Neuro-endocrinology of Growth and Energy Balance

Neuroendocrinology of Growth and Energy Balance

Guest Editors

Claude Kordon, Paris, France Iain Clarke, Monash, Vic., Australia

12 figures, 3 in color, and 4 tables, 2007

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Cover illustration Metabolic status gates reproduction to guarantee that attempts to reproduce occur only under favorable energetic conditions. See article by Steiner et al., p. 175.

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Vol. 86, No. 3, 2007

Contents

146 Introduction Kordon, C. (Paris); Clarke, I. (Monash, Vic.) 147 Heterogeneity of Ghrelin/Growth Hormone Secretagogue Receptors.

Toward the Understanding of the Molecular Identity of Novel Ghrelin/ GHS Receptors Muccioli, G.; Baragli, A.; Granata, R.; Papotti, M.; Ghigo, E. (Turin) 165 Comparative Aspects of GH and Metabolic Regulation in Lower

Vertebrates Rousseau, K.; Dufour, S. (Paris) 175 Neuropeptide Signaling in the Integration of Metabolism and

Reproduction Crown, A.; Clifton, D.K.; Steiner, R.A. (Seattle, Wash.) 183 Regulation of Food Intake by Inflammatory Cytokines in the Brain Buchanan, J.B.; Johnson, R.W. (Urbana, Ill.) 191 Adipokine Gene Expression in Brain and Pituitary Gland Wilkinson, M.; Brown, R.; Imran, S.A.; Ur, E. (Halifax, N.S.) 210 Histamine and the Regulation of Body Weight Jørgensen, E.A.; Knigge, U.; Warberg, J.; Kjær, A. (Copenhagen) 215 Central and Peripheral Roles of Ghrelin on Glucose Homeostasis Sun, Y.; Asnicar, M.; Smith, R.G. (Houston, Tex.) 229 Roles of Ghrelin and Leptin in the Control of Reproductive Function Tena-Sempere, M. (Córdoba) 242 Subject Index

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Neuroendocrinology 2007;86:146 DOI: 10.1159/000109096

Published online: September 26, 2007

Introduction

This special issue offers a timely update on molecules recently identified as key regulators of Growth and Energy Balance. These include factors that are produced in the brain such as neuropeptide Y, galanin-like peptide, histamine cytokines and products of pro-opiomelanocortin processing as well as humoral factors such as ghrelin and leptin. Recently identified factors, such as kisspeptin, also receive attention. The articles contained in the issue highlight how growth hormone and its peptide regulators exert coordinated actions to control growth, metabolism, glucose homeostasis and appetite as well as reproduction and sleep. A major focus is the arcuate nucleus that appears to receive a range of signals from the periphery as well as other centers in the brain. This allows continual adaptation to maintain metabolic balance. In order to achieve a balanced regulation of these functions, brain structures involved are organized as interactive neuronal networks integrating both internal (hormonal or immune signals) and external (environmental or psychosocial) factors. Stress hormones and cytokines

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can also modulate the function of these networks, providing adaptive mechanisms. This permits the organism to cope in times of emergency with resetting the system during periods of stress or infection. Thus, response to physiological stimuli ensures energy availability under both normal and pathological conditions. Systems controlling growth, reproduction and appetite operate in a synergic manner. Thus, particular neuronal systems within the brain are multifunctional, leading to the subordination of physiological parameters in response to endogenous and environmental challenges. The network allows adaptive processes for the entire organism, which may have evolved to overcome food shortage and emergency situations. This special issue emphasizes the central role that neuroendocrine systems play in the maintenance of homeostasis with special reference to the somatotropic axis and the means by which growth and energy balance can be coordinated. Claude Kordon Iain Clarke

Neuroendocrinology 2007;86:147–164 DOI: 10.1159/000105141

Received: January 5, 2007 Accepted after revision: May 21, 2007 Published online: July 2, 2007

Heterogeneity of Ghrelin/Growth Hormone Secretagogue Receptors Toward the Understanding of the Molecular Identity of Novel Ghrelin/GHS Receptors

Giampiero Muccioli a Alessandra Baragli a Riccarda Granata b Mauro Papotti c Ezio Ghigo b a Division

of Pharmacology, Department of Anatomy, Pharmacology and Forensic Medicine, b Division of Endocrinology and Metabolism, Department of Internal Medicine, and c Division of Pathology, Department of Clinical and Biological Sciences, University of Turin, Turin, Italy

Key Words Ghrelin  Growth hormone secretagogues  Receptor types

Abstract Ghrelin is a gastric polypeptide displaying strong GH-releasing activity by activation of the type 1a GH secretagogue receptor (GHS-R1a) located in the hypothalamus-pituitary axis. GHS-R1a is a G-protein-coupled receptor that, upon the binding of ghrelin or synthetic peptidyl and non-peptidyl ghrelin-mimetic agents known as GHS, preferentially couples to Gq, ultimately leading to increased intracellular calcium content. Beside the potent GH-releasing action, ghrelin and GHS influence food intake, gut motility, sleep, memory and behavior, glucose and lipid metabolism, cardiovascular performances, cell proliferation, immunological responses and reproduction. A growing body of evidence suggests that the cloned GHS-R1a alone cannot be the responsible for all these effects. The cloned GHS-R1b splice variant is apparently non-ghrelin/GHS-responsive, despite demonstration of expression in neoplastic tissues responsive to ghrelin not expressing GHS-R1a; GHS-R1a homologues sensitive to ghrelin are capable of interaction with GHS-R1b, forming heterodimeric species. Furthermore, GHS-R1a-deficient mice do not show evident abnormalities in growth and dietinduced obesity, suggesting the involvement of another receptor. Additional evidence of the existence of another re-

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ceptor is that ghrelin and GHS do not always share the same biological activities and activate a variety of intracellular signalling systems besides Gq. The biological actions on the heart, adipose tissue, pancreas, cancer cells and brain shared by ghrelin and the non-acylated form of ghrelin (des-octanoyl ghrelin), which does not bind GHS-R1a, represent the best evidence for the existence of a still unknown, functionally active binding site for this family of molecules. Finally, located in the heart and blood vessels is the scavenger receptor CD36, involved in the endocytosis of the pro-atherogenic oxidized low-density lipoproteins, which is a pharmacologically and structurally distinct receptor for peptidyl GHS and not for ghrelin. This review highlights the most recently discovered features of GHS-R1a and the emerging evidence for a novel group of receptors that are not of the GHS1a type; these appear involved in the transduction of the multiple levels of information provided by GHS and ghrelin. Copyright © 2007 S. Karger AG, Basel

Introduction

Ghrelin is the result of a story of reverse pharmacology, which started more than a quarter of a century ago with the discovery of synthetic, non-natural growth hormone-releasing peptides (GHRP). The latter are now Ezio Ghigo Division of Endocrinology and Metabolism Department of Internal Medicine, University of Turin Corso Dogliotti 14, IT–10126 Turin (Italy) Tel. +39 011 633 4317, Fax +39 011 664 7421, E-Mail [email protected]

classified as ghrelin-mimetics. In their pioneering works, Bowers et al. [1, 2] demonstrated that small synthetic DTrp2 pentapeptides derived from the natural opiate Met enkephalin, acted at hypothalamic and pituitary sites to stimulate the release of GH. Accordingly, it was hypothesized that the GH-releasing effect of the D-Trp2 GHRP reflected the activity of an unknown endogenous factor or hypophysiotropic hormone distinct from growth hormone-releasing hormone (GHRH). The ability of the hexapeptide GHRP-6 to synergize with GHRH with remarkable potency in men [3], prompted the development of other GHRP-6 analogs (GHRP-1, GHRP-2 and hexarelin) with high potency [4–6] and some orally active peptido-mimetic GH secretagogues (GHS) such as the spiroperidine derivative MK-0677 [7–12]. Particularly remarkable is the broad range of chemistries of the GHS developed in the last few years by several pharmaceutical groups. They consist of low-molecular-weight peptides, partial peptides and non-peptide molecules [6, 13–15]. Several GHRP/GHS candidates were studied clinically, but none have reached the market [6, 11, 12]. GHS bind to specific sites in the rat forebrain [16] and other brain regions such as the cerebral cortex, hippocampus, medulla oblongata and choroid plexus, although the greatest density of binding sites is in the hypothalamus and pituitary gland [17–20]. The GHS receptor was in fact cloned in 1996 from tissues of the hypothalamo-pituitary axis [18]: the codified protein became known as the ‘type 1a GHS receptor’ (GHS-R1a). Three years later, in 1999, Kojima et al. [21] isolated and characterized a natural bioactive ligand for this receptor, which was found to be a small polypeptide primarily secreted by the stomach. This peptide was named ‘ghrelin’ and was described as a ‘GH-releasing substance’. Ghrelin was shortly identified as a motilin homologue or the ‘motilin-related polypeptide’ [22, 23], which had been found capable of stimulating gut motility [24, 25]. Since this time, numerous other neuroendocrine effects have been attributed to ghrelin, such as stimulation of CRH, ACTH, PRL secretion and inhibition of GnRH and gonadotropin release [26–28]. In addition, non-endocrine and metabolic activities of ghrelin have also been described, notably stimulation of food intake and gut motility [27, 29–33], influences on body weight and energy balance [34], insulin release, glucose and lipid metabolism [35–43], sleep [44, 45], behavioral responses to stress [46–48], learning and memory [49], improvements of cardiovascular performances [50–54], effects on cell proliferation, differentiation, survival and fetal development [55–61], besides influencing immunological responses [62, see also for reviews 63–66]. The 148

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ghrelin gene resides on chromosome 3p25-26, has five exons and four introns and produces two distinct mRNAs, which start at position –80 or –555. The main mRNA codifies for the 117-amino-acid precursor polypeptide ‘preproghrelin’ which, through enzymatic processing, leads to ‘proghrelin’ and subsequently to mature ghrelin and a C-terminal polypeptide [67, 68]. Mature ghrelin consists of 28 amino acids and it is esterified by an octanoyl group at a serine 3 residue; this is a special feature of this hormone which is conserved across species. Acylation is necessary for ghrelin binding to GHS-R1a [69], GH-releasing activity in vivo [70] and other central and peripheral endocrine and non-endocrine actions [71–73] of the peptide. The modification also determines the extent and the direction of ghrelin transport across the blood-brain barrier [74]. Although the active site for ghrelin action appears to be 4–5 amino acids of the amino terminal end of the molecule including the acyl group [69, 75, 76], short ghrelin peptides consisting of residues 1-8 neither displace ghrelin binding to pituitary and hypothalamic membranes nor stimulate GH release in vivo [70]. Quite on the contrary, in GHS-R1a-transfected cells and in adipocytes, the acylated fragments longer than 4 amino acids all maintain affinity and potency similar to that of ghrelin [69, 77, 78]. In humans, multiple ghrelin-derived molecules differing in their acyl groups on serine 3 (decanoyl or decenoyl-esterified molecules) have also been isolated from the stomach and found in the bloodstream [79]. These acyl-modified ghrelins are capable of stimulating GH release in rats to a degree similar to that of octanoylated ghrelin [79]. In addition to the acylated form, unacylated ghrelin (UAG) is found in circulation at far greater concentrations than ghrelin (octanoylated/des-octanoylated ratio 1: 4) [80] suggesting physiological relevance. UAG, however, does not bind to GHS-R1a [80], does not displace ghrelin binding to rat hypothalamus or pituitary membranes and it is unable to stimulate GH release in vivo [70, 80], so it was initially considered inactive [21]. A conspicuous number of recent reports, however, describe non-endocrine activities of UAG [63–65] breaking a paradigm lasting from 1999. Besides ghrelin, a wide variety of polypeptides is encoded by the ghrelin gene. Alternate splicing phenomena, post-translational modifications and differential processing create a number of products, some of which, e.g. des-Gln14-ghrelin, are found in circulation with the same biological activities as ghrelin [81, 50]. Other products originating in central and peripheral tissues are devoid of any endocrine effect Muccioli /Baragli /Granata /Papotti /Ghigo

[82–87]. Amongst these is the 23-amino-acid polypeptide obestatin that may be an appetite suppressant [84], although this is widely debated [86, 87] and various exon-deleted proghrelin mRNAs, which are expressed in neoplastic tissues [82, 83]. The multiplicity of ghrelin/ GHS effects prompted the question as to whether GHSR1a is the single receptor that mediates action or whether other receptors are involved [54, 65]. In particular, UAG appears to possess biological activities arguing for the role of other receptors to mediate effects of the ghrelin family.

Type 1a GHS Receptor

The GHS receptor gene is located on chromosome 3q26.2 and encodes for two transcripts, 1a which encodes a full-length receptor (GHS-R1a) and 1b which codifies for a shortened version (GHS-R1b) [88]. In human fetuses, GHS-R1a mRNA is detected at 18 and 31 weeks of gestation, indicating that ghrelin might be active early in development [59, 89]. Significant expression of GHS-R1a mRNA is evident in the pituitary gland and several endocrine and non-endocrine tissues [90] as well in the central nervous system (CNS), where GHS-R1a is found both in the cortex and in the midbrain [17, 91–93]. This is consistent with observations that ghrelin affects synaptogenesis and development of neuronal circuits, as well as modulation of memory processes, sleep patterns and behavior [44–49, 94–96]. Within the hypothalamus-pituitary axis, GHS-R1a mediates ghrelin/GHS modulation of GH, PRL, CRH/ACTH and GnRH/gonadotropin secretion [26–28, 97–102]. Orexigenic effects of ghrelin/GHS are likely mediated by GHS-R1a expressed by hypothalamic neurons containing neuropeptide Y (NPY)/Agouti-related protein [34, 103–106]. Additionally, centrally located GHSR1a was found to mediate ghrelin activity on gastric acid secretion [107]. Peripheral GHS-R1a is responsible for ghrelin inhibitory effect on pancreatic insulin secretion [42, 43], for the prevention of oxidative stress [108, 109] and for GHS control of pro-inflammatory and immune responses [62, 110, 111]. Finally, GHS-R1a has also been found in neoplastic tissues, where it mediates the stimulatory effect of ghrelin on neoplastic cell growth [58, 83].

receptors (GPCRs) [114]. GPCRs span the membrane with seven -helix hydrophobic domains forming the receptor core, joint each other by three alternate intra- and extracellular domains, beginning with an extracellular N-terminal domain and ending with an intracellular Cterminal domain [114]. GHS-R1a possesses the three conserved residues Glu-Arg-Tyr at the intracellular end of transmembrane 3 (TM3) domain, in position 140–142 (ERY/DRY motif), which are important for the isomerization between the active and inactive conformation (see below), and the two cysteine (Cys) residues on the extracellular loop 1 and 2 forming a disulfide bond [113, 114]. Based on its deduced peptide sequence, GHS-R1a is not obviously related to known subfamilies of GPCRs, although it is often included in a small family of receptors for small polypeptides comprising the receptor for motilin (52% homology), neurotensin receptor-1 (NTS-R1) and NTS-R2 subtype (33–35% homology), neuromedin U receptor-1 (NMU-R1) and NMU-R2 subtype (;30% homology), and the orphan receptor GPR39 (27–32% homology) [115, 116], named GHS-R1a homologues. Also, the thyrotropin-releasing hormone receptor possesses high homology (56%) to GHS-R1a [113]. Comparison of the predicted human rat, pig and sheep GHS-R1a amino acid sequences reveals 91.8–95.6% sequence homology [117].

Structure The human GHS-R1a is a polypeptide of 366 amino acids with a molecular mass of approximately 41 kDa [18, 112, 113] and belongs to family A of G-protein-coupled

Ligands and Ligand Binding Domains Ghrelin and its natural acylated variants, as well as synthetic GHS bind with high affinity to the GHS-R1a. Their efficacy in displacing radiolabeled non-peptidyl GHS ([35S]MK-0677) or [125I]Tyr4-ghrelin binding from pituitary membranes or the cloned GHS-R1a correlates well with concentrations required to stimulate GH release [17, 69, 118]. By contrast, UAG does not bind GHSR1a [69, 70, 118]. Adenosine was initially proposed to be a partial agonist for GHS-R1a [119–121], but this has been questioned [122, 123]. A series of other molecules apparently unrelated to ghrelin have also been shown to bind GHS-R1a, such as the natural SRIH-like neuropeptide cortistatin, some synthetic SRIH-mimetic octapeptides (octreotide, lanreotide and vapreotide) [124–126] and the atypical L-type Ca2+ channel blocker diltiazem [127]. Our own findings support the hypothesis that cortistatin (CST) acts as an endogenous antagonist for GHS-R1a [124–126]. In fact, CST not only binds all five SRIH receptor subtypes (SRIH-Rs), but also displaces radiolabeled ghrelin from its pituitary-hypothalamic binding sites and diminishes ghrelin secretion in humans [124, 128]. Recently, a synthetic CST-derived octapeptide,

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CST-8, which does not bind to SRIH-Rs, was shown to interact with GHS-R and to remove the inhibitory action of ghrelin on gastric acid volume and acid output [107]. An inverse agonist for GHS-R1a is the synthetic D-Arg1D -Phe5-D -Trp7,9-Leu11-substance P, a substance P and bombesin antagonist [129]; this is able to reduce constitutive signalling of GHS-R1a overexpressed in COS-7 cells [130, 131], but an endogenous counterpart remains to be identified. Most recently, shorter peptides derived from D -Arg1-D -Phe5-D -Trp7,9-Leu11-substance P were shown to display inverse agonist activity on GHS-R1a as well [131]. Finally, GHS-R1a antagonists are also available, such as D-Lys3-GHRP-6, L765-867 [6, 7, 120], isoxazole, diaminopyrimidine and triazole derivatives [132–134]. Antibodies and RNA-Spiegelmers have been also developed for the blockade of GHS-R1a activity [135, 136]. Ligand binding to receptors is believed to stabilize the active conformation. While the main binding pocket for small amines is deep in the cavity created by the TM domains, small peptides also bind to extracellular epitopes. According to the general model based on the 2-adrenergic receptor and the rhodopsin receptor, either methods of binding result in an alteration of receptor molecular structure leading to a reciprocal re-arrangement of the -helices with vertical seesaw movements of TM6 and TM7 around a pivot represented by the proline residues in the middle of these helices [137]. Hence the intracellular end of TM6 and TM7 move away from the center of the receptor toward TM3, exposing the sites subsequently recognized by G-protein and -arrestin [137]. The ‘toggle switch model’, as it is known, is applicable to GHS-R1a, with a binding domain for the natural ligand ghrelin involving six amino acids located in TM3, TM6 or TM7 [131]. According to Pedretti et al. [138], ligand binding and activation of GHS-R1a by ghrelin requires the ligand to interact with one pocket formed by polar amino acids and one formed by non-polar amino acids found in TM2/TM3 and TM5/TM6, respectively. On the contrary, the inverse agonist D-Arg1-D-Phe5-D-Trp7,9Leu11-substance P requires a wider binding pocket, dispersed across the main binding crevice [131]. Concerning the synthetic peptidyl and non-peptidyl GHS, they share a common binding pocket in the TM3 region of the GHSR1a, although there are other distinct binding sites in the receptor that appear to be selective for particular classes of agonists [139]. The basic amine common to peptidyl (GHRP-6) and non-peptidyl (MK-0677) GHS likely establishes an electrostatic interaction with Glu124 in the TM3 domain [139], as substitution of glutamine for glutamic acid [Glu124Gln mutant] in human GHS-R1a in150

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activates receptor function. Also, mutating Arg283 in TM6, which interacts with Glu124 eliminates both agonist-induced activation and constitutive signalling [130, 139]. Finally, the activity of all agonists can be completely abolished by disrupting the disulfide bond between Cys116 and Cys198 in the extracellular portion of the receptor, by mutating Cys116 into alanine [Cys116Ala mutant] [117, 139]. Prototypical and Alternative Signalling Once bound to GHS, activated GHS-R1a normally binds the Gq/11 subunit of a G-protein, which leads to activation of phospholipase C (PLC) and consequent hydrolysis of membrane-bound phospholipids to generate inositol (1,4,5)-triphosphate (IP3) and diacylglycerol (DAG) [18, 19]. The intracellular free calcium (Ca2+) concentration increases because of the rapid, though transient, release of Ca2+ from IP3-responsive cytoplasmatic storage pools and because of a more sustained accumulation of Ca2+ due to the activation of L-type Ca2+ channels. Together with the blockade of potassium (K+) channels, the intracellular rise in free Ca2+ provokes depolarization of the somatotropes and release of GH [18, 19, 140]. In addition to the ‘prototypical signalling’ of GHS-R1a, species-, tissue- and ligand-specific second messenger pathways are activated by various GHS in an ‘alternative-signalling mode’. In sheep, but not in rat pituitary cells, GHRP-2 activates the adenylyl cyclase/cyclic adenosine monophosphate/protein kinase A (AC/cAMP/PKA) pathway, while GHRP-6 and non-peptidyl GHS act through the PLC pathway to enhance GH release [141]. In porcine somatotropes, ghrelin-stimulated GH secretion depends on activation of PLC/PKC, AC/PKA and extracellular Ca2+ influx through L-type voltage-sensitive channels, as well as on nitric oxide/cyclic guanosine monophosphate (cGMP) signalling, suggesting that multiple signalling pathways originate from GHS-R1a [142– 144]. In GH3 cells, ghrelin modulates K+ currents through cGMP production [145], while in primary cultures of rat pituitary cells GHRP-6 stimulates Na+ conductance [146]. Because of the opening of N-type Ca2+ channels, which are modulated by cAMP-dependent PKA activation, GHS-R1a was suggested to couple to Gs in NPY neurons [147]. Although there is evidence linking GHS-R1a to Gs, protein/protein interaction between GHS-R1a and Gs was never shown in those cells or in heterologously expressing ones. A GHS-R1a homologue also displays similar pleiotropy, NTS-R1: it preferentially couples to Gq/11, although, depending on the cell type, it is capable of increasing cGMP, cAMP and IP3, as well as modulating exMuccioli /Baragli /Granata /Papotti /Ghigo

tracellular-signal-regulated kinase-1 and -2 (ERK1/2) signalling [148–151]. Besides pleiotropy, a cross-talk between Gq and Gs might be responsible for the multiple effects of GHS-R1a, as demonstrated for diacylglycerol-activated PKC stimulation of adenylyl cyclase [152, 153]. Different ligands may also induce different conformations of GHS-R1a, favoring coupling to different G-proteins and activation of dissimilar signal transduction pathways, similarly to other GPCRs [154]; nevertheless, a more appealing hypothesis is that more than one receptor for GHS is endogenously expressed [141] and that GHS-R1a can heterodimerize with other GPCRs or membrane proteins creating new binding sites distinct in terms of pharmacological and functional properties (see below). Although ghrelin can induce ERK phosphorylation in cells overexpressing GHS-R1a [130] involving novel isoforms of PKC [155] and increase the activity of both the transcription factor cAMP-responsive element (CRE)binding protein and of serum-responsive element (SRE) [130, 156], several reports indicate a role for ghrelin in cell survival through such pathways, but independently from GHS-R1a activation [56, 60, 61, 157, 158].

