency, 121

O Ontogeny, 399 Ornithine, 166, 168, 177, 180 Oxidation, 157, 160, 165–168, 171, 173, 177 Oxidative fuels, 161, 165–166, 168, 182 Oxygen, 5–7, 18

P Papillae development, 399 Parenteral, 27, 170, 173 Peroxisomal β-oxidation, 342 Peroxisome, 223–224, 227, 230 Phosphoenolpyruvate carboxykinase, 377–378, 382–385 Pig, 353 Placenta Polyunsaturated fatty acid metabolism, 23 Polyunsaturated fatty acid transfer, 23–26 Placenta, 5–11, 14, 19 Placental lactogen, 16, 18 Polyamines, 168, 170 Portal absorption, 160 Portal-drained viscera, 158–159, 161, 164, 167, 169–170, 173, 176 Portal-drained visceral amino acid sequestration, 198–199 Post mortem, 78 Preadipocytes, 279–280 Proliferation, 44, 157, 163, 169, 173, 178–182 Proline, 10, 20, 161, 163, 166, 168–169 Protease ATP-ubiquitin-dependent, 6, 17, 71, 74 Calpain, 71, 74 Gene expression, 73 Lysosomal, 71, 74 Matrix metalloprotease, 72 Proteasome, 22, 96–97 Protein accretion 83, 83–87, 90–92, 98–100 Protein breakdown, 448, 454 Protein degradation, 3, 23, 25, 48, 51, 56, 59, 70, 83, 90–92, 96–100, 163, 172–173, 180 Determination of, 7 3-methylhistidine, 72 Difference methods, 72 In vitro approaches, 74 Isotopic tracer approaches, 73 Protein kinase B, 53–54, 57 Protein microarray, 444 Protein synthesis, 4, 7, 10, 15, 37, 40, 46–57, 59, 69, 72, 83, 86, 90–95, 100, 157–158, 162, 170, 172–182, 448, 463 Pyruvate carboxylase, 375, 378

Index R Rates, 69, 70 Real-time RT-PCR, 440 Recycling of nitrogen, 198, 207, 214 Redox status, 157, 181–182 Regulation Polyunsaturated fatty acids, 13–23 Regulation-fatty acid synthesis, 282, 284–286 Regulation-lipolysis, 282t Regulation-triacylglycerol, 288–289t, 290 Ribosomal protein S6 kinase, 26, 51–52, 54–55, 57 Ribosomes, 48–49, 57 RNA interference, 436 RNase protection assay, 439 Rumen acidosis, 395 Ruminant, 405

S Satellite cells, 39, 42, 47–48, 58–60 Sepsis, 91, 95–96, 98, 99 Shivering, 356–357, 359–360, 364 Short-chain fatty acids, 414 Skeletal muscle, 14, 19, 21, 70, 73, 83–86, 90–100 Slow-twitch (SO) muscle fibres, 38, 44, 49, 55 Slow-twitch muscle, 91 Small intestine, 107–121 Somatotrophs, 93 Somatotropic axis, 83–84, 92 Somatotropin, 289 β3-Adrenergic receptors, 7, 305, 309

T Thermogenesis, 2, 17, 311–312, 316, 318, 356–357, 368 Threonine, 169, 171, 177, 181 Thyroid hormone, 14, 16, 44–45 Thyroid hormones, 353, 367 Thyroxine, 18 Total parenteral nutrition, 168, 172–173, 177 Tracee release method, 452 Transgenic technique, 435 Translation initiation, 84, 95 Translation, 26, 28, 49–50, 52, 54–57, 59 Transsulfuration, 170, 177, 179, 182 Triacylglycerol synthesis, 21, 288t Triads, 360 Triglyceride synthesis, 461 Triiodothyronine, 18 Tumor necrosis factor binding protein, 90, 95 Tumor necrosis factor-α, 83–86, 88–91, 93, 95, 97–99 TUNEL method, 446 Two-dimensional gel electrophoresis, 443 Tyramide signal amplification, 439

Index U Ubiquitin, 51, 84, 96–97, 99 UCP, 356–357 Uncoupling protein (UCP), 309, 311, 314, 318, 312t, 312f Urea synthesis, 207

V Vagus nerve, 89 Very low-density lipoprotein, 329, 330, 332, 334, 341–342

VFA metabolism, 392–394 Visual function Docosahexaenoic acid, 15–18, 36–40

W White adipose tissue (WAT), 304, 311, 313 Whole-body lipolysis, 460 Whole-body protein kinetics, 448

483