C40: this might indicate that GHS-R1a activity may be turned off or on depending on the cellular context [140]; so far the only known ligand blocking GHS-R1a constitutive activity is D-Arg1-D-Phe5-D-Trp7,9-Leu11-substance P [130, 131]. The molecular basis of such constitutive activity appears to relate to three aromatic residues located in TM6 and TM7, namely PheVI:16, PheVII:06 and PheVII:09. This region promotes the formation of a hydrophobic core between helices 6 and 7, to ensure proper docking of the extracellular end of TM7 into TM6, mimicking agonist activation and stabilizing the receptor in the active conformation [156].

Constitutive Activity The number of GPCRs displaying constitutive activity with clear physiological implications is not vast, but it is steadily growing [159]. Several reports have now demonstrated the physiological relevance of GHS-R1a constitutive activity. In a study by Wang et al. [160], Phe279Leu (corresponding to PheVI:16) and a Ala204Glu polymorphisms were found in one obese and one short stature child. A recent study has re-stated that an Ala204Glu missense allele segregates with both short stature and obesity. Surprisingly the derived GHS-R1a receptor displayed reduced basal activity and lower expression at the plasma membrane when transfected to HEK-293 cells [161]. It should be noted that, although constitutive activity of the receptor had been lost, responsiveness to ghrelin was intact [161, 162]. Both studies indicate that constitutive activity of GHS-R1a in vivo might be mandatory for proper growth and development of the human body [162]. When overexpressed in COS-7 cells, GHS-R1a possesses a constitutive activity in terms of the turnover of IP3, which is approximately 50% of the maximal agonistinduced activity [130, 131]. In HEK-293 cells, GHS-R1a transfection led to constitutively stimulated CRE and SRE activity [130]. Intriguingly, GHS-R1a did not show any constitutive activity in the pituitary cell line RC-4B/

Modulators of Receptor Signalling In the past 2 years a blossoming of papers has addressed the issue of ‘fine tuning’ of GHS-R1a signalling through multiple mechanisms. In this section, three distinct aspects of this regulation are discussed: ago-allosteric modulation, heterodimerization and receptor internalization. Allosteric modulators increase (positive allosterism) or decrease (negative allosterism) the potency (EC50) of the agonist, shifting the dose-response curve to the left or to the right, respectively. Most recently, the term ‘ago-allosterism’ has been used to indicate compounds, which not only modulate potency, but also agonist efficacy (Emax), being classified as full or partial agonists: two non-peptidyl (L-692,429, MK-0667) and one peptidyl agonists (GHRP-6) of GHS-R1a fall into this category, L-692,429 improves ghrelin potency and efficacy (positive ago-allosterism), MK-0667 has no effect on ghrelin potency but increases its efficacy (neutral ago-allosterism), while GHRP6 reduces ghrelin potency, though increasing its efficacy (negative ago-allosterism) [163]. Since ago-allosterism has been recently described for dimeric GPCRs [164, 165], likewise GHS-R1a may behave as dimeric receptor, with one ‘orthosteric’ protomer binding ghrelin and one ‘allosteric’ protomer binding the ‘ago-allosteric’ compound resulting in a modulation of both ghrelin potency or efficacy [163, 166]. With regard to heterodimerization, the synergistic effect of ghrelin and GHRH on GH secretion [167], the lack of effect of peripheral ghrelin once GHRH antagonists are administered in men [168–170] and the modulation of GHS-R1a expression by GHRH both in hypothalamic neurons and pituitary cells [171–175], suggest a strong interplay between these two hormonal systems which may involve heterodimer formation [176]. Although GHS- and GHRH-activated intracellular signalling pathways differ in pituitary cells, in that GHS increases free

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intracellular Ca2+, while GHRH increases cAMP levels, when applied together, intracellular accumulation of cAMP raises above the GHRH-stimulated level [177]. Although PKC appears involved [153], heterodimer formation between GHRH-R and GHS-R1a mainly triggers this potentiation [177]. Similarly, Jiang et al. [178] have recently reported about the heterodimeric interaction between GHS-R1a and dopamine D1 receptor subtype, which resulted in enhanced signalling through the AC/ cAMP pathway. Thus, GHS-R1a heterodimers represent novel receptors, which bind ghrelin but may display different pharmacological and functional properties. This likely accounts for at least some of the ‘alternative signalling’ previously described. In response to agonists, GPCRs undergo desensitization through phosphorylation, uncoupling from G-proteins and internalization. This may lead, depending on the receptor, to their recycling back to the plasma membrane or passage into lysosomes for degradation [179]. In cells overexpressing GHS-R1a, Camina et al. [180] demonstrated that prolonged administration of ghrelin or hexarelin determined a rapid attenuation of receptormediated Ca 2+ accumulation and a conspicuous receptor internalization through clathrin-coated pits within 20 min. On the other hand, Holst et al. [156] reported constitutive internalization of the ghrelin receptor, which could be prevented by the presence of its inverse agonist D -Arg1-D -Phe5-D -Trp7,9-Leu11-substance P. Although modulation of constitutive receptor internalization by inverse agonists is common to other receptors [181–184], the above-mentioned experimental discrepancies suggest that additional studies are needed to clarify the situation. Conversely, in in vivo studies, GHSR1a upregulated during fasting [185, 186], and during the hyperdynamic phases of sepsis in male adult rats [187].

Non-Type 1a GHS Receptors

If heterodimeric receptor complexes may partially account for the GHS-R1a ‘alternative signalling’ in response to ghrelin, it is likely that other, as yet unidentified receptors exist. In fact, remarkable differences in the binding profile among ghrelin, synthetic peptidyl (hexarelin) and non-peptidyl (MK-0677) GHS have been reported [188– 191], mostly in tissues that do not express GHS-R1a or express the receptor at a low level. For instance, the heart possesses GHS-binding sites specific for peptidyl GHS only [190–192]. The existence of various GHS-R1a homo152

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logues and of a splice variant GHS-R1b, the lack of a clear phenotype in GHS-R1a knockout mice and female rats genetically deficient of GHS-R1a, as well as the presence of multiple endogenous ghrelin-like ligands, strongly suggest the existence of multiple receptors for ghrelin and GHS. GPCR Homologues of GHS-R1a All GHS-R1a homologues bind gastrointestinal or neuronal peptides except for GPR39, for which a ligand is still unknown [87, 116]. The highest homology is shared by GHS-R1a and the GPR38 receptor for motilin [113, 115]. The ghrelin and motilin genes are also structurally similar, despite the encoded polypeptides being different. GRP38 is less widely expressed in the neuroendocrine tissues than GHS-R1a, being mostly confined to the thyroid gland, bone marrow, stomach and gastrointestinal smooth muscles. Nevertheless, both receptors mediate pulsatile biological effects upon continuous stimulation and increase gastrointestinal motility. In response to motilin, GRP38 increases cytosolic free Ca2+, consistently with IP3-dependent Ca2+ release, mediated by its coupling to Gq/G13 [193]. The other GHS-R1a homologue, GRP39, remains an orphan receptor since the recent controversy whether or not it binds obestatin, a sub-product of the ghrelin gene, has been solved [84, 85, 194–197]. Its structure is obviously similar to the GHS-R1a with the fundamental amino acids being conserved in the key sites for receptor function [115]. GRP39 transcripts are detected in many tissues, but mostly in brain regions [115]. In contrast to GHS-R1a and GRP38, GRP39 has a very long C-terminal and two potential palmitoylation sites (C360-1), which creates a 4th intracellular loop [115, 198]. Activation of GRP39 by zinc (Zn2+) leads to PLC signalling, activation of CRE- and SRE-dependent transcriptional activity, and also cAMP production [87]. Besides receptor sequence homology with GHS-R1a, GPR39’s relationship with the ghrelin system remains elusive and unexplored yet. Neuromedin U is a 23- to 25-amino-acid polypeptide which is the ligand for other members of this receptor family, the NMU-R1 and NMU-R2 receptors. NMU-R1 and NMU-R2 have a 40–50% homology and both are about 30% homologous with GHS-R1a and the neurotensin receptors [199]. While the NMU-R1 subtype is mostly expressed in the periphery, NMU-R2 is mostly expressed in the brain [199]. Both receptors are implicated in smooth muscle contraction, in the regulation of gastric acid secretion, insulin secretion, ion transport in the gut, feeding behavior and stress [199]. Both NMU-R1 and Muccioli /Baragli /Granata /Papotti /Ghigo

NMU-R2 signal through activation of Gq/11, PLC and Ca2+, with the R2 subtype stimulating arachidonic acid production through activation of PLA2 [199]. NMU is highly conserved among species, is widely distributed in the body and expressed at high levels in the brain where it mediates effects on food intake opposite to those of ghrelin [200], probably by cross-talking with the anorectic polypeptide leptin system [200, 201]. NMU also appears to play some function in the growth of neoplastic cells with expression being downregulated in cancer [202]; in fact it was demonstrated to inhibit esophageal squamous cell carcinoma cell growth [203]. A most intriguing discovery was that in absence of its receptors, neuromedin was capable of stimulating the heterodimeric receptor GHS-R1b/NTS-R1 in order to induce lung cancer cell proliferation [204]. Although it is difficult to make conclusions on the basis of one single work, the hypothesis of heterodimeric assortment between ghrelin receptor homologues as a mean to create pharmacological and functional diversity is an extremely appealing one. Neuromedin might thus function as a mediator of the cross-talk between ghrelin receptor homologues, which, at least in cancerous cells, leads to aberrant signalling. The other group of receptors included in the ‘ghrelin superfamily’ are those for NTS, namely NTS-R1 and NTS-R2. NTS-R1 mainly functions through Gq/11-PLC, but it is also capable of activating cGMP, stimulating cAMP, inositol phosphate signalling and ERK1/2 phosphorylation [148–151, 205, 206], while NTS-R2 activation signals via increased Ca2+ accumulation and ERK phosphorylation [207–209]. Similar to GHS-R1a, a splice variant of the NTS-R2 has been recently found, bearing only the first five TM domains, albeit pharmacologically and functionally active [210]. NTS is a 13-amino-acid polypeptide, with biological function being ascribed to the C-terminal portion. The peptide has high sequence homology with neuromedin N. In the CNS, NTS-Rs have been found in the hypothalamus, amygdala and nucleus accumbens, being involved in modulation of the dopaminergic system, but neurotensin also acts in the small intestine endocrine cells where it increases acid secretion and regulates smooth muscle contraction [205]. Besides their sequence homology and signalling similarities, to date heterodimerization of GHS-R1b and NTS-R1 is the only available evidence for a functional cross-talk amongst GHS-R homologues with physiological implications [204].

Type 1b GHS Receptor Type 1b GHS receptor (GHS-R1b) is a splice variant of the GHS-R1a. GHS-R1b is a truncated receptor, containing 298 amino acids corresponding to the first five TM domains (encoded by exon 1), plus a unique 24-amino-acid ‘tail’ encoded by an alternatively spliced intronic sequence. In GHS-R1b-transfected cells, the receptor did not bind ghrelin or GHS and the cells did not respond to these ligands [19]. It was concluded, therefore, that this receptor was not of biological significance. Nevertheless, since it is widely expressed in many normal GHSR1a-positive or -negative tissues [90], it is likely that this receptor possesses biological functions. A recent report has revealed that GHS-R1b acts as a repressor of the constitutive activity of GHS-R1a when overexpressed in HEK-293 cells [211]; thus GHS-R1b may represent an endogenous candidate for GHS-R1a modulation. The presence of the 1b form in neoplastic tissues and its overexpression in growing and differentiating cells [57, 212– 214] supports a significant role in tumor progression. In lung cancer-derived cell lines, NMU induced cell proliferation through binding to the heterodimeric receptor GHS-R1b/NTS-R1 [204]; it is then apparent that GHSR1b is at least capable of forming heterodimers with fulllength GPCRs receptors, causing altered biological properties compared to the original receptor, a fascinating behavior which is indeed shared by neurotensin truncated receptor 2 [210].

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Ghrelin/GHS-Binding Sites Shared by Non-Acylated Ghrelin An early report on the physiological role of non-acylated ghrelin (UAG) demonstrated that, together with ghrelin and other GHS, this natural ghrelin variant prevented cell death of cultured cardiomyocytes and endothelial cells by activation of ERK1/2 and kinase B/AKT [56]. This evidence implies that UAG is a biologically active peptide, which exerts its function through a receptor that is not GHS-R1a [56]. UAG is also active on isolated guinea pig papillary muscle where exerts negative inotropic effects similar to those of ghrelin and peptidyl GHS [50]. The potency of these compounds correlates with their ability to compete with [125I]Tyr4-ghrelin, indicating a common binding site [50]. In the past 3 years an increasing number of ghrelin-sensitive tissues or cell lines have been shown to be UAG-responsive, occasionally with different pharmacological and functional profiles. Both ghrelin and UAG were shown to stimulate neurogenesis of rat fetal spinal cord [215] and to augment osteoblast proliferation through the ERK/PI3K pathways 153

during the differentiation period, despite the undetectable expression of GHS-R1a [61]. Similarly, both ghrelin and UAG modulate the survival and proliferation of pancreatic  cells and human islet cells [60]. Interestingly, in bone marrow of GH-deficient rats, both ghrelin and UAG, but not the potent GHS-R1a agonists such as L163255, stimulated adipogenesis, thus excluding a ghrelin/ GHS-R1a-mediated effect [39]. Similarly, rat adipocytes isolated from epididymal adipose tissue that did not express mRNA for GHS-R1a, specifically bound radiolabeled ghrelin, which was equally displaced by both unlabelled ghrelin and UAG and slightly less potently by hexarelin and MK-0667 [78]. Moreover, UAG inhibited isoproterenol-stimulated lipolysis in rat epididymal adipocytes in a manner similar to ghrelin [78]. If ghrelin, UAG and GHS mostly mediate cell proliferation and/or differentiation of normal tissues by activating this ‘shared receptor’, they exert opposite effects on the growth of some neoplastic cell lines derived from human prostate carcinomas [58], raising the issue as to whether the receptor is the same but the environment has evidently changed, or if a different receptor is involved [57]. With a comparable pattern, ghrelin, UAG and some GHS (MK0667 and hexarelin) displace 125I-Tyr4-ghrelin binding to membranes of cells from human prostate carcinomas and the PC-3 prostate cancer cell line, which do not express GHS-R1a or GHS-R1b. This pattern of competition, however, is also seen in DU-145 prostate cancer cells, which do express GHS-R1a and GHS-R1b [58]. These binding studies indicate the presence of a specific binding moiety, common for ghrelin, UAG and GHS, which is likely to be involved in mediating the antiproliferative effects of ghrelin/GHS molecules, which is not the 1a or 1b receptor. The involvement of non-GHS-R1a receptors in the control of neoplastic cell growth is consistent with their presence only in the tumoral stages of tissues derived from organs that normally do not express them, e.g. breast [55]. In breast tumors, the highest binding activity is present in well-differentiated invasive breast carcinomas and is progressively reduced in moderately to poorly differentiated tumors [55, 57]. GHS-R are also present in both estrogen-dependent (MCF7 and T47D) and estrogen-independent (MDA-MB231) breast cancer cell lines, in which ghrelin, synthetic GHS and EP-80317 (a hexarelin analog devoid of GH-releasing effect) inhibit cell proliferation at concentrations close to their binding affinity [55]. The above evidence clearly demonstrates that at least one receptor exists, different from 1a or 1b which is shared by the acylated and non-acylated form of ghrelin, eventually recognized by GHS. 154

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Receptors for Non-Acylated Ghrelin That Are Not Shared by Ghrelin The existence of receptors which distinguish between ghrelin, UAG and selective GHS was suggested by the finding that in isolated pig primary hepatocytes, lacking GHS-R1a mRNA expression, ghrelin increased glucose release, hexarelin was ineffective, while UAG antagonized not only ghrelin-induced but also glucagon-induced glucose release [40]. In humans, the combination of ghrelin and UAG prevented ghrelin-induced reduction in the peripheral sensitivity to insulin [216] and eliminated the negative ghrelin effect on insulin release and elevation of glucose levels [217]. These findings suggest potentially antagonistic roles of the two molecules. Two receptors may be responsible for such opposite effects, nevertheless it is possible that both ghrelin and UAG bind to the same receptor in a competitive manner, activating two different signalling pathways. The existence of a UAG receptor not shared by ghrelin was strongly suggested by Gauna et al. [218]. These data indicated that both ghrelin and UAG stimulated insulin release in insulinoma INS-1E cells, but while ghrelin acted through GHS-R1a because its effect was completely blocked by the GHS-R1a antagonist D-Lys3-GHRP-6, UAG appeared to interact with a different receptor, since its effect on insulin secretion was not blocked by D-Lys3-GHRP-6 [218]. Recently, the capability of GHS-R1a only (and consequently of acylated ghrelin only) to influence food intake has been revised since UAG was shown capable of modulating appetite itself in several experimental models. If centrally administered, UAG stimulates appetite in GHSR1a knockout mice insensitive to the ghrelin orexigenic activity, probably through stimulation of an unidentified receptor localized on orexin neurons [219]. On the contrary, UAG has been reported to prevent ghrelin-induced food intake in goldfish [220]. GHS-R not only include receptors for ghrelin and/or UAG, but there is evidence of receptors which do not bind ghrelin/UAG and are specific for synthetic peptidyl GHS only. The only one of these receptors that is well characterized is CD36 (see below). Nevertheless, it is worth mentioning the existence of binding sites for hexarelin shared by other peptides structurally related to hexarelin such as GHRP-6 and EP-80317, but not by ghrelin, MK0667 and EP9399 (a cyclic derivative of hexarelin), which were found in CALU-1 lung carcinoma cell line, and may be capable of reducing cell growth stimulated by insulinlike growth factors (IGFs) [221].

Muccioli /Baragli /Granata /Papotti /Ghigo

CD36 Receptor for Synthetic Peptidyl GHS Specific binding sites for Tyr-Ala-hexarelin and other peptidyl GHS, which appear to exist in greater amounts than GHS-R1a, have been found in some endocrine glands (pituitary, thyroid and adrenal) and in a wide range of non-endocrine peripheral animal and human tissues such as heart, lung, arteries, skeletal muscle, kidney, liver, uterus and adipose tissue [190, 191]. These binding sites are presumably different to GHS-R1a because they show a very low binding affinity for non-peptidyl GHS such as MK-0677 and no binding affinity for ghrelin. Studies utilizing a hexarelin derivative, Tyr-BpaAla-hexarelin, that is photoactive and has the same biological activities as the native molecule, demonstrated the existence of a GHS-R subtype in human and bovine pituitary with a molecular weight of 57 kDa [188] and a second receptor subtype for peptidyl GHS in rat heart with a molecular weight of 84 kDa [192]. The tripeptide EP-51389 is as effective as hexarelin in stimulating GH secretion in the rat, but is far less effective in protecting the heart from ischemia [222]. Binding experiments revealed that EP-51389 effectively displaced hexarelin from hypothalamic binding sites, but poorly from cardiac membranes [223], confirming that different GHS-R are present at the two levels. Recently, CD36, a multifunctional class B scavenger receptor expressed in many tissues, including microvascular endothelium, skeletal and smooth muscle cells and monocytes/macrophages [224], was identified as a peptidyl GHS cardiac binding site [192, 225]. CD36 is implicated in multiple physiological functions (i.e. antigen presentation, cellular adhesion, fatty acid/lipid transportation and modulation of vascular tone), as well as pathophysiological processes related to the formation of macrophage foam cell and atherosclerotic lesions [224]. In a perfused heart preparation, hexarelin elicited vasoconstriction perhaps via CD36, since no similar effect occurred in CD36 null mice and rats [225]. Since ghrelin and MK-0677 do not share all the cardiotropic actions of peptidyl GHS [222, 226–228], it has been suggested that the reason for this discrepancy could reflect a different interaction of ghrelin, hexarelin and MK-0677 with a heterogeneous population of cardiovascular GHS-R: some (GHS-R1a) recognized by ghrelin, hexarelin and MK-0677 [19, 21], some specific for ghrelin, UAG and hexarelin [50, 56, 226, 227] and others specific (CD36) for hexarelin alone [188, 190–192]. It has also been demonstrated recently that hexarelin inhibits accumulation of cholesterol as oxidized low-density lipoprotein (oxLDL) in macrophages through CD36 by interfering with the binding of oxLDL on the same interaction

site on CD36 [229]. In addition, hexarelin acting through both CD36 and GHS-R1a enhances expression of the ABCA1 and ABCG1 transporters which improve cholesterol efflux from macrophages [229]. In a manner similar to hexarelin, EP-80317, a hexarelin analog devoid of GHreleasing effect, also reduced internalization of oxLDL and increased cholesterol efflux in macrophages, resulting in a decreased number of atherosclerotic lesions in apolipoprotein E-deficient mice fed with atherogenic diet [230].

The physiological role of the ghrelin system has been matter of debate for at least three decades. The GHS and ghrelin appear to play a major role in the control of the GH/IGF-I axis, as well as in the regulation of appetite and energy expenditure. The first studies in mouse knockout models for either ghrelin or its receptor (GHS-R1a) showed that these animals were not anorectic dwarves as expected. This evidence contributed to re-dimension the expectations that ghrelin and its analogs, acting as agonists or antagonists, might be potential therapeutical agents for treatment of GH deficiency, eating disorders, cachexia or obesity. Quite apart from these potential clinical applications, ghrelin physiology has continued to attract the interest of many researchers, progressively incrementing the knowledge about the molecular aspects underlying its involvement in different physiological and pathophysiological conditions. From the basic point of view, the past few years have witnessed the recognition of a number of endogenous ligands (des-Glu14-ghrelin, decanoyl ghrelin) originating from the ghrelin gene, which act on a pleiotropic receptor, the GHS-R1a. In addition, the possible existence of an endogenous inverse agonist has been suggested by the fact that GHS-R1a possesses a high constitutive activity, modulable by synthetic inverse agonists [131] (see also table 1). Most of all, the re-evaluation of the role played by the non-acylated form of ghrelin, which shares some of ghrelin effects or elicits opposite ones, has strongly indicated the presence of still unidentified multiple receptors subtypes (table 1). Although the cloning of the gene(s) for such receptors has proven difficult, uncovering their molecular identity would represent a major step forwards in this field. On the other hand, GHS-R1a has been demonstrated capable of heterodimerization and susceptible to allosteric modulation by synthetic GHS. These characteristics may explain, at least in part, the GHS-R1a pleiotropy in different tissues

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Conclusions

155

Table 1. Survey of ghrelin/GHS receptor types Receptor type and molecular features GHS-R1a GPCR family A Heptahelical (TM1–7) with 366 amino acids [18, 112, 113] Constitutive activity [130, 131, 156] Homo*/heterodimeric with full-length GPCRs [163, 177, 178, 211]

Ligands

Signalling

Agonists: ghrelin and short ghrelin fragments [69] synthetic peptidyl (GHRP-6) [3, 102, 223, 234], partial peptidyl and non-peptidyl GHS [6, 7, 15, 120, 127]

Gq: d Ca2+, PLC, IP3, DAG, PKC [18, 19]

Partial agonists: adenosine* [119–121]

blockade of K+ channels [145]

Antagonists: D-Lys3-GHRP-6 [234], L765–867 [120], isoxazole, diaminopyrimidine and triazole derivatives [132–134], cortistatin and its octapeptide analogue CST-8 [107]

d ERK1/2 , CREB and SRE [130]

d cAMP [141, 147] Heterodimeric formation with GHRH and dopamine D1 receptors [177, 178]

Inverse agonists: D-Arg1-D-Phe5-DTrp7,9-Leu11-substance P [130, 131] GHS-R1b Truncated (TM1–5) with 298 amino acids [88]. Heterodimeric with full-length GPCRs [211, 204]

Biological significance

d GH, PRL, CRH, ACTH and glucocorticoid secretion [21, 26, 27] f GnRH and gonadotropin release d Appetite [30, 105] and imagination of food [27] d Synaptogenesis and memory performance [49, 96], dopaminergic neurotransmission, locomotor activity and motivation to feed [95, 96] d Neoplastic cell growth [57] d Gastrointestinal motility and gastric acid secretion [25] d Hepatic glucose output [40] f Glucose-stimulated insulin secretion [36*, 42*] f Peripheral insulin sensitivity [72*, 41*] f Pro-inflammatory and immune responses [111]

Agonists: neuromedin in the neurotensin receptor 1 (NTS-R1)/GHS-R1b heterodimer [204]

Heterodimer formation with GHS-R1a and NTS-R1 [211, 204]

f GHS-R1a constitutive activity [211] d Neoplastic cell growth [204]

Agonists: ghrelin, UAG and synthetic GHS [50, 56, 58, 60, 61, 78, 215]

d cAMP/PKA, PI3K/AKT, ERK1/2 [56, 60, 61]

d Neurogenesis [215] d Cell proliferation [60, 61] and survival under pro-apoptotic conditions [56, 60] d Adipogenesis [39] f Lipolysis under stimulated conditions [78] f Neoplastic cell growth [55, 57, 58]

GHS-Rxb Uncloned

Agonists: UAG [40, 218, 219]

Unknown

d Food intake [219] f Hepatic glucose output [40, 217] d Glucose-stimulated insulin secretion [217, 218] d Peripheral insulin sensitivity [216]

CD36 Scavenger receptor family class B glycoprotein, large extracellular domain with two TMs and 471 amino acids

Agonists: oxidized low-density lipoproteins [224] Antagonists: peptidyl GHS (hexarelin and its analog EP-80317) [229, 230]

Uptake of oxidized low-density lipoproteins [224]

d Foam cell formation and pro-atherogenic processes [224] d Angiogenesis and immune cells recognition [224]

GHS-Rxa Uncloned

* Supposed; d and f indicate increased or decreased activity. TM = Transmembrane domain; UAG = unacylated ghrelin; CREB = cAMP-responsive element-binding protein; SRE = serum-responsive element. References are shown in brackets.

and in response to different ligands. Thus, far from being simply a ‘saginary hormone’ [231], ghrelin and its natural and synthetic analogs possess a wide variety of activities, sometimes ligand- and receptor-specific (table 1). Recently, transgenic and knockout animal models, as well as in vitro and in vivo studies, provided evidence for a major role of the ‘ghrelin/GHS orchestra’ in peripheral metabolism. For instance, it is noteworthy to remind that 156

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knocking out ghrelin improves glucose tolerance in ob/ ob mice [43], while mice lacking ghrelin or GHS-R1a are resistant to diet-induced obesity [232, 233]. There is great interest in the hypothesis that both the acylated and nonacylated ghrelin may influence endocrine pancreatic activity, by improving -cell survival and regulating insulin secretion [86, 218], thus revealing useful in the treatment of diabetes mellitus and metabolic syndrome. Muccioli /Baragli /Granata /Papotti /Ghigo

Interestingly, a number of studies are also focusing on the role of the ghrelin system in the control of neoplastic cell growth through heterodimerization of the truncated splice variant GHS-R1b with full-length GPCRs [204], as well as in modulating immune responses, cardiovascular performances and brain functions (table 1). Finally, we should not forget that ghrelin was discovered as a ‘motilin-related gastric peptide’, and ghrelin as well as ghrelinmimetic agents have potential clinical applications in the treatment of gastrointestinal motility disorders. Almost 30 years after the ‘invention’ of synthetic peptidyl GHS by Cyril Bowers [234], the ghrelin system is yet not com-

pletely understood. We may expect exciting aspects of ghrelin biology to emerge, including the identification of multiple receptors for multiple ligands.

Acknowledgments This review was supported by grants to G.M. (Regione Piemonte A58/2004), to E.G. (European Community Sixth Framework Programme – LSHM-CT-2003-503041) and to E.G. and G.M. (MIUR, Rome, Italy – project No. 2005060517, year 2005) and to E.G. (European Community Sixth Framework Programme – LSHM-CT-2003-503041).

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Neuroendocrinology 2007;86:165–174 DOI: 10.1159/000101029

Received: February 9, 2007 Accepted after revision: February 12, 2007 Published online: March 22, 2007

Comparative Aspects of GH and Metabolic Regulation in Lower Vertebrates Karine Rousseau Sylvie Dufour MNHN, Département des Milieux et Peuplements Aquatiques, USM 0401, UMR 5178 CNRS, Paris, France

Key Words Growth hormone  Growth hormone regulators  Metabolism  Lower vertebrates

Abstract In all vertebrates, the regulations of growth and energy balance are complex phenomena which involve elaborate interactions between the brain and peripheral signals. Most vertebrates adopt and maintain a life style after birth, but lower vertebrates may have complex life histories involving metamorphoses, migrations and long periods of fasting. In order to achieve the complex developmental programs associated with these changes, coordinated regulation of all aspects of energy metabolism is required. Somatotropic axis (somatostatin (SRIH) growth hormone (GH) and insulin-like growth factor 1 (IGF1), is known to be involved in the regulation of growth and energy balance. Interestingly, recent studies showed that additional factors such as pituitary adenylate cyclase-activated polypeptide (PACAP), corticotropin-releasing hormone (CRH), ghrelin and leptin could also have major roles in the control of growth and metabolism in lower vertebrates (fish, amphibians and reptiles). This minireview will survey the function of GH and metabolic regulation in lower vertebrates. Copyright © 2007 S. Karger AG, Basel

© 2007 S. Karger AG, Basel 0028–3835/07/0863–0165$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/nen

Introduction

Growth hormone (GH), prolactin (PRL) and somatolactin (SL) belong to a family of pituitary hormones which share many similarities in structure, function and gene organization. The recent finding that GH was the only member of this family to be present in an agnathan, the sea lamprey [1], suggests that it is the ancestral hormone of this family, present in all classes of vertebrates, and that its gene duplicated during the early evolution of gnathostomes to form PRL and/or SL [for review, see 2]. GH exerts pleiotropic functions in all vertebrates. It is better known for its essential role in the regulation of body growth and development, but it can also influence reproduction, immunity, and osmoregulation in teleosts. Concerning its growth-promoting effect, GH acts at different levels, directly on tissues such as central nervous system or muscles and indirectly via endocrine or local production of insulin-like growth factor 1 (IGF1). IGF1 is highly conserved during vertebrate evolution and is believed to be of more ancient origin than GH, appearing in various classes of invertebrates. GH is also involved in the regulation of metabolism via its lipolytic action and anabolic effect on protein metabolism. GH regulation has been extensively studied in mammals, but studies of lower vertebrates (fish, amphibians Karine Rousseau, Muséum National d’Histoire Naturelle Département des Milieux et Peuplements Aquatiques USM 0401 UMR 5178 CNRS, 7, rue Cuvier, CP 32 FR–75231 Paris Cedex 05 (France) Tel. +33 1 40 79 36 11, Fax +33 1 40 79 36 18, E-Mail [email protected]

and reptiles) have revealed similarities as well as important phylogenetic differences in the neuroendocrine control of this hormone [for review, see 3]. Interestingly, many GH regulators are also directly or indirectly involved in the regulation of energy balance. The aim of this mini-review is to provide a comprehensive survey of the physiological role of GH and its regulators in the control of growth and metabolism.

GH Regulation of Growth and Metabolism

Animal growth is largely genetically controlled in vertebrates, but is also strongly influenced by environmental and nutritional factors, especially in ectotherms. Indeed, these animals rely on temperature, photoperiod and food availability to trigger developmental processes such as metamorphosis and reproduction. Information from both external stimuli and internal state is processed and integrated by the brain. A central point of convergence in this endocrine pathway is the somatotropic axis. As in other vertebrates, numerous studies have shown GH to have a growth-promoting action in fish [for reviews, see 4, 5], as demonstrated by direct GH injection [for review, see 6] and transgenic technology [7]. Conversely, GH immunodepletion [8] and hypophysectomy [9, 10] lead to reduced growth, which is reversible after the end of the treatment or after GH injection. In contrast to mammals, growth continues throughout adult life in fish [11]. This can be related to the strong spontaneous basal activity of somatotropes, persisting in vitro in the absence of secretagogues or serum, as shown in various teleosts, differently from mammals [12, 13; for review, see 3]. The increase in weight associated with growth in fish appears to be due to a combination of increased appetite [14, 15] and increased feed conversion [14, 16, 17; for review, see 18]. In fish, both GH administration [19, 20] and GH transgenesis [21] result in increased growth that might be attributed to increased feeding and improved food assimilation [20, 22]. However, the role of GH on the control of food intake is still controversial. Indeed, central injections of GH did not change feeding in rainbow trout [23], whereas peripheral GH injections were able to increase appetite [coho salmon: 14 ; channel catfish: 24 ; rainbow trout: 15]. As in teleosts, some studies indicate that GH is a principal regulator of growth in amphibians and reptiles. Dodd and Dodd [25] reported that hypophysectomy of amphibians slowed growth rate significantly and implantation of the pituitary gland at an ectopic site could re166

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store the growth rate. The first study explicitly demonstrating a key role of GH in the control of overall growth in tadpoles and frogs used transgenic Xenopus that overexpress the cDNA-encoding GH [26]. In reptiles, GH has also been shown to increase appetite and growth [lizard: 27; turtle: 28]. Fish are the most numerous and diverse group of vertebrates and display varying degrees of tolerance to food deprivation, ranging from some in which a mammal-like pattern (unable to resist long periods of starvation) occurs to others that can tolerate prolonged periods of fasting (6 months or longer). In teleosts as in most other vertebrates (human, sheep, dogs, chickens, but not rats), food deprivation causes significant elevation of plasma GH concentrations [salmonids: 29–32 ; eel: 33; sea bream: 34 ; tilapia: 35, 36]. In this regard, most species models are applicable to the human with the obvious exception being rats [for review, see 37]. Available data in fish emphasize the key metabolic role of GH in conditions where energy availability for growth is limited. Two major metabolic effects of GH in fish appear to be catabolic with the stimulation of lipid mobilization and anabolic with the induction of protein synthesis [for reviews, see 18, 38]. The catabolic, lipolytic effects of GH in rainbow trout have both been shown in vivo by elevated fatty acids (FA) levels [39] and in vitro by the stimulation of FA and glycerol release from liver slices [40]. This appears to be due to increased triacylglycerol (TG) lipase activity, as seen in studies on coho parr [41]. In contrast, in physiological state in which plasma GH levels are elevated (such as smoltification), GH treatment fails to raise plasma FA levels [42] or hepatic lipid mobilization [41]. Similarly, hypophysectomy of coho smolts decreases hepatic TG lipase activity, which can partly be restored by GH treatment [for review, see 18]. The anabolic effects of GH on protein metabolism have been studied in some detail in rainbow trout. In this species, ovine GH significantly enhances whole-body growth rates as a result of the stimulatory effect on protein synthesis rates and little action on protein degradation [43]. The rates of change in body weight and length, as well as the rate of protein synthesis in white muscle were enhanced after injection of bovine GH [44]. Similarly, in hypophysectomized eels, in vivo administration of ovine GH increased [14C]leucine incorporation into protein of the liver, skeletal muscle and opercular muscle [45]. In summary, GH (via its lipolytic action) may increase energy supply in a state of negative energy balance (such as fasting) and may also promote growth (via its anabolic action on protein metabolism) when energy is available. Rousseau/Dufour

Regulators of GH Involved in Energy Balance

In lower vertebrates as in mammals, the regulation of metabolism is a complex phenomenon which involves many neuropeptides and hormones including neuropeptide Y, cholecystokinin, galanin and insulin [for reviews, see 46, 47]. A brief overview of recent findings concerning peptides involved in regulation of growth hormone as well as of energy balance and feeding is presented below.

Neuroendocrine Inhibitor of GH: Somatostatin

content [63]. Collectively, these data suggest that SRIH peptides play a role in the control of energy balance either directly or indirectly via GH or insulin. Moreover, GH and insulin were recently shown to stimulate SRIH receptor expression in liver, raising the question as to whether regulation at this level may be important for the coordination of growth-development and metabolism in vertebrates [64]. While SRIH is able to influence lipid and carbohydrate metabolisms, there are in return several feedbacks from nutrients to SRIH in fish. Indeed, lipids increase plasma SRIH levels in rainbow trout in vivo and stimulate the secretion of SRIH from isolated perfused rainbow trout Brockmann bodies (endocrine pancreas) [65]; glucose stimulates the secretion of SRIH from isolated perfused Brockmann bodies [66] as well as the expression of mRNAs encoding PPSS [67, 68].

The different forms of somatostatin (SRIH) in mammals (e.g., SRIH-14 and SRIH-28) are all derived from a single precursor, preprosomatostatin I (PPSS-I), which contains SRIH-14 at its C-terminus. Lampreys, numerous teleost fish, and frogs possess other PPSS in addition to PPSS-I, which give rise to other forms of SRIH (SRIH22, 25, 28, etc.) [for reviews, see 3, 48]. SRIH is a potent inhibitor of GH release and this inhibitory control is conserved during vertebrate evolution [12, 13; for review, see 3]. The potential metabolic actions of SRIH are widespread, ranging from regulation of feeding behavior and nutrient assimilation to modulation of energy allocation [for review, see 49]. SRIH peptides are expressed in brain nuclei known to be involved in the control of pituitary function as well as food intake [50]. Rainbow trout and goldfish display postprandial elevation of plasma SRIH14 concentrations [51], suggesting a possible role for SRIH peptides in the regulation of feeding [52]. Treatment of teleost fish and lampreys with exogenous SRIH promotes lipid mobilization, with elevated plasma FA, by depletion of stored lipid via activation of TG lipase [coho salmon: 53, 54 ; rainbow trout: 55, 56 ; lamprey: 57]. Induction of SRIH deficiency in juvenile salmon undergoing parrsmolt transformation (smoltification) results in reduced hepatic lipolysis [58]. Sheridan and Kao [59] suggested that the increase in plasma SRIH levels during smoltification may contribute to the observed lipid depletion in two ways; by working to reduce insulin levels and by directly promoting lipid mobilization from fat depot. SRIH peptides also appear to directly mediate carbohydrate metabolism, stimulating the breakdown of glycogen and the release of glucose in agnathans [lamprey: 60, 61] and teleosts [eel: 62 ; coho salmon: 54 ; rainbow trout: 55]. Induction of SRIH deficiency in juvenile salmon undergoing smoltification results in increased hepatic glycogen

PACAP PACAP is a member of the VIP/secretin/glucagon/ GHRH/GIP superfamily [for review, see 70]. It is a polypeptide that was first isolated from ovine hypothalamus for its ability to stimulate adenylate cyclase activity in rat pituitary cells and is highly conserved throughout evolution [for reviews, see 70, 71]. Interestingly, PACAP and GHRH are encoded by the same gene in non-mammalian species such as teleosts, amphibians and birds [for review, see 70]. Several studies, including those of the European eel [72] and turbot [13] demonstrated that PACAP was effective in stimulating GH release in teleosts [for review, see 3]. These data suggested that PACAP, instead of GHRH, may represent the ancestral GH-releasing factor and that GHRH may have progressively acquired its major role in control of GH synthesis and release during later tetrapod evolution [for review, see 3, 71]. Recently,

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Neuroendocrine Stimulators of GH In contrast to higher vertebrates, such as mammals in which the positive neuroendocrine control of GH is exerted by growth hormone-releasing hormone (GHRH) [for review, see 69], GH can be stimulated by a variety of neuropeptides in lower vertebrates [for review, see 3]. Such stimulatory neuropeptides include pituitary adenylate cyclase activating polypeptide (PACAP) and corticotropin-releasing hormone (CRH), which may play important coordinating roles in the control of both growth and energy balance. Further studies are required to confirm this hypothesis.

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PACAP was also shown to inhibit food intake in goldfish [73], like in mice [74] and chickens [75]. It was also demonstrated that expression of mRNA for PACAP and its receptors were increased by excessive feeding [76]. Moreover, i.c.v. PACAP administration reduced locomotor activity [76], suggesting that it may have a positive effect on energy balance through hypomotility, like it was shown in rats [77] and mice [74]. PACAP-deficient mice show a dysfunction of lipid and carbohydrate metabolism, leading to postnatal death [78] indicating a similar role in mammals. Studies in rats and mice demonstrated that PACAP may also be involved in energy homeostasis, through induction of hyperthermia [79, 80], but this effect must have been acquired later in evolution as is unlikely to occur in poikilothermic vertebrates such as reptiles, amphibians and fish. CRH Another neuropeptide with interesting potential in the control of both growth and energy balance is CRH. This neuropeptide is known as the major hypothalamic factor mediating stress-induced adrenocorticotropin (ACTH) secretion from the anterior pituitary in mammals. Its sequence, as well as its corticotropic action, has been highly conserved throughout vertebrate evolution [for review, see 81]. We demonstrated that CRH was also able to stimulate GH production in the European eel, and that the CRH receptor antagonist, -helical CRH (9–41), significantly inhibited this stimulatory effect [82]. We suggested that the activity of CRH on GH production in the European eel could be related to the special biological cycle of this species, in which mobilization of energy stores is specially required to fulfill requirements for gonadal growth and reproductive migration [3]. Thus, in the eel, CRH could act as a potential coordinator for activating both corticotropic and somatotropic axes during critical developmental or physiological events, such as silvering, fasting and reproductive migration. Such an action of CRH on GH has also been observed in reptiles [83–85], and in humans under stress situations and in some pathological conditions such as depression or acromegaly [for reviews, see 3, 82]. Recently, CRH-related peptides were shown to be potent anorexigenic signals in fish, as in mammals [for review, see 86]. In goldfish, i.c.v. administration of either ovine CRH [87], rat/human CRH, or carp/goldfish urotensin I (UI) [88] inhibits food intake. CRH and UI (a peptide found in the urophysis of teleost fish and structurally related to CRH; [81]) were already shown to be 168

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potent inhibitors of food intake in mammals [89]. This anorectic effect can be reversed by pretreatment with the CRH receptor antagonist, -helical CRH (9–41) [88, 90]. Together, these data suggest a coordinator role of CRH in the combined activation of neuroendocrine axes during physiological or pathological situations and this role may have been partially conserved during evolution.

Feedback Regulators of GH: IGF1 and Thyroid Hormones IGF1 IGF1 is a major growth factor secreted by the liver under the control of GH, in species ranging from mammals to lamprey. In a classical feedback manner, IGF1 exerts inhibitory control over pituitary GH production in all teleost species studied so far [for review, see 3] and among them, the European eel [12] and in the turbot [91]. IGF1 has been highly conserved throughout vertebrate evolution. In fish, as is the case in mammals, IGF1 is expressed in a wide variety of tissues with the highest levels being found in the liver. Hepatic IGF1 expression is under the regulation of GH [for reviews, see 3, 92]. While GH overproduction or deficiency indicates the critical role of GH in postnatal growth, nutritional deprivation (which causes growth arrest in most juvenile vertebrates) leads to an increase in circulating GH levels. These observations indicated that the primary cause of the growth arrest is resistance to GH action at the tissue level. Some authors suggest that this growth arrest is primarily due to a decline in the production of IGF1 [for review, see 92]. Indeed, nutrition is another major regulator of IGF1, as food deprivation causes reduction of circulating levels [rainbow trout: 93; coho salmon: 94 ; tilapia: 36 ; 95; chinook salmon: 31; channel catfish: 96] and mRNA levels [eel: 97; coho salmon: 29; barramundi: 98 ; Chinook salmon: 31; channel catfish: 96 ; grouper: 99; tilapia: 36] of IGF1. The starvation-induced increase in plasma GH could be due to a reduced IGF1 feedback on the pituitary resulting, in turn, from a reduction in hepatic GH receptor (GH-R) [coho salmon: 100 ; salmon: 29; masu salmon: 101; black seabream: 102]. However, other recent studies showed that starvation did not induce changes in hepatic GH-R expression [tilapia: 36 ; rainbow trout: 32], suggesting post-receptor mechanisms of resistance to GH. Compared with rats [37], a much longer period of starvation is required to observe significant changes in IGF1 levels in fish and this slower response may be a reflection of the Rousseau/Dufour

generally slower metabolism of ectothermal animals [29]. In addition, many fish live in cold water and are metabolically adapted to long periods of food deprivation during their life cycle. A particular example is the stunted salmon, which despite its retarded growth and abnormal development, shows increased secretory activity of GH cells and elevated plasma GH concentrations [103]. These fish also display reduced GH receptor and IGF1 expression in the liver, which suggests that they are GH-resistant. These changes in the GH-IGF1 axis in stunted salmon are similar to those in starved fish. The nutritional regulation of the GH-IGF1 axis, which is an interface between nutrients and hormones acting in concert to control animal growth and development, seems to be important and well conserved throughout vertebrate evolution.

changes in lipid metabolism came from studies on coho salmon [for review, see 59]. Immersion of parr, which have low endogenous levels of TH and high tissue lipid concentration, in T4-containing water for 12 days elevated plasma T4 to levels normally seen in smolts and resulted in enhanced lipid depletion. Smolts, which already possess low lipid levels, were refractory to T4 treatment.

Thyroid Hormones Thyroid hormones (TH: triiodothyronine, T3 and thyroxine, T4) and GH are thought to play synergistic roles in the control of growth and developmental processes in vertebrates. Hypothalamic thyrotropin-releasing hormone (TRH) (and CRH in some vertebrates and some developmental or physiological situations such as metamorphosis in amphibians) stimulates the synthesis and secretion of thyrotropin (TSH) by the pituitary. TSH, then, acts on the thyroid to induce the synthesis of T4, which is peripherally deiodinated into biologically active T3. In the European eel, it was demonstrated that GH was able to increase circulating T3 by stimulation of peripheral 5-monodeiodination [104] and that both T3 and T4 could inhibit GH release and synthesis in vivo and in vitro [105]. In other teleosts, studies reported either stimulatory or no effects of TH on GH, and this species-related variation was also observed among mammals [for review, see 3]. Few studies have examined the effect of starvation on TH levels. While T4 concentrations were relatively unaffected by food deprivation and refeeding in coho salmon [106], reduced levels of T3 and T4 were observed in chronically starved rainbow trout [107] and short-term starved tench [108]. In rainbow trout, chronic fasting induced a downregulation of the response of thyroid tissue to bovine TSH challenge and of the GH stimulation of T3 production in vivo [107]. TH generally promote lipid depletion from depot sites of salmon parrs not undergoing smoltification. Injection of T4 reduced stored body fat and elevated plasma fatty acids [60]. TH deficiency induced by radiothyroidectomy caused fat accumulation in rainbow trout [109]. Direct evidence that T4 plays a role on smoltification-associated

Ghrelin In 1999, information about the isolation, characterization and some in vitro and in vivo biological actions of ghrelin were first reported in rat [110]. This novel acylated protein was successfully purified using the GH secretagogues-receptor (GHS-R), an orphan receptor, and the approach of reverse pharmacology [110]. In fish, GHS-R was identified in pufferfish [111] and in black seabream [112], and shared good homology with human GHS-R. Among teleosts, ghrelin has been identified in the goldfish [113], in the Japanese eel [114], in the tilapia [115, 116], in the rainbow trout [117] and in the channel catfish [118]. There is a postprandial reduction in ghrelin mRNA expression in the hypothalamus and gut of goldfish, and fasting increases ghrelin mRNA expression in these tissues [119, 120]. On the other hand, 2 weeks of fasting reduced ghrelin-like immunoreactivity in the plasma of burbot [121]. Recently, the development of a specific RIA against acylated eel ghrelin led to the demonstration that acylation of the peptide in the stomach of this species is quite low [122] compared to that in the rat stomach [123, 124]. These authors concluded that the differences in energy metabolism between homeotherms (mammals) and poikilotherms (fish) may be related to the contrasting ghrelin levels [122]. Using mammalian and piscine ghrelins, it was demonstrated that ghrelin could stimulate GH release in vitro in the tilapia [125, 114, 115] and in the goldfish [126]. In goldfish, ghrelin also stimulates GH mRNA expression [119]. In vivo studies on ghrelin and GH release support the in vitro data. Intraperitoneal injections of ghrelin stimulated plasma GH levels in rainbow trout [117], in goldfish [127] and in channel catfish [118]. In addition to the peripheral

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Ghrelin and Leptin Recently, two hormones, which have opposite effects on both GH and metabolism, have received major attention in mammals. Increasing numbers of studies in lower vertebrates (mainly in fish) suggest that their actions may be conserved during evolution.

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effects of ghrelin, Unniappan and Peter [127] found that i.c.v. injection of goldfish ghrelin stimulated serum GH release. In goldfish, i.c.v. and i.p. administration of ghrelin peptides stimulates food intake [119]. Ghrelin has also been characterized in bullfrog [128] and in turtle [129] and it stimulates GH and PRL secretion from dispersed bullfrog pituitary cells [128]. Leptin Leptin is an adipocyte-derived hormone, member of the class-I helical cytokine family, discovered in 1994 by positional cloning of the murine obese (ob) gene and its human homologue [130]. Zhang et al. [130] hybridized genomic DNA of representative vertebrate species with a murine ob probe and also obtained positive signals with eel DNA. In addition, leptin-like immunoreactive material has been found in blood, brain, heart, liver and stomach of various fish species [121, 131–134]. Using immunocytochemical staining with anti-human leptin antibodies, Vegusdal et al. [135] showed that a leptinlike protein was present in salmon differentiated adipocytes. Nevertheless, the presence of leptin in fish remained controversial until the first identification and cloning in fish of a homologue to mammalian leptin, recently reported for the pufferfish [136]. The authors used genomic synteny (positional cloning) around the human leptin gene and found that the deduced amino acid sequence of pufferfish leptin had only 13.2% similarity to human leptin. More recently, two similar leptin proteins encoded by duplicate ob genes were identified from common carp [137]. In contrast to mammals in which leptin is found mainly in adipose tissue, fish leptin seems to be mostly produced in the liver [puffer: 136 ; common carp: 137]. Brain leptin levels correlate positively with adiposity in bluegill and white crappie [131] and fasting lowers circulating leptin concentrations in green sunfish [131]. However, a recent study in common carp demonstrated that leptin mRNA expression changes acutely following food intake, but not after fasting or feeding to satiation, suggesting that leptin may not be involved in the longterm regulation of food intake and energy metabolism in this species [137]. In other lower vertebrates, leptin-like immunoreactivity was also detected in stomach of amphibians [132] and reptiles [132] and in plasma, liver and fat bodies of the lizard [138]. Since 2000, many studies investigated the possible role of leptin in metabolism in fish species. In catfish, peripheral administration of leptin did not modify food intake in either fasted or fed conditions [139]. Similarly, after 170

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leptin implants in the peritoneal cavity of fed and fasted immature coho salmon for 2 weeks, there were no effects on body weight, growth or energy stores [140]. Leptin injections for 2 weeks also did not change food intake and body weight in green sunfish [141]. However, more recently, in goldfish, both acute (i.c.v. or i.p.) and chronic (i.p.) administration of leptin was shown to reduce food intake, body weight gain, specific growth rate and food efficiency ratio [46, 142]. Moreover, leptin can stimulate both lipid and carbohydrate metabolism in goldfish [142] and fat metabolism in green sunfish [141]. Peripheral injections of murine leptin reduce food intake and increase metabolic rates in lizards [143]. In another lizard species, treatment with leptin increased insulin, glucagon and glucose levels in blood, and reduced glycogen levels [144], which suggests the involvement of this hormone in glucose metabolism of reptiles. In conclusion, the diversity of species and life cycles, and the large adaptability to different environments make lower vertebrates ideal comparative models for studying the evolution of regulatory mechanisms in growth and metabolism of vertebrates. Compared to mammals, lower vertebrates are more directly subjected to environmental conditions, which they face thanks to a large plasticity of the regulation of their growth and metabolism. As in higher vertebrates, GH is able to modulate both lipid and carbohydrate metabolisms, as well as body growth. The different GH regulators presented in this review can by themselves or via GH act on energy balance through central and peripheral actions, including many types of feedback signaling. This leads to coordinated and fine regulations, necessary for the complex developmental programs occurring during their life histories.

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Neuroendocrinology 2007;86:175–182 DOI: 10.1159/000109095

Received: November 30, 2006 Accepted after revision: December 11, 2007 Published online: September 26, 2007

Neuropeptide Signaling in the Integration of Metabolism and Reproduction Angelena Crown c Donald K. Clifton b Robert A. Steiner a, b Departments of a Physiology and Biophysics, b Obstetrics and Gynecology, and c the Undergraduate Program in Neurobiology, University of Washington, Seattle, Wash., USA

Key Words Kiss1 ⴢ Kisspeptin ⴢ Neuropeptide Y ⴢ Galanin-like peptide ⴢ Proopiomelanocortin ⴢ Metabolism ⴢ Reproduction ⴢ Neuropeptide ⴢ Arcuate nucleus ⴢ Leptin ⴢ Insulin ⴢ Thyroid hormone ⴢ Adiponectin

Abstract Fertility is gated by nutrition and the availability of stored energy reserves, but the cellular and molecular mechanisms that link energy stores and reproduction are not well understood. Neuropeptides including galanin-like peptide (GALP), neuropeptide Y (NPY), products of the proopiomelanocortin (POMC; e.g., ␣-MSH and ␤-endorphin), and kisspeptin are thought to be involved in this process for several reasons. First, the neurons that express these neuropeptides all reside in the hypothalamic arcuate nucleus, a critical site for the regulation of both metabolism and reproduction. Second, these neuropeptides are all targets for regulation by metabolic hormones, such as leptin and insulin. And third, these neuropeptides have either direct or indirect effects on feeding and metabolism, as well as on the secretion of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH). As the target for the action of metabolic hormones and sex steroids, these neuropeptides serve as molecular motifs integrating the control of metabolism and reproduction. Copyright © 2007 S. Karger AG, Basel

© 2007 S. Karger AG, Basel Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

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Energetics and Reproduction

Species viability is predicated on reproductive success. For individual animals, the caloric demands of reproduction must be weighed against those deemed essential for immediate survival, which include thermoregulation, sensory function, and locomotor activity. Any extra energy is stored primarily as fat and glycogen, which are titered along with glucose to allow growth and reproduction. Reproducing without enough energy reserves to insure the survival of offspring would be counterproductive. To prevent this from happening, the reproductive system must monitor energy status and limit fertility to times of adequate energy reserves. Furthermore, since the time between conception and the emergence of viable offspring can take many months, some species incorporate predictive mechanisms to increase the probability of success. These mechanisms limit reproductive activity to times of the year when birth and nourishment of offspring are optimal. These strategies prevent the expenditure of energy on reproductive efforts that are risky and costly, conserving energy so that reproductive outcomes are maximized [1]. An animal’s energy stores depend not only on the availability of energy sources (food), but also on energy expenditure. Conditions that lead to excessive locomotor activity drain energy reserves and generally suppress reRobert A. Steiner Department of Physiology and Biophysics, Health Sciences Building (G-424) School of Medicine, University of Washington, Box 357290 1959 NE Pacific Street, Seattle, WA 98195-7290 (USA) Tel. +1 206 543 8712, Fax +1 206 685 0619, E-Mail [email protected]

production [2]. Lactation, like locomotion, is also energetically demanding, and delivering milk to hungry offspring taxes the metabolic system. The postpartum period is calorically expensive, and the caloric requirements of lactation tap the reserves of stored adipose tissue, which are typically augmented during gestation. It is energetically disadvantageous to incur the energetic demands of pregnancy while lactating, and, although there are examples of animals becoming pregnant while nursing, these pregnancies are usually suspended in either the preimplantation or earlier embryonic stage. One reproductive strategy that resolves this conflict between the energetic demands of pregnancy and lactation is ‘lactational infertility’. In most female mammals, ovulation, mating, and pregnancy are blocked during lactation [3]. Although the basic principle that reproductive activity is carefully guarded by physiological mechanisms that couple fuel availability to sexual activity is generally accepted, the molecular mechanisms that mediate this process remain poorly understood [2, 4–6].

Metabolic Hormones Linking Metabolism and Reproduction

Several metabolic hormones are recognized as important signals that link fuel reserves and reproduction. Leptin is an adipocyte-derived hormone, which has profound effects on feeding, thermogenesis, glucose and lipid metabolism, as well as physical activity. Plasma levels of leptin are directly proportional to fat reserves, such that declining levels of leptin trigger feeding behavior, slow metabolism, and help to conserve metabolic reserves. Animals with congenital deficiencies in either leptin or its receptor eat voraciously and become obese and hypothermic. The administration of exogenous leptin can readily reverse this phenotype. Leptin is not only important in the control of feeding and metabolism, but also appears to serve as an important signal to the reproductive system [7, 8]. In well-fed animals with normal circulating levels of leptin, the effects of additional leptin on the reproductive axis are subtle; however, in malnourished animals, the effects of leptin on reproduction can be profound [9]. Animals lacking either leptin or its receptor fail to undergo normal pubertal maturation and remain sexually infantile for their entire lives [10–12]. Leptin stimulates gonadotropin-releasing hormone (GnRH) and gonadotropin secretion, and administration of exogenous leptin to leptin-deficient animals (e.g., ob/ob mice), which are 176

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reproductively incompetent, can rescue their impaired sexual function [13–15]. These findings suggest that the body interprets circulating levels of leptin as an indicator of a metabolic state, which may then act as a gate to control the activity of the reproductive axis. Although leptin’s ability to rescue metabolic and reproductive function in animals and humans with leptin deficiency is well documented [9, 13, 14], the cellular and molecular targets of leptin’s action are still not completely known. Studies aimed at elucidating the targets of leptin action have shown that GnRH antiserum blocks the effect of leptin effect on LH secretion, but GnRH neurons themselves do not express the leptin receptor [16, 17]. Thus, leptin has little or no direct effect on LH secretion in the absence of GnRH, and it would appear that the action of leptin on the neuroendocrine reproductive axis is mediated by one or more populations of afferent inputs to GnRH neurons that do express the leptin receptor. Insulin has also been directly implicated in the integration of metabolism and reproduction. Insulin is an anabolic hormone produced and secreted by the pancreas in response to glucose. Insulin promotes cellular intake and storage of energy from ingested food, while simultaneously inhibiting the utilization of stored energy. The secretion of insulin fluctuates throughout the day, with plasma levels increasing immediately following a meal and decreasing between meals. Although plasma levels of insulin vary periodically, the basal plasma level of insulin is proportional to the amount of adipose tissue. Because this steady-state level of insulin reflects the status of stored energy reserves, circulating insulin levels may be used to communicate information about longterm metabolic conditions to the reproductive axis [6]. Insulin has been shown to regulate GnRH and LH secretion. In obesity, animals become insulin-resistant, which disrupts insulin signaling. Pulsatile LH secretion is inhibited in insulin-deficient states (e.g., fasting and diabetes), and central (into the cerebral ventricle) administration of insulin can reverse the deficiency in LH under these conditions [4, 5, 18, 19]. As in the case of leptin or leptin receptor deficiency, animals that lack proper insulin signaling become obese and have disordered metabolism and reproduction [20]. Diabetic animals display a panoply of reproductive deficiencies – including delayed pubertal maturation, reduced ovulation, infertility, disrupted estrous cycles, absent or delayed LH surges (and pulsatile GnRH/LH secretion), as well as impaired sexual behaviors [21–27]. These deficits can be either reversed or ameliorated by insulin administration [23, 24, 28–30], thus testifying to the imCrown /Clifton /Steiner

Fat Pancreas

Leptin

Thyroid

T3/T4

Insulin

Gonads

Sex Steroids Arcuate Nucleus

NPY POMC GALP Kiss1 Fig. 1. Metabolic status gates reproduction

to guarantee that attempts to reproduce occur only under favorable energetic conditions. Kisspeptin, NPY, GALP, and POMC neurons in the hypothalamic arcuate nucleus are targets for metabolic hormones, such as leptin and insulin, and these circuits regulate metabolism, feeding and reproduction to ensure efficiency and success.

Feeding and Metabolism

portance of insulin signaling for normal reproductive function. Thyroid hormone is also critical for growth, metabolism and reproduction. Abnormalities in circulating levels thyroid hormone, hyperthyroidism and hypothyroidism, are associated with metabolic and reproductive deficiencies [31–33]. Hyper- and hypothyroid individuals have reduced levels of available energy [6]. Hyperthyroidism inhibits sexual behavior in rodents [31, 32], and lambs that are hyperthyroid have stunted growth, decreased LH and testosterone secretion, and impaired gonadal function [34]. Hypothyroidism is associated with impaired menstrual cyclicity and disrupted follicular development in females [33, 35]. These conditions resulting from hypothyroidism are ameliorated by the administration of thyroid hormone, suggesting that thyroid hormone, along with leptin and insulin, should be counted among those humoral factors that relay important information about metabolism to the reproductive axis. Thyroid hormone has also been implicated in the seasonal regulation of reproduction, with thyroid hormone secretion increasing during the breeding season in some species [36] – further evidence that metabolic status and reproductive activity are inexorably intertwined. It is also plausible that other metabolic hormones, besides leptin, insulin, and thyroid hormone – such as adiponectin – may influence the neuroendocrine axis, posNeuropeptide Signaling in Metabolism and Reproduction

GnRH Neuron

Reproductive Function

sibly through direct actions on the pituitary [37]. The complete constellation of metabolic hormones that influence the brain and pituitary has yet to be fully elucidated.

Neuropeptides as Central Processors for Integrating Metabolism and Reproduction

GnRH neurons are the final common pathway through which the brain regulates reproduction (fig. 1), although these cells are not direct targets for metabolic signals [6]. Gonadal steroids also regulate GnRH secretion through an indirect action on sex steroid-sensitive afferent inputs to GnRH neurons. Neurons that contain receptors for metabolic hormones and send afferent inputs to GnRH neurons are likely to be responsible for sensing the metabolic milieu and controlling GnRH secretion as a function of fuel availability and fat reserves. Candidates for serving this integrative function include neurons that express galanin-like peptide (GALP), neuropeptide Y (NPY), proopiomelanocortin (POMC) and its processed derivatives (e.g., ␣-MSH and ␤-endorphin), kisspeptin, and possibly orexin [38, 39], as well as catecholaminergic neurons that reside in the brainstem and project to the hypothalamus [40].

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177

GALP is expressed in the arcuate nucleus of the hypothalamus, and its expression is regulated by leptin and insulin [41–44]. GALP neurons express the leptin receptor, ObRb, and thus would appear to be direct targets for the action of leptin. The central administration of GALP stimulates GnRH/LH secretion in the rat, mouse, and monkey, and GALP-containing fibers are found in close proximity to GnRH neurons [45, 46]. The expression GALP mRNA is reduced by fasting and diabetes, and the deleterious effects of diabetes on reproductive function can be reversed (or attenuated) by the administration of GALP [8, 47, 48]. Thus, GALP neurons are poised to serve as cellular conduits coupling the physiological systems that regulate metabolism and reproduction. NPY is among the most abundant peptides in the central nervous system, and it plays a key role in energy homeostasis. NPY neurons in the hypothalamus are activated by fasting, stimulate hunger and food-seeking behavior and also regulate thermogenesis, peripheral insulin secretion, and hepatic glucose output [49]. The observation that antiserum to NPY and NPY mRNA anti-sense oligodeoxynucleotides (administered centrally) diminish food consumption and reduce body weight is consistent with an orexigenic role for NPY [50, 51]. The expression of NPY mRNA in the hypothalamus is reduced by leptin [52, 53], and NPY-expressing neurons also express the leptin receptor, suggesting that they are direct targets for the action of leptin [16, 54]. Mice bearing targeted deletions of NPY have only subtle phenotypic abnormalities [55, 56], but leptin-deficient ob/ob mice also bearing targeted deletions of NPY eat less and are leaner than ordinary ob/ob mice. This testifies to the complex involvement of NPY in modulating feeding behavior. The activity of NPY neurons is also regulated by insulin, which like leptin, inhibits the expression of NPY mRNA [57, 58]. Moreover, elevations in circulating levels of insulin reverse the increase in NPY mRNA associated with fasting [57]. Thus, NPY neurons serve as an important relay center in the brain for the regulation of feeding and metabolism. NPY also influences GnRH and gonadotropin secretion, although effects depend upon the steroidal milieu. For example, in intact animals and steroidprimed ovariectomized animals, the central administration of NPY administration stimulates GnRH and LH secretion [59, 60]. The expression of NPY is increased in intact female rats during the afternoon and evening of proestrus [61], and similarly, NPY gene expression is increased in ovariectomized animals just before the steroid-induced LH surge [62]. The stimulatory effects of NPY on GnRH and LH secretion are limited in scope, as 178

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chronic administration of NPY causes delayed sexual maturity and disruption of estrous cycles [63, 64], and in some species (e.g., sheep), NPY has a predominantly negative effect on reproductive function [65]. The importance of the role of NPY in the regulation of reproduction is reaffirmed by a study of NPY KO mice. The pituitary glands of these animals are less responsive to GnRH than normal controls [66]. Both male and female NPY KO mice display reduced mating behaviors [67], but remain fertile and do not manifest abnormalities in body weight, feeding behavior, or endocrine function [68]. This apparent lack of significant metabolic and reproductive abnormalities may reflect compensatory adaptation during development of the existence of redundant pathways for controlling these complex processes [55, 56]. It is also possible that NPY plays a significant role in metabolism and reproduction only in states of low energetic reserves. In this regard, it is notable that, although serum levels of LH are reduced by fasting in normal animals, a similar response is not seen in NPY KO mice [69]. Thus, NPY neurons are likely to act as central integrators of metabolism and reproduction. POMC-expressing neurons in the arcuate nucleus may also contribute to the governance of the intersection of metabolism and reproduction. The expression of POMC mRNA is reduced by fasting, and other leptin and insulin-deficient states, such as diabetes and impairment of leptin signaling [70–74]. Additionally, POMC neurons in the arcuate nucleus are direct targets for the action of leptin [7, 74], which stimulates POMC gene expression. The various POMC gene products have differential effects on the feeding and reproductive axes, but precisely how different peptide products of the POMC precursor are drafted into their respective functions remains unknown. For example, on the one hand, ␣-MSH reduces food consumption and stimulates lordosis behavior in female rats [75, 76]. On the other hand, ␤-endorphin stimulates food consumption and inhibits GnRH/LH secretion [77–81]. Despite the complexity, POMC neurons in the arcuate nucleus reside at the anatomical and physiological juncture where metabolism and reproduction are coordinated – and learning more about the processing of this complex protein could shed light on the enigmatic mechanisms by which metabolism and reproduction are controlled.

Crown /Clifton /Steiner

Kiss1-Kisspeptin-GPR54 Pathway as a Linchpin Coupling Metabolism and Fertility

Another possible link between metabolism and reproduction is the neuropeptide, kisspeptin. Kisspeptin is encoded by the Kiss1 gene and signals through the GPR54 receptor. The kisspeptin/GPR54 signaling system is necessary for normal reproduction. Mutations in GPR54 result in complete disruption of reproductive function in both humans and mice [82, 83]. Centrally administered kisspeptin stimulates GnRH neurons in the mouse, rat, sheep, and primate [84–86]. GPR54 is expressed by virtually all GnRH neurons, indicating that the cells are direct targets for kisspeptin [85, 87]. Kiss1 neurons are also direct targets for the action of sex steroids, which regulate the expression of Kiss1 mRNA [88, 89]. The activation of Kiss1 gene expression is likely to play an important role in timing the onset of puberty, sexual differentiation of the GnRH/LH surge mechanism, and the preovulatory GnRH/LH surge itself (in females) [84, 86, 90–94]. Thus, Kiss1/kisspeptin/GPR54 signaling is critically involved in virtually all aspects of neuroendocrine reproductive function. Kiss1/kisspeptin neurons also have direct links to the mechanisms that regulate metabolism. Fasting is associated with inhibition in the expression of Kiss1 mRNA (and decreased circulating levels of leptin) [95]. Central injections of kisspeptin can reverse the fasting-induced inhibition of GnRH secretion [95]. Likewise, kisspeptin administration rescues GnRH decline in rats that are treated with leptin antibodies [96]. These findings suggest that kisspeptin is involved in relaying metabolic signals to the neuroendocrine reproductive axis. The expression of Kiss1 mRNA is also regulated by leptin. Leptin-deficient ob/ob mice have reduced levels of

Kiss1 mRNA compared to wild-type controls, and the central administration of leptin partially reduces this effect [97]. Further support of the notion that the cells are regulated by leptin is the observation that 40% of Kiss1 cells in the arcuate nucleus express the signaling version of the leptin receptor, ObRb [97]. Kisspeptin also interacts with the insulin signaling pathway in the hypothalamus. Rats that are rendered diabetic have diminished expression of Kiss1 mRNA, which can be reversed with insulin therapy [96]. Thus, together with GALP, POMC, and NPY neurons, Kiss1 neurons are likely to serve as cellular conduits for relaying information about circulating levels of leptin and insulin to the neuroendocrine reproductive axis. The cellular and molecular basis for the integration of metabolism and reproduction involves a complex interaction of hypothalamic neuropeptides with metabolic hormones, fuels, and sex steroids. Kisspeptin is unlikely to be the last of the neuropeptides discovered having relevance to both metabolic regulation and reproductive function – just as leptin, insulin, and thyroid hormone are not the full cast of metabolic hormones with actions on the neuroendocrine reproductive axis. Understanding this integrative process will require careful mapping of hypothalamic and brainstem circuitry, cataloguing of receptor expression profiles within these circuits, and a detailed analysis of the action of metabolic hormones on these pathways. There is much work ahead.

Acknowledgement This work was supported by grants from the National Institutes of Health [R01 HD27142, SCCPRR (U54) HD12629, and R01 DK61517].

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75 Gonzalez M, Celis M, Hole D, Wilson C: Interaction of oestradiol, ␣-melanotrophin and noradrenaline within the ventromedial nucleus in the control of female sexual behaviour. Neuroendocrinology 1993; 58: 218– 226. 76 Scimonelli T, Medina F, Wilson C, Celis M: Interaction of ␣-melanotropin (␣-MSH) and noradrenaline in the median eminence in the control of female sexual behavior. Peptides 2000;21:219–223. 77 Bruni J, Van Vugt D, Marshall S, Meites J: Effects of naloxone, morphine and methionine enkephalin on serum prolactin, luteinizing hormone, follicle-stimulating hormone, thyroid-stimulating hormone and growth hormone. Life Sci 1977;21:461–466. 78 Gilbeau P, Almirez R, Holaday JW, Smith C: Opioid effects on plasma concentrations of luteinizing hormone and prolactin in the adult male rhesus monkey. J Clin Endocrinol Metab 1985;60:299–305. 79 Leadem C, Kalra S: Reversal of ␤-endorphin-induced blockade of ovulation and luteinizing hormone surge with prostaglandin E2. Endocrinology 1985; 117:684–689. 80 Leadem CA, Kalra S: Effects of endogenous opioid peptides and opiates on luteinizing hormone and prolactin secretion in ovariectomized rats. Neuroendocrinology 1985; 41: 342–352. 81 Wardlaw S, Ferin M: Interaction between ␤endorphin and ␣-melanocyte-stimulating hormone in the control of prolactin and luteinizing hormone secretion in the primate. Endocrinology 1990; 126:2035–2040. 82 De Roux N, Genin E, Carel J, Matsuda F, Chaussain J, Milgrom E: Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc Natl Acad Sci USA 2003;100:10972–10976. 83 Seminara S, Messager S, Chatzidaki E, Thresher R, Acierno JJ, Shagoury J, Bo-Abbas Y, Kuohung W, Schwinof K, Hendrick A, Zahn D, Dixon J, Kaiser U, Slaugenhaupt SA, Gusella J, O’Rahilly S, Carlton M, Crowley WJ, Aparicio S, Colledge W: The GPR54 gene as a regulator of puberty. N Engl J Med 2003;349:1614–1627. 84 Gottsch M, Cunningham M, Smith J, Popa S, Acohido B, Crowley WF, Seminara S, Clifton DK, Steiner RA: A role for kisspeptins in the regulation of gonadotropin secretion in the mouse. Endocrinology 2004; 145: 4073– 4077. 85 Irwig M, Fraley G, Smith J, Acohido BV, Popa S, Cunningham M, Gottsch ML, Clifton DK, Steiner R: Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the male rat. Neuroendocrinology 2004;80:264–272. 86 Shahab M, Mastronardi C, Seminara S, Crowley WF, Ojeda S, Plant T: Increased hypothalamic GPR54 signaling: a potential mechanism for initiation of puberty in primates. Proc Natl Acad Sci USA 2005; 102: 2129–2134.

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Crown /Clifton /Steiner

Neuroendocrinology 2007;86:183–190 DOI: 10.1159/000108280

Received: November 13, 2006 Accepted after revision: December 11, 2007 Published online: September 7, 2007

Regulation of Food Intake by Inflammatory Cytokines in the Brain Jessica B. Buchanan a Rodney W. Johnson a, b a b

Laboratory of Integrative Immunology and Behaviour, Division of Nutritional Sciences and Department of Animal Sciences, University of Illinois Urbana-Champaign, Urbana, Ill., USA

Key Words Inflammatory cytokines ⴢ Feeding ⴢ Cachexia ⴢ Hypothalamus

Abstract A number of inflammatory cytokines are synthesized and released after activation of the immune system. In addition to other biological effects, these cytokines can potently inhibit food intake. Cytokine-mediated inhibition of food intake is of particular importance because excessive production of peripheral inflammatory cytokines is often associated with the cachexia-anorexia syndrome seen in some chronic diseases. The weight loss in cachexia is associated with an increase in morbidity and mortality. Understanding how cytokines regulate food intake may be crucial in enhancing quality of life and facilitating recovery in patients exhibiting cachexia. This review describes the main inflammatory cytokines that influence food intake and explores how peripheral cytokines communicate with hypothalamic nuclei to influence feeding. Copyright © 2007 S. Karger AG, Basel

Introduction

Upon activation, cells of the innate immune system (i.e. monocytes, macrophages, microglia) synthesize and release cytokines such as interleukin (IL)-1␤, IL-6 and © 2007 S. Karger AG, Basel 0028–3835/07/0863–0183$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/nen

tumor necrosis factor-␣ (TNF␣) that serve as major mediators of the immune system. They are produced both peripherally and centrally in response to immune stimulation and orchestrate CNS-specific responses including fever, sleep, activation of the hypothalamic-pituitary-adrenal axis, decreased social interaction, and anorexia. The effects of cytokines on food intake are of particular interest because cachexia frequently accompanies aging and chronic diseases that are characterized by excessive production of inflammatory cytokines. The weight loss in cachexia is due to a reduction in appetite, increased metabolic rate, and a preferential loss of lean body mass [1]. In a number of diseases such as AIDS, cardiovascular disease, rheumatoid arthritis, and certain cancers, the loss of lean body mass is closely associated with morbidity and mortality. Therefore, restoring appetite and subsequently increasing lean body mass may enhance the quality of life and facilitate recovery in chronically ill patients. A number of cytokines inhibit food intake after peripheral or central administration (table 1) and can exert their anorectic effects through a myriad of ways. They can act directly in the brain (e.g., the melanocortin system in the hypothalamus), modulate gastrointestinal activities that inhibit feeding, induce the release of hormones (such as leptin and cholecystokinin) that modulate feeding, and induce alterations in metabolism that impact food intake regulation [2, 3]. In addition to their anorectic effects under pathophysiological conditions, it Rodney W. Johnson Laboratory of Integrative Immunology and Behavior Department of Animal Sciences, University of Illinois Urbana-Champaign 1207 W. Gregory Drive, Urbana, IL 61801 (USA) Tel. +1 217 333 8811, Fax +1 217 333 8286, E-Mail [email protected]

Table 1. Cytokines shown to inhibit food intake

Cytokine

Route of administration

References

IL-1␣, IL-1␤

i.p. i.v. i.c.v.

79, 90 15 8, 17

IL-6

i.c.v.

37, 38

IL-8

i.c.v

17

IL-11

i.c.v.

91

IL-18

i.c.v.

92

TNF␣

i.p. i.v. i.c.v.

14 93 17, 30

IFN␣, IFN␥

i.p. i.c.v.

94 95

CNTF

s.c. i.p. i.c.v.

47, 96 44 41, 45

BDNF

i.p. i.c.v.

97 98

GM-CSF

i.c.v.

99

FGF

i.c.v.

100, 101

LIF

i.c.v

40, 102

HMGB-1

i.p. i.c.v.

103

is increasingly recognized that cytokines play a role in the control of food intake and energy homeostasis in the absence of infection and/or disease [4, 5]. An in-depth description of how each of these cytokines exerts their effects is well beyond the scope of this review. Here, we describe the main inflammatory cytokines affecting food intake regulation and discuss how cytokines in the periphery gain access to key neuronal systems in the hypothalamus to influence feeding.

Inhibition of Food Intake by Inflammatory Cytokines: Who Are the Key Players?

Interleukin-1␤ The IL-1 system consists of two agonists (IL-1␣ and IL-1␤), a naturally occurring receptor antagonist (IL1ra), the type I receptor (IL-1RI) responsible for signal184

Neuroendocrinology 2007;86:183–190

ing, the type II receptor (IL-1RII) that cannot functionally signal, and the IL-1 receptor accessory protein (IL-1R AcP), which increases the binding affinity for IL-1RI [6]. IL-1␤ is involved in all aspects of the acute-phase response including fever and sickness behavior [7–9], influences learning and memory [10, 11], and is increasingly thought to play a role in regulating normal physiological processes such as sleep and food intake [4, 5, 12]. IL-1␤ is a potent inducer of anorexia when administered peripherally [13] or centrally [14, 15], and can be affected by nutritional state [4, 5, 16]. It appears to suppress food intake by reducing meal size and duration, without affecting meal frequency [17]. In addition to suppressing feeding behavior, IL-1 inhibits the maturation of adipocytes as well as the synthesis of fatty acid transport proteins in adipocyte tissue in vitro [18, 19]. It also decreases gastric emptying and motility, and increases circulating levels of leptin, glucagon and insulin [2, 20]. The role of IL-1␤ in food-intake suppression is supported by studies with IL-1ra. Lipopolysaccharide (LPS) potently suppresses food intake and increases the expression of inflammatory cytokines in numerous brain areas including the hypothalamus [21–23]. Administration of IL-1ra attenuates LPS-induced anorexia as well as LPSinduced hypothalamic expression of IL-1␤, IL-6 and TNF␣ in mice [24]. In addition, IL-1␤-induced appetite suppression can be completely blocked by IL-1ra [25]. However, mice deficient in IL-1␤ converting enzyme or IL-1␤ still decrease food intake after peripheral injection of LPS or inoculation with influenza virus [26, 27]. LPSinduced suppression of food intake also does not differ between IL-1RI knockout mice and wild types [28]. Moreover, IL-1␤ antisera do not affect LPS-induced suppression of food intake [29]. This suggests that while IL-1␤ is undeniably an important mediator of the suppression of food intake, it does not act alone. Indeed, as noted below, several other inflammatory cytokines share with IL-1␤ an ability to reduce appetite. Tumor Necrosis Factor- ␣ There are two types of TNF␣ receptors (p55 and p75) that are believed to elicit distinct responses. TNF␣ suppresses food intake when administered either peripherally or centrally, but its anorectic effects are less potent than those of IL-1␤ [17, 30]. Adipose tissue secretes TNF␣ and synthesis of this cytokine within adipose tissue occurs in response to both nutritional and immunological regulators [31]. An increase in basal plasma TNF␣ is associated with adiposity [32].

Buchanan /Johnson

LPS-induced suppression of food intake can be attenuated by TNF-binding protein [33]. The anorectic effect of TNF␣ can also be attenuated by administration pentoxifylline, a phosphodiesterase inhibitor that blocks TNF␣ production [34]. However, food intake suppression was not attenuated after either local (turpentine) or systemic (LPS) immune activation in TNF␣ receptor knockout mice [28]. Nor was it affected by co-administration of TNF antisera and subcutaneous (s.c.) LPS [29]. TNF␣, when administered with IL-1␤, acts synergistically to suppress food intake [17].

The hypothalamus is considered the main site of integration for factors involved in regulating energy expenditure and food intake. This was first established in 1951 by Anand and Brobeck [49] who showed that lesions to the ventromedial hypothalamus (VMH) result in hyperphagia and obesity, while lesions to the lateral hypothalamus (LH) cause aphagia and weight loss. Since then, there

have been numerous studies providing further support for the integral role of the hypothalamus, implicating several different nuclei within the hypothalamus such as the arcuate (ARC) and paraventricular (PVN) nuclei, as well as the VMH and the LH. All of these nuclei contain receptors for cytokines, and in some cases, receptors for hormones (such as leptin) that can be induced by cytokines [50]. But if inflammatory cytokines act directly in the hypothalamus as would be suggested by results of studies where cytokines are administered into the third cerebral ventricle, how do they get there? Inflammatory cytokines are large hydrophilic proteins and thus unable to passively diffuse from the blood into the brain across an intact blood-brain barrier (BBB). Active transport systems into the brain have been established for a number of cytokines including IL-1␤, IL-6 and TNF␣ [51]. However, the BBB associated with several hypothalamic nuclei is, by design, imperfect. This lack of integrity, in the form of circumventricular organs (CVO), enables neurons in the ARC to ‘see’ a variety of signals from the periphery including inflammatory cytokines and convey information to other hypothalamic nuclei that are involved in energy balance (fig. 1). Although there is no direct evidence that cytokines can diffuse into the ARC, IL-1␤-immunoreactive cells have been described in numerous CVOs [52–54] and cells of the CVOs can be activated by inflammatory cytokines [55]. This mechanism of communication is convenient if blood concentration of inflammatory cytokines is elevated, but a reduction in food intake is not always associated with a detectable increase in circulating inflammatory cytokine(s). In such instances, peripheral inflammatory cytokines may act locally to induce other humoral responses that can reach the relevant brain areas via the circulatory system, or induce neural mechanisms that ultimately activate microglial cells and/or astrocytes in the brain to produce inflammatory cytokines. One of these mechanisms of communication is through the vagus nerve (fig. 1). Cytokines, specifically IL-1␤, are able to activate afferent neurons of the vagus [56, 57]. Peripheral administration of IL-1␤ and LPS induces c-fos expression in the NTS, the predominant termination site of the vagal afferent fibers [57], and injecting IL-1␤ into the hepatic portal vein increases the activity of vagal afferent fibers in a dose-dependent manner [58]. Moreover, electrical stimulation of the afferent vagus nerve increases IL-1␤ expression in the hypothalamus and hippocampus, as well as increasing hypothalamic mRNA for corticotrophin-releasing hormone (CRH), a factor known to suppress food intake [59].

Cytokines and Food Intake

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Interleukin-6 IL-6 suppresses food intake after central but not peripheral administration [35–37]. When chronically administered into the brain, IL-6 causes anorexia, increases energy expenditure and decreases body fat without inducing an acute-phase reaction [38]. Mice deficient in IL-6 show attenuated inhibition of food intake after both turpentine and influenza virus inoculation [39]. Further, administration of IL-6 antisera attenuates LPS-induced suppression of food intake [29]. Other members of the IL-6 family also show potent inhibitory effects on food intake. These include IL-11, leukemia inhibitory factor, and ciliary neurotrophic factor (CNTF) [17, 40, 41], with CNTF receiving the most attention. Like IL-6, CNTF induces fever [42] as well as inducing other aspects of the acute-phase response [43]. CNTF is predominantly produced by astrocytes in the CNS and chronic administration (peripherally or centrally) reduces food intake and causes weight loss [44, 45]. While all cytokines discussed herein inhibit food intake and result in weight loss, only CNTF appears to have a long-term effect; in obese humans and rodents, weight loss is maintained even after treatment is stopped [46, 47]. There is recent evidence that this long-term effect is due to CNTF’s ability to induce neurogenesis within areas of the hypothalamus involved in energy balance [48].

How Do Cytokines Impinge upon the Central Food Intake Regulatory Centers?

185

2 nd order neurons in other hypothalamic nuclei

fFood intake FMetabolic rate CRH Release IL-1␤

IL-1R MC4-R

Microglia

Y1-R

Y1-R AgRP/ NPY

1 st order neurons in arcuate nucleus

POMC MC3-R

IL-1R

IL-1␤

IL-1R

Microglia

Fig. 1. Model for cytokine communication

to, and action in, the brain. Peripherally induced cytokines act at the level of the BBB and/or activate vagal afferents that project via the NTS to hypothalamic nuclei involved in feeding regulation. Inflammatory cytokines such as IL-1␤ bind to receptors on POMC-containing neurons in the ARC that activate second-order neurons through the interaction of ␣-MSH with MC4 receptors, ultimately resulting in decreased food intake. Inflammatory cytokines may also influence NPY/AgRPcontaining neurons by decreasing NPY signaling through the NPY Y1 receptor, as well as decreasing AgRP antagonism of MC4 receptors. This model was adapted from DeBoer and Marks [86].

IL-1␤

Vagal afferents

IL-1␤

Liver

Macrophage

Subdiaphragmatic vagotomy has been reported to attenuate behavioral as well as neural effects induced by LPS and IL-1␤ such as fever [60, 61], induction of c-fos within the CNS [62, 63], food-motivated behavior [64], and induction of IL-1␤ mRNA in the brain [65, 66]. However, the evidence supporting the role of the vagus in the inhibition of food intake is mixed. While subdiaphragmatic vagotomy attenuated the effects of i.p. IL-1␤, it did not block the effects of i.v. or s.c. administration [67]. Moreover, others have shown that i.p. administration of IL-1␤ and LPS still reduced food intake after subdiaphragmatic vagal deafferentation alone [68] or in combination with surgical transection of splanchnic afferent 186

Vagal afferents projecting to the NTS are activated by peripheral cytokines. NTS neurons that project to hypothalamic nuclei can stimulate microglia.

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pathways [69]. Taken together, the evidence suggests that the vagus plays a role in anorexia, but this role depends on several factors including dose and route of administration. It is likely that the vagus influences feeding behavior via secondary mechanisms such as eliciting taste aversion [70], suppressing food-motivated behavior [64] or through humoral mechanisms. Cytokine mediation of the suppression of food intake could also result from the induction of hormones involved in feeding regulation such as leptin, a potent inhibitor of food intake. Although predominantly produced in the periphery by adipocytes, leptin’s main actions are in the brain. Leptin is not only induced by Buchanan /Johnson

cytokines but can, in turn, induce cytokine expression in the brain [71–73]. In rodents, LPS, IL-1␤ and TNF␣ increased leptin in plasma and induced expression of leptin in adipocytes [74–77]. Administration of leptin antisera reversed LPS-induced suppression of food intake [29]. However, there is evidence that leptin is not the major cause of the suppression of food intake during infection. Mice lacking leptin (ob/ob) or leptin receptor (db/db) are responsive to LPS-induced suppression of food intake [75]. Obese Zucker rats that have only some functional leptin receptors show greater sensitivity to the anorecticinducing effects of i.c.v. IL-1␤ despite showing no differences in brain IL-1 compared to their lean counterparts [78]. Further, they show no differences in the anorectic response compared to lean rats after i.p. LPS, TNF␣ or IL-1␤ [79]. How Do Cytokines Influence Hypothalamic Food Intake Regulatory Centers?

Peripheral immune stimulation activates microglia in the brain. This is accomplished by the actions of peripheral cytokines at the level of the BBB and/or activation of projections of the vagus nerve to the NTS. The question then becomes what are the actions of cytokines once they reach, or are induced in the hypothalamus? The ARC possesses neurons containing pro-opiomelanocortin (POMC), a precursor of ␣-melanocyte-stimulating hormone (␣-MSH) which is a potent inhibitor of food intake [80]. POMC-containing neurons project to several different nuclei that express the melanocortin MC4 receptor (to which ␣-MSH binds) and are involved in appetite regulation, including the PVN, the LH and the VMH. These second-order neurons synthesize CRH that, as mentioned earlier, also inhibits food intake. Inhibition of food intake through melanocortin signaling is tempered by another class of neurons in the ARC associated with increased feeding behavior that contain neuropeptide Y (NPY) and agouti-related protein (AgRP), a natural antagonist of MC3 and MC4 receptors. Neurons in the ARC express IL-1RI [81] as well as receptors for the gp130 family such as CNTF [41] and peripheral administration of IL-1␤ activates ARC neurons expressing POMC as well as decreasing feeding behavior [15]. CNTF activates POMC neurons and can suppress NPY mRNA levels [41, 82] and peripheral administration of LPS induces POMC mRNA expression in the ARC while leaving NPY mRNA levels unchanged [83]. Central administration of IL-1␤ upregulates POMC and decreasCytokines and Food Intake

es food intake [84–86] and this inhibition of food intake can be significantly attenuated by blocking melanocortin receptors [87]. Blocking melanocortin receptors also blocks the LPS-induced decrease in food consumption as well as causing resistance to tumor-induced anorexia in rats [84]. POMC- and NPY/AgRP-containing neurons also express functional leptin receptors; however, the melanocortin-signaling system remains active even when leptin levels are suppressed due to weight loss [88, 89] or abolished in leptin-deficient mice [75]. For a recent review on cytokine influence of the melanocortin system, see DeBoer and Marks [86]. Given the data discussed above, it is possible that one of the main ways inflammatory cytokines regulate food intake is through their actions on POMC-containing neurons. Figure 1 shows a schematic view of cytokine communication to, and action in the brain. Inflammatory cytokines reach, or are induced within the hypothalamus and bind to receptors on POMC-containing cells in the ARC. These cells in turn signal to other neurons within the hypothalamus containing MC4 that affect outputs related to anorexia, loss of lean body mass and increased energy expenditure. In summary, inflammatory cytokines predominantly suppress food intake via mechanisms that are CNS-specific. It is essentially the central actions of cytokines that are responsible for changes in feeding behavior during an immune response. It is clear that no single cytokine is responsible for the modulation of food intake. Instead, each cytokine along with other factors involved in metabolic function, such as insulin, CCK and ghrelin, plays a role in a concerted response that ultimately leads to the inhibition of food intake through their actions on neurons in the hypothalamus. Cachexia is associated with excessive production of inflammatory cytokines and in a number of diseases and certain cancers the loss of lean body mass is closely associated with morbidity and mortality. Therefore, understanding how these inflammatory cytokines regulate food intake may be a crucial step in restoring appetite and lean body mass, which could enhance the quality of life and facilitate recovery in patients exhibiting cachexia.

Acknowledgements This work was supported by NIH grants AG16710, AG023580, and MH069148 (to R.W.J.). J.B.B. is supported by the NIH under Ruth L. Kirschstein National Research Service Award (T32 DK59802).

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65 Laye S, Bluthe RM, Kent S, Combe C, Medina C, Parnet P, Kelley K, Dantzer R: Subdiaphragmatic vagotomy blocks induction of IL-1␤ mRNA in mice brain in response to peripheral LPS. Am J Physiol 1995; 268: R1327–R1331. 66 Hansen MK, Taishi P, Chen Z, Krueger JM: Vagotomy blocks the induction of interleukin-1␤ (IL-1␤) mRNA in the brain of rats in response to systemic IL-1␤. J Neurosci 1998; 18:2247–2253. 67 Bluthe RM, Michaud B, Kelley KW, Dantzer R: Vagotomy attenuates behavioural effects of interleukin-1 injected peripherally but not centrally. Neuroreport 1996;7:1485–1488. 68 Schwartz GJ, Plata-Salaman CR, Langhans W: Subdiaphragmatic vagal deafferentation fails to block feeding-suppressive effects of LPS and IL-1␤ in rats. Am J Physiol 1997;273: R1193–R1198. 69 Porter MH, Hrupka BJ, Langhans W, Schwartz GJ: Vagal and splanchnic afferents are not necessary for the anorexia produced by peripheral IL-1␤, LPS, and MDP. Am J Physiol 1998;275:R384–R389. 70 Schwartz MW, Woods SC, Porte D Jr, Seeley RJ, Baskin DG: Central nervous system control of food intake. Nature 2000; 404: 661– 671. 71 Luheshi GN GJ, Rushforth DA, Loudon AS, Rothwell NJ: Leptin actions on food intake and body temperature are mediated by IL-1. Proc Natl Acad Sci USA 1999;96:7047–7052. 72 Hosoi T, Okuma Y, Wada S, Nomura Y: Inhibition of leptin-induced IL-1␤ expression by glucocorticoids in the brain. Brain Res 2003; 969:95–101. 73 Bariohay B, Lebrun B, Moyse E, Jean A: Brain-derived neurotrophic factor plays a role as an anorexigenic factor in the dorsal vagal complex. Endocrinology 2005; 146: 5612–5620. 74 Grunfeld C, Zhao C, Fuller J, Pollack A, Moser A, Friedman J, Feingold KR: Endotoxin and cytokines induce expression of leptin, the ob gene product, in hamsters. J Clin Invest 1996;97:2152–2157. 75 Faggioni R, Fantuzzi G, Fuller J, Dinarello CA, Feingold KR, Grunfeld C: IL-1␤ mediates leptin induction during inflammation. Am J Physiol 1998;274:R204–R208. 76 Finck BN, Kelley KW, Dantzer R, Johnson RW: In vivo and in vitro evidence for the involvement of tumor necrosis factor- ␣ in the induction of leptin by lipopolysaccharide. Endocrinology 1998; 139:2278–2283. 77 Sato T, Laviano A, Meguid MM, Rossi-Fanelli F: Plasma leptin, insulin and free tryptophan contribute to cytokine-induced anorexia. Adv Exp Med Biol 2003; 527: 233– 239. 78 Ilyin SE, Plata-Salaman CR: Molecular regulation of the brain interleukin-1␤ system in obese (fa/fa) and lean (Fa/Fa) Zucker rats. Brain Res Mol Brain Res 1996;43:209–218.

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Neuroendocrinology 2007;86:191–209 DOI: 10.1159/000108635

Received: November 13, 2006 Accepted after revision: December 4, 2006 Published online: September 19, 2007

Adipokine Gene Expression in Brain and Pituitary Gland Michael Wilkinson a–c Russell Brown a, b Syed A. Imran a, c Ehud Ur a Departments of a Obstetrics and Gynaecology, b Physiology and Biophysics, and c Division of Endocrinology and Metabolism, Faculty of Medicine, Dalhousie University, Halifax, N.S., Canada

Key Words Leptin ⴢ Resistin ⴢ Adiponectin ⴢ Fasting-induced adipose factor ⴢ Blood-brain barrier ⴢ Hypothalamus ⴢ RNA interference ⴢ Cephalokines

Abstract The brain has been recognized as a prominent site of peptide biosynthesis for more than 30 years, and many neuropeptides are now known to be common to gut and brain. With these precedents in mind it is remarkable that adiposederived peptides like leptin have attracted minimal attention as brain-derived putative neuromodulators of energy balance. This review outlines the evidence that several adipose-specific genes are also expressed in the central nervous system and pituitary gland. We, and others, confirmed that the genes for leptin, resistin, adiponectin, FIAF (fastinginduced adipose factor) and adiponutrin are expressed and regulated in these tissues. For example, leptin mRNA was readily detectable in human, rat, sheep and pig brain, but not in the mouse. Leptin expression in rat brain and pituitary was regulated through development, by food restriction, and following traumatic brain injury. In contrast, hypothalamic resistin mRNA was unaffected by age or by fasting, but was significantly depleted by food restriction in mouse pituitary gland. Similar results were seen in the ob/ob mouse, and we noted a marked reduction in resistin-positive hypothalamic nerve fibres. Resistin and fiaf mRNA were also upregulated in hypoxic/ischaemic mouse brain. Our studies on the regulation of neuronal adipokines were greatly aided by the availability of clonal hypothalamic neuronal cell lines. One of

© 2007 S. Karger AG, Basel 0028–3835/07/0863–0191$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/nen

these, N-1, expresses both rstn and fiaf together with several other neuropeptides and receptors involved in energy homeostasis. Selective silencing of rstn revealed an autocrine/ paracrine regulatory system, mediated through socs-3 expression that may influence the feedback effects of insulin and leptin in vivo. A similar convergence of signals in the pituitary gland could also influence anterior pituitary hormone secretion. In conclusion, the evidence is suggestive that brain and pituitary-derived adipokines represent a local regulatory circuit that may fine tune the feedback effects of adipose hormones in the control of energy balance. Copyright © 2007 S. Karger AG, Basel

Introduction

Background That the brain is an important site of peptide biosynthesis was originally suggested more than 30 years ago [1]. The seminal studies of Pearse and co-workers [2], and others [3], proposed the now readily accepted view that multiple neuropeptides are common to gut and brain. It followed that peptides found in peripheral, e.g. gastrointestinal or respiratory, systems could reasonably be predicted to be biosynthesized in the brain and to exert physiological effects there [4]. Ghrelin is a recent example of this principle. First isolated from the stomach, ghrelin is now known to be expressed in the brain as well [5]. With these precedents in mind it is remarkable that adipose-derived peptide hormones such as leptin and resistin have attracted little attention as Michael Wilkinson Department of Obstetrics and Gynaecology, IWK Health Centre 5980 University Avenue, PO Box 9700 Halifax, N.S. B3K 6R8 (Canada) Tel. +1 902 470 7198, Fax +1 902 470 7192, E-Mail [email protected]

Leptin/18S (arbitrary units)

A a 1.5

**

0.5 0

Fed

Leptin/18S (arbitrary units)

Fast

Refed

***

b 1.5

*

1.0 0.5 0

Fed

Fast

Refed

***

c 4 Leptin (ng/ml)

*

1.0

3 2

*

1

B

1.0

Leptin/18S (arbitrary units)

0

0.8

Fed

Fast

Refed

**

0.6 0.4 0.2 0

Arcuate nucleus

Remainder of hypothalamus

Fig. 1. A Arcuate nucleus ob expression regulated by food intake.

Weight-matched adult female rats were divided into three groups and either: (1) fed ad libitum, (2) fasted for 48 h, or (3) fasted for 42 h and refed a normal diet for 6 h. Microdissected arcuate nucleus (a) and visceral fat (b) were collected for semiquantitative RT-PCR analysis of ob mRNA. Blood was collected for serum leptin levels (c; RIA; Linco). Values are means 8 SEM; n values were 8–10 per group. * p ! 0.05; ** p ! 0.01; *** p ! 0.001; ANOVA with Newman-Keuls post-hoc test. B Ob mRNA is enriched in the arcuate nucleus. Analysed by semiquantitative RT-PCR (n = 4 per group). ** p ! 0.01.

putative central neurotransmitters or neuromodulators. In this review we will provide evidence that several socalled adipose-specific hormones are also expressed in the brain and pituitary gland of numerous species, including man. 192

Neuroendocrinology 2007;86:191–209

A singular event in the now prodigious ongoing study of energy balance and body weight regulation was the discovery of leptin by J.M. Friedman’s laboratory [6]. Subsequent investigations by several research groups revealed that leptin is but one of a large family of factors secreted by adipocytes (adipokines [7–9]). These include the familiar (resistin [10]; adiponectin [11]), the new (FIAF (fasting-induced adipose factor) [12]; visfatin [13]; vaspin [14]) and the unexpected (nerve growth factor [15]). Application of mass spectrometry-based proteomic techniques will certainly enlarge this list of secreted proteins [16, 17]. In the case of leptin, its receptors were reported early on to be widely distributed in the rodent and human brain [18–20] and pituitary [21, 22]. The abundance of leptin receptors in brain regions such as cerebellum, hippocampus and cerebral cortex suggested that leptin probably subserves functions in addition to, and distinct from, those that control energy homeostasis. Since leptin is a large peptide (16 kDa) that may not readily enter the brain, we hypothesized that many of these receptors, with the exception of those in the basal hypothalamus, would be accessible only to a brain-derived ligand, and that this may be leptin of cephalic origin. We subsequently demonstrated that the rat brain did indeed express leptin mRNA [23, 24]. Leptin mRNA levels were enriched in the arcuate nucleus, and expression was regulated by fasting and refeeding (fig. 1). Further studies by ourselves, and others, revealed that leptin mRNA was readily detectable in the brain of many species, including human, sheep, pig and fish, and in human neuroblastoma and rat glioblastoma cells (table 1). Moreover, in vivo investigations by Esler and co-workers [25, 26] demonstrated that leptin was secreted from the human brain. In the mouse, leptin mRNA was undetectable by Northern analysis in whole brain [6] and we could not detect it by RT-PCR in discrete brain regions, including the arcuate nucleus. However, an early report by Bennett et al. [27] included data on leptin expression in C57BL6 mouse fetal brain. We were unable to reproduce this in the CD-1 mouse, but we did obtain a clear signal in neonatal brain, suggesting that the unusual expression of leptin in mouse brain might occur in a strain- and developmental-dependent fashion. This may reflect the unique energy regulation mechanisms in the mouse detailed by Himms-Hagen [28] and by Arner [29]. The lack of success in detecting leptin mRNA in murine brain has profoundly influenced current thinking of the physiology of leptin. Thus all of the central effects of this adipokine are assumed to result from circulating peripheral leptin entering the brain, via a saturable transport mechanism, and Wilkinson /Brown /Imran /Ur

binding to leptin receptors. The widespread acceptance of such a view, based mainly on mouse data, neglects the findings obtained in other species that suggest brain-derived leptin may have neurotransmitter, neuromodulator or neurotropic properties. At present we do not know why leptin expression appears to be suppressed in the murine brain.

Table 1. Leptin gene expression in brain

Species

References

Rat

Morash et al., 1999 [23] Beretta et al., 2002 [169] C6 glioblastoma: Morash et al., 2000 [78] Li et al., 2001 [79] Brown et al., 2005 [83]

Sheep

Ehrhardt et al., 2002 [170]

Pig

Smolinska et al., 2004 [130] Kaminski et al., 2006 [151]

Mouse

No expression: Zhang et al., 1994 [6] (but see Bennett et al., 1996 [27]; fetal brain)

Monkey

Not known

Human

Knerr et al., 2001 [85] Eikelis and Esler, 2005 [26] Neuroblastoma cells: Russo et al., 2004 [84]

Tiger salamander

Boswell et al., 2006 [171]

Triturus tadpole

Buono and Putti, 2004 [172]

Xenopus laevis

Crespi and Denver, 2006 [132]

Do Adipokines Cross the Blood-Brain Barrier? The impetus to search for brain-derived adipokines came from our conviction that such large peptides would not readily cross the blood-brain barrier (BBB) to permit binding to their central receptors. Nevertheless, several groups have provided evidence for a transport mechanism that allows leptin entry into the brain [30–33]. This transport mechanism is partially saturated over a wide physiological range [34] and there remain doubts as to whether leptin can enter the brain to target neurons directly [35]. We argued previously that paradoxically the leptin receptors localized in brain microvessels might serve to facilitate degradation of leptin (a ‘metabolic’ BBB) rather than permit its entry into the brain [24]. The issue is complicated by methodological questions. For example, the brain perfusion technique reported by Banks et al. [36] and Hileman et al. [37] quantified tissue-bound [125I]leptin as a measure of brain penetration, i.e. radioactivity was quantified by direct counting of microdissected brain nuclei. It is possible that some, if not all, the tissue radioactivity could represent binding of leptin to brain capillaries rather than to brain cells [38]. Even the more sophisticated technique of brain perfusion with an oxygenated artificial plasma [33] cannot fully represent the situation in the intact animal, where leptin is known to circulate as a leptin-leptin binding protein complex [39]. This binding protein serves to suppress leptin action by inhibiting specific leptin binding to leptin receptors [40] and might well prevent brain uptake of leptin. Thus the brain perfusion technique could overestimate brain uptake of leptin. The detection of leptin in cerebrospinal fluid (CSF) suggests that leptin might reach brain cells from the peripheral circulation. However, and as noted for the BBB, the transport mechanism of leptin from the blood to CSF is also saturated at physiological concentrations [41]. Furthermore, the concentration of leptin in CSF, at normal plasma levels, appears to be too low to activate the long form of the leptin receptor. CSF concentrations of leptin (approx. 1 ng/ml; 0.06 nmol/l) [41, 42] are significantly below the KD value of 0.3 nmol/l for the leptin receptor [43].

Of further concern is the paucity of evidence showing that peripherally-derived leptin can bind to extrahypothalamic leptin receptors and modify signaling pathways [44]. For example, we predicted that peripheral injection of leptin should increase c-fos expression in basal hypothalamus but not, for example, in hippocampus and cerebral cortex. This is indeed the case, since we observed leptin-induced c-fos expression only in basal hypothalamus [24], and these data are largely in agreement with other reports [45]. However, a lack of c-fos expression does not necessarily imply an absence of leptin-induced cell activation. Equally, c-fos may not be expressed in all cells that have leptin receptors. Leptin target neurons in brain contain STAT-3-ir (immunoreactivity) [46] and leptin injection activates STAT-3 in hypothalamus [47]. Our experiments showed that leptin increases STAT-3-ir in hypothalamus but not in hippocampus, cerebral cortex or substantia nigra [48]. This again implies either that leptin does not readily penetrate the BBB, or that cortical and hippocampal neurons possess unusual leptin-signaling pathways. A critical experiment would be to determine whether STAT-3 mRNA, or SOCS-3 mRNA, another leptin-sensitive gene [45], is increased following

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microinjection of leptin into, for example, hippocampus or cerebral cortex. STAT-3 and SOCS-3 are known to be expressed throughout the brain [49, 50]. The case for a brain-derived adiponectin is also strong: (a) adiponectin does not cross the BBB [51, 52]; (b) adiponectin receptor expression is present in brain tissue [53, 54]; (c) adiponectin mRNA is readily detected in chicken brain [55], and we have confirmed the presence of adiponectin mRNA in mouse brain by real-time RT-PCR. Thus, like leptin, brain-derived adiponectin requires further study as a putative neuromodulator, particularly since adiponectin reduced body weight and stimulated c-fos expression following injection into the brain [53]. As we outline elsewhere (see below), there is also good evidence for resistin and FIAF as brain-derived adipokines. In summary, while it seems likely that limited amounts of leptin do cross the BBB to gain access to the brain [31, 33, 38, 56], it is also possible that in the mouse, whose brain does not express ob, the BBB may allow greater penetration of leptin when compared to the rat. This could also account for the failure of peripherally administered leptin to reduce body weight in several clinical trials [57, 58], whereas leptin-deficient obese children and adults respond readily to leptin treatment [59, 60]. We recognize, of course, that such an effect of leptin in leptin deficiency might also be due to modified leptin receptor signaling. A Dual System? Our evidence that brain cells are also a source of leptin raises an interesting question: Why would circulating leptin (and possibly other adipokines) need to gain access to brain sites where leptin is already biosynthesized? Such a dual system is not without precedent. There are several well-described examples of neurotransmitter/hormonal dualisms. For example, catecholamines are synthesized as neurotransmitters within the brain, and as hormones in the periphery, as are gastrointestinal hormones such as somatostatin and cholecystokinin. The question does not apply only to leptin. Pan et al. [61] reported that ‘… the central nervous system effects of peripheral growth hormone can be attributed to the permeation of the BBB …’, a claim refuted by the demonstration of growth hormone gene expression and regulation in the hippocampus [62]. Our evidence, that adipokines are not produced exclusively by adipocytes, suggests they may be part of a brain system that operates independently, or in conjunction with, the peripheral systems. This is not to discount the well-described central effects of circulating adipokines 194

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[34, 44]. It is possible that endogenous brain adipokines might modulate the feedback effects of adipose hormone signals originating in the periphery or may provide the brain with an independent adipokine circuitry. For example, leptin-deficient, or leptin-insensitive, rodents have impairments in hippocampal function [63], and direct injection of leptin into the hippocampus improves memory performance in mice and can facilitate longterm potentiation via NMDA receptors [64, 65]. There is also new evidence that peripheral injection of leptin can facilitate learning and memory performance and longterm potentiation [66]. However, it seems to make little sense that human cognitive performance should be dependent on adipose tissue secretion. These values vary widely (e.g. after meals) and may be chronically high in obese individuals, or very low in periods of prolonged food deprivation. A more considered view is that leptin made within the hippocampus, in close proximity to leptin receptors, is responsible for its effects on cognition. On the other hand, aging obese and diabetic patients, whose BBB might be compromised, could be more sensitive to changing leptin secretion [67]. This paradox, of why a leptin target tissue should also be capable of expressing leptin, is also demonstrable in the pituitary gland. The pituitary of course is not protected by the BBB and is chronically bathed in blood-borne leptin. In this case our hypothesis is that pituitary-derived leptin might ‘tune’ the leptin-signaling pathways to incoming leptin signals from adipose tissue. In addition, pituitary leptin expression might be maintained under conditions of starvation when there is minimal, if any, leptin secretion from adipose tissue.

Leptin Gene Expression in Brain

Introduction It is obvious from table 1 that the brain of most, if not all, species is capable of expressing the leptin gene (ob). However, as noted above, the mouse appears to be a highly significant exception. Following the discovery of leptin [6], the absence of leptin mRNA in brain became the accepted view. Thus ‘… little, if any (leptin) is produced in the CNS …’ [34]. This is clearly not the case for species other than the mouse. The assertion that low expression levels in the brain, compared to adipose tissue, preclude a significant role for adipokines including leptin and resistin is also incorrect [68, 69]. As a general principle, low mRNA abundance does not equate to lack of function. Two examples will serve to illustrate this. First, leptin reWilkinson /Brown /Imran /Ur

Fig. 2. Leptin-ir is localized to neuronal nuclei. Confocal images of leptin immunofluorescence (A) and NeuN immunofluorescence (neuronal marker; B) in sections of rat piriform cortex. Arrows indicate double-labeled neurons. Leptin staining of nuclei is clearly evident. Scale bar: 50 ␮m [adapted from 72].

ceptor mRNA is undetectable by in situ hybridization in neurons that have high levels of receptor protein immunoreactivity [18, 70]. Second, messenger RNA for the neurotropins NT4 (neurotropin 4) and NGF (nerve growth factor), which are indisputably essential for developmental and adult brain plasticity, are below the level of detection via in situ hybridization [71]. However, NGF mRNA was readily quantified by RT-PCR (36 cycles) in cerebral cortex [71]. We determined that the CT (threshold cycle) values for leptin mRNA, as a measure of mRNA abundance, were 38 (rat hypothalamus), 36 (rat cortex) and 20 (rat adipose tissue). For resistin mRNA the corresponding CT values were, 36, 36 and 36, respectively. In the mouse, CT values for resistin mRNA were 30, 34, 31 and 17 (hypothalamus, N-1 neurons, pituitary and fat, respectively). A very similar pattern for fiaf mRNA was also observed (i.e., CT values 32, 28, 31 and 24). Thus, low abundance values for central leptin and resistin mRNAs are not unusual, and are comparable to the accepted state of affairs for NT4 and NGF. Moreover, and similar to the detection of NGF protein, leptin and resistin protein immunoreactivity is readily seen in those brain areas where the mRNAs are detectable [72, 73]. Detection and Regulation of Leptin mRNA in the Rat Brain We have provided convincing evidence that ob mRNA is readily quantified in the rat brain and in the rat-derived C6 glioblastoma cell line (table 1). Leptin mRNA was widely distributed in the brain, including the cerebral cortex, cerebellum, hypothalamus, pineal gland, retina, and in the posterior and anterior pituitaries. Two sets of intron-spanning primers (217 and 495 bp) gave identical results. The low abundance of ob mRNA precluded

the use of in situ hybridization to localize ob mRNA, although signals were detected in sections of cerebellum and anterior pituitary. Nevertheless, tissue microdissection revealed a marked enrichment of ob mRNA in the arcuate nucleus when compared to the rest of the hypothalamus (fig. 1B). We used double-label immunofluorescence confocal microscopy to reveal that leptin immunoreactivity (ir) was colocalized with the neuron-specific marker NeuN in various brain regions including the arcuate nucleus, piriform cortex and hippocampus [72]. In the neurons of the paraventricular and supraoptic nuclei, most oxytocin- and vasopressin-positive cells contained leptin-ir. We made two further interesting findings: (a) leptin is also present in non-neuronal cells in the arcuate nucleus, and (b) there is clear evidence that leptin-ir is confined to the cell nucleus in some neurons (fig. 2). This localization suggests the existence of a so-far undescribed nuclear leptin receptor. We have provided evidence that central leptin gene expression can be regulated. For example, we observed striking tissue-dependent developmental changes in ob mRNA in the rat cerebral cortex and pituitary, but not in the hypothalamus [74, 75]. In cortex, ob mRNA increased markedly from birth to puberty in male and female rats. In contrast, pituitary levels were high at birth but declined to minimal levels post-weaning. In adults, fasting and refeeding significantly modified leptin expression in the hypothalamus (fig. 1). In recent studies we implicated brain-derived leptin in the consequences of traumatic brain injury. Since leptin is known to be involved in the etiology of cachexia, and may also possess neuroprotective properties. We tested the hypothesis that the brain responds to injury by increasing leptin gene expression [76]. Twelve hours post-injury (fluid

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Fig. 3. Effects of unilateral fluid percussion injury on brain ob

gene expression. Adult male Sprague-Dawley rats were subjected to unilateral fluid percussion brain injury as previously described in detail [168]. Total RNA was isolated from ipsilateral and contralateral cortex, hippocampus and thalamus, reverse transcribed and then subjected to real-time PCR to quantify mRNA levels as previously described [83]. 12 h post-injury ob mRNA levels were significantly increased in the ipsilateral cortex (A) and thalamus (B), but not in ipsilateral hippocampus (C). No effects were seen in the contralateral brain sites. Values are means 8 SEM, expressed as a percentage of the sham control values (n = 6–8). *** p ! 0.001; ** p ! 0.01. TBI = Traumatic brain injury.

percussion) ob mRNA was significantly elevated in the ipsilateral thalamus (227%; p ! 0.01) and cortex (255%; p ! 0.001) (fig. 3). No effects were seen in the contralateral sites [77]. These data suggest that increased leptin expression may be confined to the site of injury and therefore may play a role in neuroprotection or recovery from injury. Detection and Regulation of ob mRNA in C6 Glioblastoma Cells We sought a cell line to further our studies on the regulation of ob gene expression in brain. Rat C6 glioblastoma cells are immortalized glial cells that express recep196

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tors for insulin, glucocorticoids and noradrenaline, all of which are known to regulate adipose-derived leptin levels. C6 cells proved to be a valuable model system to investigate leptin gene expression [78, 79]. Ob mRNA and leptin protein were readily detectable, but control of gene expression was unexpectedly the opposite of that reported for adipose tissue. For example, dibutyryl cAMP stimulated ob mRNA in C6 cells, whereas corticosterone was inhibitory [80]. In addition, insulin and IGF-1 also stimulated rather than inhibited leptin expression in C6 cells. Our subsequent studies on the regulation of the leptin gene promoter [79] confirmed our previous results that there are tissue-specific differences in the control of ob transcription in adipose vs. neuroectodermal tissues. Two additional points should be mentioned: (a) these results indicate that leptin expression in the brain may occur in glial cells (but keeping in mind that C6 cells may not fully represent differentiated glial cells), in addition to neurons, and this is consistent with our double-label immunohistochemistry data; (b) we subsequently found no evidence that C6 cells secrete leptin (radioimmunoassay; Imran, Brown, Ur and Wilkinson [unpubl. data]). The fact that leptin-ir is predominantly localized in the nucleus in C6 cells (also in neurons [72]) suggests that leptin may act as an autocrine or intracrine factor. Additional studies in neuronal cells are now necessary (see below: Leptin Gene Expression in the Human Brain). Silencing of Brain Leptin Gene Expression At minimum, our data emphasize the need for further study of brain-derived leptin. In order to definitively establish a physiological or pathological role for brain leptin, a specific disruption, or silencing, of central leptin gene expression is necessary. We hypothesized that RNA interference (RNAi), a powerful post-transcriptional silencing technique [81, 82], could selectively silence rat brain ob expression and provide important insights into potential physiological roles for brain leptin. We used the C6 cells as a model system to establish this technique [83]. Briefly, we successfully reduced ob mRNA levels by 50% and leptin protein levels by 55%. Silencing leptin unexpectedly induced a 2-fold increase in cell death, suggesting that endogenous brain leptin could be a critical factor for cell survival in the brain. This finding is also consistent with the report that leptin reduced apoptosis in human neuroblastoma cells [84]. The consequences of silencing ob expression in intact rat brain in vivo are currently being determined. We hypothesize that silencing of hypothalamic ob expression will increase food intake and body weight. Wilkinson /Brown /Imran /Ur

Leptin Gene Expression in the Human Brain Ob gene expression has been reported in the human brain and in a human neuroblastoma cell line [26, 84, 85]. Knerr et al. [85] used RT-PCR to detect low levels of ob mRNA in astrocytomas and glioblastomas, but also in normal temporal lobe tissue. Similarly, leptin mRNA was quantifiable, again in low concentration, in samples of hypothalamus [26, 86]. These limited reports suggest that the human brain, like the rat brain, can express the leptin gene. Additional evidence that the human brain is a source of leptin comes from studies measuring simultaneous peripheral arterial and internal jugular venous blood at the level of the base of the brain (to exclude the possibility of contamination with facial venous drainage) [25, 87, 88]. These studies suggest that brain-derived leptin constitutes a surprising 40% of the whole body leptin secretion and is gender and weight dependent, being higher in women and obese individuals. In a further report from this group, patients with major depressive disorder released significantly less leptin from the brain compared to healthy controls (–70%, p ! 0.05) [86]. Whether this leptin is entirely produced within the brain, or that the brain along with producing some leptin also acts as a storage-and-release facility for adipose-derived leptin, is not clear [89]. If the former, this would imply that leptin is secreted from cells within the brain, possibly neurons. At present we have no evidence from animal studies that this is the case, but experiments designed to test such a hypothesis are relatively easy to perform, keeping in mind that leptin is contained within anterior pituitary secretory granules (see below: Leptin Gene Expression in Pituitary Gland). There are also data indicating leptin is secreted from some pituitary tumours [90]. Our work on rat C6 glioblastoma cells suggests that human brain tumour cells may also express leptin. Russo et al. [84], working on a human neuroblastoma cell line (SH-SY5Y), demonstrated that these cells express leptin and its receptors. Further, leptin was capable of stimulating cell proliferation. The mechanism appears to be mediated via leptin receptors located in the cell membrane. However, the authors do not speculate on the release mechanism that permits leptin to reach these receptors. Whether this is a secretory pathway remains to be determined. The SH-SY5Y neuroblastoma cells exhibit many features of mature sympathetic neurons, including the secretion of noradrenaline [91]. It would be interesting to determine whether leptin is also released from these cells by depolarization. These collective data suggest that brain-derived leptin, and possibly other adipokines, may

Regulation of Resistin Expression in Brain Rstn expression in mouse brain was evaluated in adult C57BL6 and CD-1 mice using RT-PCR analysis using two alternate sets of resistin primers designed to span 278 and 330 bp of the cDNA [101]. Products of the expected size were reproducibly observed in fat, hypothalamus and cortex using both sets of primers in both strains of mice. Rstn expression was readily detectable even after 30 cycles. Since low-level expression of rstn has been reported in human monocytes [102], we compared rstn mRNA levels in saline perfused and non-perfused tissues. Our results indicate that rstn expression was identical in both perfused and non-perfused mouse brain and pituitary indicating that blood cells are not the source of rstn mRNA in our experiments. This was confirmed by RTPCR [97]. Rstn expression in microdissected basal hypothalamus (MBH) was compared to expression in the remainder of the hypothalamus using semiquantitative RTPCR analysis (278-bp primers). Resistin expression was significantly higher (⬃3-fold; p ! 0.005) within the MBH compared to the remainder of the hypothalamus, consistent with the immunolocalization of resistin protein (see below). Enrichment of rstn mRNA in MBH was subsequently confirmed by in situ hybridization [103]. These

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play a more complex role in the human brain than originally anticipated. Further studies are required to elaborate the central role of other adipokines in humans and to unravel the complex interactions of these hormones.

Resistin Gene Expression in Brain

Introduction The adipokine resistin, like leptin, is generally considered to be exclusively produced by adipose tissue [92]. Nevertheless, there is little doubt that the resistin gene (rstn) is expressed in multiple non-adipose sites. Resistin expression is abundant in human macrophages [93], leukemia cells [94], placenta [95], and pancreatic islets [96], in several rat tissues [97] and in mouse hepatic cells [98]. This abundance of non-adipose tissue sites for rstn expression has complicated the original hypothesis that resistin might be an important link between adipocytes and insulin resistance, and this issue is yet to be resolved [68, 99]. We hypothesized that resistin would be produced by the brain and pituitary gland and this proved to be correct [100, 101]. The regulation of rstn expression in mouse pituitary gland is described below (see below: Resistin and Pituitary).

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data revealed discrete localization of rstn mRNA to the arcuate and ventromedial nuclei of the hypothalamus, and to the hippocampus and cerebral cortex. Resistin mRNA was also detected in human fetal brain [104]. Hypothalamic resistin gene expression was unaffected by age (postnatal day (PD) 3 to PD 40) [101] or by fasting [100]. Studies by Nogueiras et al. [97] also showed that rstn mRNA was reduced by fasting in fat tissue, but not in a variety of other tissues including stomach and muscle. Also, and in marked contrast to changes seen in fat and pituitary, hypothalamic rstn mRNA in ob/ob mice was not different from control, non-obese, values. In situ hybridization may be required to determine fasting-induced changes in discrete hypothalamic nuclei. We considered the alternative possibility that changes in resistin protein levels might be detectable using immunohistochemistry since we observed a complex network of resistin +ve fibres extending rostrally from the arcuate nucleus of the hypothalamus (ARC) to the preoptic area. Double-label immunofluorescence revealed that these fibres colocalized with ␣-MSH-ir, suggesting that rstn was expressed by POMC neurons [73]. However, in keeping with our RT-PCR data, hypothalamic resistin-ir was unaffected by fasting (48 h) or by high-fat diet, but periventricular staining was greatly increased in the lactating mouse. In contrast, marked reductions in resistin +ve fibres were seen in brain tissue from: (a) ob/ob mice and (b) young mice made underweight for their age by raising them in large litters. This pattern of reduced resistin-ir in ob/ob mice is reminiscent of the results described by Bouret et al. [105, 106]. They reported that in ob/ob mice, ␣-MSH-positive axonal projections from the arcuate nucleus were severely disrupted when compared to those in wild-type mice. These deficits were reversible by neonatal treatment with leptin [107]. Similar experiments are clearly required to determine the influence of leptin replacement on resistin-ir in ob/ob mice. We previously reported the colocalization of leptin-ir with oxytocin and vasopressin in the rat paraventricular and suproptic nuclei [72], and our data on hypothalamic resistin +ve fibres revealed a further example of an adipokine colocalized with hypothalamic neuropeptides. It remains to be determined what role resistin might play in these neurons. Nevertheless, our data suggest that resistin is present within a key hypothalamic circuit responsible for energy balance [108]. An important question is whether the resistin gene is expressed by POMC neurons, or alternatively, is resistin taken up by these cells from the peripheral circulation? Regardless of whether resistin is biosynthesized or merely accumulated by POMC neurons, our 198

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data indicate that the hypothalamus is a target for resistin (see below). Resistin circulates as a large hexamer and, like adiponectin [51, 52], is unlikely to cross the BBB. At present, resistin receptors remain to be identified, although the first resistin binding protein was recently reported [109]. Nonetheless, evidence for an effect of exogenous resistin is now available. First, Brunetti et al. [110] reported that human resistin (0.1–10 nM) inhibits the depolarizationinduced secretion of catecholamines, but not serotonin, from rat hypothalamic synaptosomes. Resistin was without effect on basal secretion. It is not clear from this report how much hypothalamic tissue was used to prepare the synaptosomes, i.e. were the nerve endings from the median eminence and therefore accessible to circulating resistin, or were they from sites protected by the BBB? Second, Tovar et al. [103] provided further evidence that resistin influences neuronal activity. Microinjection of mouse resistin (10 ␮g per rat, aa 26–49 or aa 23–42) into the lateral ventricle transiently reduced food intake in both fasted and satiated adult male rats. This effect was reproducible over several days, but these repeated treatments did not reduce body weight. Third, resistin increased expression of c-fos in the arcuate nucleus of fasted, but not fed, rats. To summarize, resistin mRNA and resistin immunoreactivity are present in hypothalamus, and resistin appears to exert neurochemical effects on hypothalamic synaptosomes (nerve endings) and in the rat brain in vivo. We have also determined that resistin mRNA is readily detectable in a novel hypothalamic neuronal cell line. This clonal cell line, N-1, expresses a variety of neuropeptides and receptors that are typical of mouse hypothalamic neurons [111]. As such, they appear to be a valuable resource to extend our studies on brain adipokines. Resistin Gene Expression in a Novel Hypothalamic Neuronal Cell Line So far, we have been unsuccessful in detecting alterations in hypothalamic adipokine expression in vivo, especially with regards to fasting-induced changes in hypothalamic-derived resistin and FIAF (see below: FastingInduced Adipose Factor). We speculate that changes in gene expression are likely occurring in discrete cellular populations that would not be detected via RT-PCR [100], and progress here will require other methods such as in situ hybridization. Recently, Belsham et al. [111] developed a cohort of immortalized hypothalamic neuronal cell lines that express a variety of hypothalamic neuropeptides and receptors that are involved in numerous Wilkinson /Brown /Imran /Ur

rstn mRNA expression (% of controls) fiaf mRNA expression (% of controls)

A

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processes, most notably central energy homeostasis. We hypothesized that rstn and fiaf would be expressed to varying degrees in the various hypothalamic cell lines and this proved to be correct. For our studies on rstn regulation we employed the N-1 cell line, which expresses both rstn and fiaf, permitting us to study their expression at the single cell level. N-1 cells do not express glial fibrillary acidic protein, a glial cell maker [111], but are immunopositive for the neuronal marker NeuN [112], confirming their neuronal nature. We showed in preliminary studies that valproic acid, an antiepileptic drug that is commonly associated with weight gain, inhibits both resistin and FIAF expression in N-1 cells [112]. Of greater interest, treating N-1 cells with resistin reduced socs-3 expression, an inhibitor of leptin and insulin signaling [113]. This result is the opposite to the effect of resistin in adipocytes, where resistin was reported to induce socs-3 gene expression [92]. Thus, resistin’s actions are tissuespecific. Conversely, specifically silencing resistin gene expression using RNAi induced significant increases in both fiaf and socs-3 expression as detected by real-time RT-PCR (fig. 4) [114]. Thus despite the low levels of resistin expression in N-1 cells, resistin appears to modulate the expression of other genes (i.e. socs-3) implicated in central energy homeostasis. The data obtained from N-1 cells indicate that rstn and fiaf, and possibly other adipokine genes, are transcribed in the same neurons. In this respect they begin to resemble adipocytes in the range of their gene products. Since there is evidence for complex autocrine/paracrine interactions of adipokines in adipocytes [115], this may also be true for neuron-derived adipokines as well. Although the N-1 neurons are immortalized cells, they represent a valuable resource to investigate the physiological relevance of adipokine expression in neurons.

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Fig. 4. Silencing rstn expression increases socs-3 and fiaf mRNA in N-1 hypothalamic neurons. A Rstn gene expression was reduced by 60% in serum-starved N-1 cells transfected with a StealthTM RNAi (STH R4). B, C Fiaf and socs-3 expression was increased by 46 and 65%, respectively, in the STH4-treated cells, relative to cells treated with STH CTL. Values are expressed as a percentage of the control 8 SEM (triplicate experiments; n = 10– 12). * p ! 0.005.

Adipose tissue is the primary source of FIAF, a secreted protein also known as angiopoietin-like protein 4 (ANGPTL4) [12, 116]. We demonstrated that fiaf mRNA is present in high abundance in mouse brain and pituitary [117]. It was also detectable in human glioblastoma and oligodendrocyte tissue [118] and in a human glioblastoma cell line [119]. Unexpectedly, we observed no change in hypothalamic fiaf expression following a 24hour fast in male mice. However, in the same mice, fiaf mRNA was significantly increased approximately 2.5fold in both visceral fat and in the pituitary gland [117]

(also see below: Other Pituitary Adipokines). In order to investigate whether fiaf might be regulated in hypothalamic neurons, we initiated both in vivo and in vitro studies. FIAF is associated with inflammation in several tissues. For example, hypoxia induced fiaf mRNA in endothelial cells [120], human cardiomyocytes [121] and in human glioblastoma [119]. We demonstrated that fiaf gene expression was upregulated in the neonatal hippocampus and cerebral cortex after hypoxic/ischaemic brain injury. Fiaf was upregulated ipsilateral to the lesion 2 days after onset of injury and remained elevated at 7 days, but returned to basal levels after 21 days [122]. Whether increased adipokine production is beneficial or deleterious to the outcome of cerebral ischaemic insult remains to be elucidated. FIAF is reported to exert strong

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pro-angiogenic effects in renal carcinoma [118], and the time-course of fiaf upregulation observed in our experiments coincides with that of cerebrovascular angiogenesis after focal brain ischaemia in adult mice [123] or cortical cold injury in adult rats [124]. In additional experiments we observed that a lipopolysaccharide-induced inflammatory response inhibited cortical fiaf expression in neonatal mice. These data therefore indicate that brain adipokine genes are sensitive to peripheral inflammatory stimuli. The impact of obesity-related low-grade inflammation [125] on the central control of the endocrine system now needs to be addressed. Studies in N-1 neuronal cells have also confirmed that LPS is capable of inducing fiaf expression, but not rstn. The role of hypothalamicderived FIAF remains elusive at best. In order to elucidate potential roles in the hypothalamus we undertook studies in the N-1 cell line, as described above for resistin (see Resistin Gene Expression in a Novel Hypothalamic Neuronal Cell Line). We have previously confirmed that both fiaf and rstn are expressed in fairly high abundance in N1 cells by RT-PCR. Although controversial, FIAF has been shown to improve glucose tolerance via a hepatocyte-dependent mechanism [126]. However, the hypothalamus is also regarded as a glucose-sensing system [127], whose function might be modulated, in part, by local fiaf expression. We were able to detect FIAF secretion from N-1 cells that were overexpressing fiaf ; however, there were no detectable changes in rstn or socs-3 expression. Conversely, reduction of fiaf expression using RNAi failed to affect rstn or socs-3. Although resistin appears to modulate fiaf expression, as discussed previously, the reverse does not hold true and rules out a potential crosstalk between the two adipokines. As mentioned above, fiaf expression in the central nervous system is extremely sensitive to inflammation. Treatment of N-1 cells with LPS, a potent inflammatory molecule, induced a rapid and robust increase in fiaf that is sustained for 124 h. Studies are now underway to elucidate possible signaling mechanisms, and transcription factors, that are responsible for its induction. We suggest that like rstn, hypothalamic-derived fiaf is likely acting in a paracrine manner in order to maintain hypothalamic homeostasis.

Pituitary Adipokines

Adenohypophyseal hormone secretion is regulated by integrating various inputs from hypothalamic releasing factors, negative feedback signals from target tissues, as well as autocrine/paracrine loops. Adipokines may also 200

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be involved in the modulation and maintenance of pituitary function, especially with regards to autocrine/paracrine-signaling events. Leptin Gene Expression in Pituitary Gland We were amongst the first to show that leptin is expressed in the rat pituitary [23], a finding confirmed in numerous other species including the mouse [128], human [21, 129], pig [130], cow [131] and frog [132]. Western blot analysis also confirmed the existence of leptin peptide in the rat pituitary [23, 133]. Pituitary leptin content is very high at birth but is rapidly reduced to adult levels between PD1 and PD15 [134], and is coincident with a peak in leptin mRNA expression between PD7-PD14 in the adenohypophysis [74]. The localization of pituitary leptin by immunohistochemistry provided insight into its potential function within the gland. We demonstrated that leptin-ir was present in most rat anterior pituitary cells, but not in the posterior lobe [23]. The staining, abolished by preadsorption of the antibody, appeared to be nuclear. In contrast, using different leptin antisera, Jin et al. [128] observed cytoplasmic leptin-ir in a restricted number of rat anterior pituitary cells, but staining was much more widespread in human pituitaries [21]. High levels of staining were also reported by McDuffie et al. [135] in cultured rat pituitary cells, and these results were confirmed by in situ hybridization. This latter study firmly established that anterior pituitary cells express ob mRNA. Discrepancies in leptin immunostaining patterns may reflect different antibody specificities, and/or the possibility that bound leptin from the circulation is also being detected. Immunohistochemical analysis of pituitary cells in culture would resolve this problem (see below: human pituitary cells in vitro). Colocalization studies revealed leptin was expressed in TSH- or LH/FSH-expressing cells in adult rat pituitaries [128, 136]. However, a conflicting study suggested that leptin mRNA, and protein, colocalized with GH as assessed by in situ hybridization and immunohistochemistry [135]. These differences might reflect the use of different strains of rats of slightly different ages (i.e. adult male and female Wistar Imamici rats [133] vs. female Wistar-Furth rats [128] vs. female Sprague-Dawley rats [135]). The age-related decreases in pituitary leptin levels [134] may account for some of the reported differences in immunolocalization. An additional perspective on information transfer within the pituitary gland comes from studies on a sixth type of pituitary cell, the folliculostellate cell [137]. This Wilkinson /Brown /Imran /Ur

cell type forms an extended paracrine network that communicates throughout the gland via gap junctions and movement of calcium ions. Lloyd et al. [138, 139] used laser capture microdissection to isolate rat folliculostellate cells and clearly demonstrated the expression of leptin mRNA and protein therein [21]. This group suggested that since most, if not all, pituitary cells express leptin receptors [140], the folliculostellate cells might communicate widely throughout the gland with all cell types. Note however that other work from the same group indicated that leptin-ir was undetectable in folliculostellate cells using electron microscopy [141]. These authors speculated on reasons for this result, and concluded that fixation of tissue for electron microscopy might alter antigenicity. Leptin expression within the human pituitary is also well described. Leptin-ir was readily detectable in about 25% of healthy human pituitary cells, but staining was weaker in pituitary adenomas [21]. However, leptin mRNA was barely detectable in normal human pituitaries, but could be amplified in some adenomas by RT-PCR [129]. It is noteworthy that leptin mRNA appears to be particularly labile in the pituitary [129], and it may be inappropriate to compare mRNA samples obtained from postmortem controls that have been exposed to hypoxic conditions for several hours. This may explain the discrepancy between low leptin mRNA and protein levels in the pituitary. Leptin-ir appears more widespread in the human pituitary, and colocalizes predominantly with cells expressing ACTH (70%), but is also detectable in TSH, LH and FSH (⬃30%), and to a lesser extent with GH (⬃20%) expressing cells [21, 129]. In addition PCR analysis of various human pituitary adenomas revealed a similar expression profile [129]. However, since the pituitary is composed of 40–50% somatotropic cells, compared to only 15–20% corticotropic cells, the somatotropes might actually be the largest source of pituitary-derived leptin in humans, despite the fact that a smaller proportion of these cells appear to biosynthesize the peptide. This pattern of distribution suggests that locally produced leptin may influence GH and ACTH secretion by directly targeting the appropriate cellular populations. As noted, leptin appears to colocalize with pituitary hormones in the same cells [136, 141]. Further, immunoelectron microscopy revealed that leptin was colocalized with GH, ACTH, TSH, and LH/FSH, but not PRL, in the same secretory granules [141]. However, less than 20% of somatotropes and thyrotropes were so labeled. Corticotropes had the highest level of leptin-positive granules (70–80%). There is evidence from adipocytes that leptin

is released from secretory vesicles via a calcium- and insulin-dependent pathway [142]. However, there is little if any evidence that leptin is secreted from normal pituitary tissue, and this may reflect the small number of leptinpositive vesicles (5–25% [141]) and the consequent low, perhaps undetectable, levels of leptin secretion. For example, we were unable to detect leptin secretion from rat pituitary explants even under depolarizing conditions (whole pituitary glands, ages PD14 and PD60; Imran et al. [unpubl. results]). In contrast, some secretion was detected from 16 of 47 adenoma samples, but there was no correlation with tumour type or with the pituitary hormone released [90]. It is possible that very small amounts of leptin are released, along with anterior pituitary hormones, not into the bloodstream but to act in an autocrine/paracrine fashion on closely apposed leptin receptors on pituitary cells. This hypothetical mode of action for locally-released leptin in the pituitary should be detectable via activation of leptin receptor-signaling pathways. These signals are well described in the hypothalamus and include the JAK2-STAT-3 and the PI3 kinase pathways [69, 143]. There is also good evidence that PI3 kinase is expressed in high abundance in the anterior pituitary [144] as is the STAT-3/SOCS-3 signaling system [145–147]. Leptin receptors are present in all anterior pituitary cell types [140] and we reported a homogeneous distribution of leptin receptor-ir throughout the prepubertal rat pituitary [134]. There is evidence that leptin can activate the pituitary JAK-STAT pathway. Using the immortalized HP75 human pituitary cell line, Tsumanuma et al. [145] reported that leptin stimulated the expression of socs-3 mRNA and phosphorylation of SOCS-3. Studies in isolated porcine somatotropes revealed that leptin increased intracellular calcium ion levels via JAK-STAT and MAPK pathways [148]. In total, the evidence indicates that: (a) leptin is produced in pituitary cells; (b) leptin is localized in secretory vesicles, often with other pituitary hormones, and (c) the pituitary possesses leptin receptor-coupled signaling pathways that are activated by exogenous leptin. It remains to be determined whether pituitary-derived leptin is able to act via a paracrine/autocrine mechanism. If this is the case, what might be the functional physiological role of such a system? As noted already (see Do Adipokines Cross the Blood-Brain Barrier?), the pituitary is chronically exposed to blood-borne, adipose-derived leptin. It is conceivable that the endogenous pituitary leptin-leptin receptor system needs to be primed under circumstances where circulating leptin levels are low. Such a circumstance could occur during fetal development.

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Pituitary Leptin and Pituitary Development Although the exact role of pituitary-derived leptin remains elusive, current evidence suggests it might be involved in the growth and differentiation of certain pituitary cell populations. We established that leptin gene expression is age-dependent in the rat pituitary, with a peak level of expression occurring around PD7 [74] and declining to low levels in the adult [134]. Similarly, pituitary leptin content is very high at birth, but falls rapidly to adult levels by around PD30, just before puberty [134]. Leptin promoter analysis confirmed binding sites for Pit1, a critical transcription factor implicated in adenohypophyseal development and maturation [79]. Thus, increased levels of leptin in the developing pituitary could be the result of increased Pit-1 activity, pointing to a developmental role for pituitary-derived leptin. Treatment of GH3 cells, a somatomammotrope cell line, or HP75 cells, with leptin reduced cell growth and increased apoptosis [21, 145]. In contrast, leptin had no effect on the growth of L␤T2 or ␣T3-1 gonadotropic cell lines [21] or folliculostellate cells [128], suggesting that leptin stimulates apoptosis in a cell-type-specific manner. Moreover, leptin and OBRb gene expression was detected in somatotropes [133], as well as in rat GH3 cells [128], raising the possibility that endogenous pituitary leptin modulates cell survival in somatotropic cells. This could be clarified by specifically silencing leptin gene expression in GH3 cells using RNAi, as we have done in rat C6 glioblastoma cells [83], and then monitoring cell survival. Likewise, leptin-ir appears to be reduced in human pituitary adenomas relative to normal pituitaries [21]. Therefore, the low levels of endogenous leptin in adenoma cells may contribute to their uncontrollable growth. Immortalized cells may not be the ideal model system on which to base conclusions, but it remains plausible that the neonatal surge in pituitary leptin could inhibit the proliferation, and promote the differentiation, of certain cellular populations in the adenohypophysis. Pituitary Leptin and Hormone Secretion The effects of circulating leptin on pituitary hormone secretion are outlined in two excellent reviews [149, 150]. Is it possible, or even likely, that pituitary-derived leptin might also exert some control over the release of pituitary hormones? At present there is no direct evidence for this, but the use of RNAi to silence leptin gene expression in cultured pituitary cells is clearly a practical method for testing this hypothesis. We are particularly interested in the putative role of pituitary leptin in the reproductive system. For example, we demonstrated that pituitary 202

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leptin mRNA was markedly sensitive to treatment with testosterone. Leptin mRNA levels in female rat pituitary was double the amount seen in males [75]. Moreover, treatment of neonatal female rats with testosterone severely downregulated ob mRNA in PD14 and PD22 pituitary. Interestingly, we saw no effect of testosterone in hypothalamus or in adipose tissue. Studies in the pig indicate that estrogen does not affect pituitary ob mRNA during the estrous cycle, though levels were significantly reduced during pregnancy [130]. Further investigation revealed that this reduction occurred at a time when hypothalamic ob mRNA was increased [151]. Since pituitary and hypothalamic leptin receptor expression is also reduced by estrogen during pregnancy, leptin signaling within the pituitary may be regulated by different reproductive states [151–154]. Further studies are needed to establish whether the intrapituitary leptin-leptin receptor system exerts any control over the reproductive system. As noted already (see A Dual System?), the pituitary is bathed in blood-borne circulating leptin and it is not intuitively obvious why a leptin target tissue such as the pituitary should also be capable of expressing the leptin gene. We suggest that pituitary-derived leptin might ‘tune’ leptin-signaling pathways to incoming adipose-derived leptin signals. A similar argument can be proposed for other pituitary adipokines (see below). Pituitary Adiponectin Adiponectin is regarded as an adipose-specific gene [11] in spite of evidence to the contrary. In addition to brain, the pituitary gland is also a site for adiponectin gene expression [55]. Moreover, the two isoforms of the adiponectin receptor AdipoR1/R2 were detected in numerous mouse tissues including liver, skeletal muscle, and brain [54] and in the chicken anterior pituitary [55, 155]. Fasting induced a significant decrease in both adiponectin and Adipo R1 expression in the pituitary, providing an intriguing link between adiponectin and metabolic status in the pituitary [55, 155]. We also detected AdipoR1/R2 expression in the mouse pituitary, and in the AtT20 corticotrope cell line. Adiponectin and its receptors are also expressed in the rat pituitary [156], a result we have confirmed using real-time RT-PCR. Treatment of isolated rat anterior pituitary cells with adiponectin inhibited GH release, but increased the expression of the GH-releasing hormone receptor and GH secretagogue receptor, the ghrelin receptor [156]. Likewise, adiponectin reduced the expression of the GnRH receptor and decreased LH hormone secretion from rat gonadotropes [156]. These authors also detected an adiponectin-induced increase in Wilkinson /Brown /Imran /Ur

adiponectin mRNA, and a reduction in Adipo R1 gene expression. In contrast to these data, Lu et al. [157] reported that adiponectin increased the secretion of LH from an immortalized pituitary gonadotrope cell line, LbT2. These cells express both adiponectin receptors and respond to adiponectin through an increase in phosphoAMPK. In total the data suggest that locally produced adiponectin may regulate pituitary hormone secretion. Resistin and Pituitary Resistin was initially cloned from adipose tissue in 2001, and was reported to induce insulin resistance in mice [68]. Although a description of its receptor remains elusive, there is increasing evidence suggesting that resistin acts at various tissue sites. We reported that resistin is expressed in the mouse pituitary, as well as in AtT20 corticotropes [101, 112], and more recently we confirmed its expression in the rat pituitary by real-time RT-PCR. In addition, we should note that resistin-ir colocalized with ␣-MSH, a corticotrope marker, in the mouse pituitary. In vitro and in vivo analysis suggested that pituitary resistin expression is directly regulated by glucocorticoids, i.e., adrenalectomy attenuated pituitary resistin expression, but was restored by exogenous glucocorticoid treatment [158]. Experiments are needed to establish whether resistin regulates the secretion of ACTH. We also demonstrated that pituitary resistin expression was age-, sex-, and leptin-dependent. Resistin mRNA levels were lowest at birth and increased to a maximum at weaning (females) and puberty (males) [100, 101]. Male values were 2- to 3-fold higher than in female pituitaries, but in contrast to the effects seen for leptin mRNA, neonatal androgenization did not modify resistin mRNA. This suggested that the sexual dimorphism in pituitary resistin expression may be androgen-dependent. However, short-term androgen depletion (castration; 10 days) did not modify resistin expression [unpubl. data], indicating that neonatal programming of males by testosterone could be responsible for the sex difference in rstn expression. The development of resistin gene expression is also leptin-dependent. Values in pubertal leptin-deficient ob/ob mice were 3- to 4-fold lower than in lean controls, though this differential was absent in adult pituitaries. Reduction of circulating leptin by food restriction (24 and 48 h) in pubertal CD-1 mice significantly inhibited pituitary rstn mRNA. Our data indicate that some of the factors known to regulate adipose tissue resistin expression (i.e. gender, age, fasting) also control resistin mRNA in the pituitary gland. The role of leptin in the regulation of rstn mRNA needs to be studied further. Adipokine Expression in Brain

There was a marked neonatal peak in pituitary resistin gene expression, raising the possibility that it is also involved in the development and maturation of the brainpituitary system [100]. However, there was no detectable spike in leptin-deficient (ob/ob) mice, and this peak could be abolished by neonatal MSG treatment in CD-1 mice [100]. Therefore, leptin appears to modulate pituitary resistin via central leptin signaling. Fasting also reduced hypophyseal resistin expression suggesting it is also sensitive to metabolic changes in the mouse [101] and rat [Brown et al., unpubl. observations]. However, unlike leptin, resistin expression was greater in the adenohypophyses of male mice. Recently, resistin was shown to inhibit cytokine signaling in adipocytes via the induction of SOCS-3, an inhibitor of leptin and insulin signaling. Perhaps resistin also modulates cytokine signaling in the pituitary, and could have a profound impact on corticotrope function, as well as the signaling events in other pituitary cells. Other Pituitary Adipokines Our data on the detection of leptin, adiponectin and resistin gene expression in the pituitary gland strongly suggest that expression of additional adipose genes will be found there. Our preliminary studies revealed that three of these, namely adiponutrin, FIAF, and peroxisome proliferator-activated receptor ␥ (PPAR␥), are expressed and regulated in mouse pituitary tissue [117]. Adiponutrin and ppar␥ mRNA, but not fiaf, are also abundantly expressed in the AtT20 corticotrope tumour cell line. Food restriction increases fiaf and decreases ppar␥ mRNA in both pituitary and adipose tissue. In contrast, adiponutrin expression is increased in pituitary, but decreased in fat by fasting. This effect of fasting on pituitary gene expression is intriguing. There is a remarkable convergence of adipokine gene expression with leptin and insulin-signaling pathways in the anterior pituitary [144, 146, 159]. For example, insulin receptors and insulin receptor substrates are present in pituitary cells. It is conceivable that pituitary adipokine expression serves as a link between peripheral metabolic signals and the regulation of pituitary hormone secretion. Conclusions We have outlined a growing body of evidence indicating that not only are adipokines expressed in the pituitary, but they may also have an effect on normal pituitary growth and secretion. For example, leptin was shown to induce apoptosis in certain types of pituitary cells, as well as inhibit GH secretion. Despite the emphasis on pituNeuroendocrinology 2007;86:191–209

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itary leptin, simply because it has received the most attention thus far, we should not overlook the emerging role(s) of resistin, adiponectin, and other adipokine genes, that are also expressed in the pituitary. Adipokines appear to link metabolic and developmental status with the normal functioning of the adenohypophysis. Moreover, the emerging data linking adipokines to pituitary secretions highlight the complicated network of integrative signals that are necessary to maintain its normal function. Although the pituitary is bathed in circulating adipokines, local expression may be necessary when plasma levels fall in order to maintain adequate signaling within the pituitary. Our data suggest that the convergence of insulinsignaling and adipokine pathways in pituitary cells may provide a putative, but neglected, link between peripheral metabolic signals and the regulation of anterior pituitary hormone secretion. For example, polycystic ovary syndrome – characterized by anovulation, obesity and insulin resistance – could be a clinical entity in which abnormal pituitary adipokine expression and insulin signaling cause dysregulation of the reproductive system.

Conclusions: Are There More Adipokines in Brain and Pituitary?

We have provided evidence that several adipose-specific genes are also expressed in the brain. In total we confirmed that leptin, resistin, FIAF, adiponectin and adiponutrin were all expressed centrally. Our studies in the N-1 neuronal cell line indicated that, for example, resistin of hypothalamic origin exerted paracrine/autocrine control over local fiaf and socs-3 expression. We speculated that such a pathway may contribute to the feedback effects of leptin in the control of energy homeostasis. A logical question is whether other adipokines might also be found in the brain. A brief literature search revealed several candidate genes. Thus, visfatin [13], apelin

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[160] and adipsin genes [161] are all expressed in the brain. Nerve growth factor (NGF), long accepted as a central neurotropin [162], is now regarded as an adipokine that is secreted from white adipose tissue [163]. Intuitively one might suppose that the converse could also be true, i.e. are hypothalamic peptides present in adipose tissue? Gene expression profiling revealed a plethora of unexpected transcripts, including several appetite-regulating peptides and receptors, e.g. NPY, CRF, NPY receptors, MCH and cholecystokinin [17, 164, 165]. Of these, CRF is the best studied [166, 167], particularly in human adipose tissue. Nevertheless, a note of caution over the interpretation of gene expression profiling was offered by Trayhurn and Wood [125], who failed to find some of these transcripts using RT-PCR. Similar doubts were raised with respect to adipokine expression in the brain, though we feel that the accumulated evidence, outlined in this review, is sufficient to merit continued investigation. An issue that is yet to be resolved concerns the appropriateness of the word ‘adipokine’ to label brain (or pituitary) derived peptides such as leptin. The definition of adipokine: ‘… a protein that is secreted from, and synthesized by, adipocytes’ [125] is clearly inappropriate for those adipokines found in non-adipose sites. We suggest that the adipokines discovered in the brain and pituitary gland, and in brain tumour cells, should more appropriately be termed cephalokines.

Acknowledgements The studies in our laboratory were made possible by financial support from the NSHRF, the IWK Health Centre, the Atlee Endowment, UIMRF and Capital Health. R.B. is the recipient of a NSHRF Graduate Studentship, and E.U. is a Dalhousie University Senior Clinical Scholar. We are indebted to Dr. D.D. Belsham (University of Toronto) for the generous gift of N-1 hypothalamic neurons, and to D. Wilkinson and P. Wilkinson for their invaluable support and technical assistance.

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Neuroendocrinology 2007;86:210–214 DOI: 10.1159/000108341

Received: November 11, 2006 Accepted after revision: December 4, 2006 Published online: September 11, 2007

Histamine and the Regulation of Body Weight Emilie A. Jørgensen a, b Ulrich Knigge a, c Jørgen Warberg a Andreas Kjær a, b a

Cluster for Molecular Imaging, University of Copenhagen, b Department of Clinical Physiology, Nuclear Medicine and PET, and c Department of Surgery C, Rigshospitalet, Copenhagen, Denmark

Key Words Appetite, neuroendocrine regulation ⴢ Histamine ⴢ Histamine, body weight regulation ⴢ Histaminergic system ⴢ Leptin ⴢ Receptor antagonists

Abstract Energy intake and expenditure is regulated by a complex interplay between peripheral and central factors. An exhaustive list of peptides and neurotransmitters taking part in this complex regulation of body weight exists. Among these is histamine, which acts as a central neurotransmitter. In the present article we review current evidence pointing at an important role of histamine in the regulation of appetite and metabolism. Studies using both knockout mouse models as well as pharmacological studies have revealed that histamine acts as an anorexigenic agent via stimulation of histamine H1 receptors. One effect of histamine in the regulation of appetite is to act as a mediator of the inhibitory effect of leptin on appetite. It seems that histamine may attenuate and delay the development of leptin resistance in high-fatdiet-induced obesity. Furthermore, histamine may also act to accelerate lipolysis. Based on the current evidence of the involvement of histamine in the regulation of body weight, the histaminergic system is an obvious target for the development of pharmacological agents to control obesity. At

© 2007 S. Karger AG, Basel 0028–3835/07/0863–0210$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/nen

present, H3 receptor antagonists that stimulate the histaminergic system may be the most promising histaminergic drugs for antiobesity therapy. Copyright © 2007 S. Karger AG, Basel

Introduction

Obesity is a major public health problem in the Western world as it increases the risk of developing life-threatening conditions such as type 2 diabetes, hypertension, cardiovascular diseases and some forms of cancer [1]. Elucidation of potential pharmacological targets to treat obesity is therefore of great interest. The increased body weight of obese people is basically the result of energy intake exceeding energy expenditure. Energy intake and expenditure are regulated by a complex interplay between peripheral and central factors. An exhaustive list of peptides and neurotransmitters taking part in this complex regulation of body weight exists. Among these is histamine, a neurotransmitter released by neurons originating exclusively in the tuberomammillary body in the posterior hypothalamus [2]. It was initially discovered that the histaminergic system plays a role in the regulation of body weight when some antipsychotics and antidepressants, proved to be potent histamine H1 receptor Andreas Kjær Cluster for Molecular Imaging, Building 12.3 The Panum Institute, University of Copenhagen Blegdamsvej 3 C, DK–2200 Copenhagen N (Denmark) Tel. +45 3532 7504, Fax +45 3532 7546, E-Mail [email protected]

antagonists, were found to have profound side effects in the form of appetite stimulation and weight gain [3]. Later, several studies supported the involvement of the histaminergic system in appetite and body weight regulation. The purpose of this minireview is to give a brief overview of these studies and thereby evaluate the potential of the histaminergic system as a target for antiobesity treatment.

The Histaminergic System

Histamine was discovered by Sir Henry Dale at the beginning of the 20th century. This amine, consisting of an imidazole ring and an amino group connected by a short chain of two carbon atoms, is stored in and released from various types of cells. These include the IgE receptor bearing mast cells and basophils, as well as endocrine cells and neurons in the central and peripheral nervous system [4]. Histamine is synthesized from the essential amino acid, L-histidine, which is actively transported into the cells. It is formed by decarboxylation of histidine, a reaction catalyzed by the enzyme histidine decarboxylase (HDC). The newly formed histamine is transported into secretory vesicles, from which it is released upon stimulation of the cell. In the central nervous system, histamine is found in mast cells as well as in neurons. Histamine derived from mast cells is possibly involved in vascular control and immune responses. On the other hand, histamine derived from neurons functions as a neurotransmitter [5, 6]. Histamine does not cross the blood-brain barrier, so brain histamine must be synthesized in situ [7]. As already stated, neuronal histamine originates exclusively in neurons of the tuberomammillary body in the posterior hypothalamus. From there, the histaminergic neurons project to, more or less, every part of the brain. The highest density of fibers is found in the hypothalamus [2]. Within the brain, three subclasses of histamine receptors exist. These are designated H1, H2 and H3 receptors. The first two receptor types are present on the postsynaptic membrane and mediate the actions of histamine. The third type is present on the presynaptic membrane of the histaminergic neuron and is a so-called autoinhibitory receptor [8]. The H1 receptor has a widespread distribution throughout the brain, with a high density in the hypothalamus [9]. The distribution of H2 receptors resembles that of the H1 receptors, although a much lower density is found in the hypothalamus, compared to H1 Histamine and Body Weight

receptors [10]. As the H3 receptor is an autoinhibitory receptor localized to the presynaptic membrane, binding of histamine to these receptors results in inhibition of synthesis and release of histamine. This feedback mechanism resembles that of many other neurotransmitters, e.g. noradrenaline, serotonin and dopamine [4]. A special feature of the H3 receptor is that it is a heteroreceptor. This means that besides its localization on the histaminergic neurons, it is also found on serotonin-, noradrenaline-, dopamine-, GABA-, and acetylcholine-containing neurons, indicating that it modulates the release of these neurotransmitters as well [11].

The Histaminergic System and Neuroendocrine Regulation of Appetite

Histamine and Appetite In 1973, Clineschmidt and Lotti [12] were the first to show that administration of histamine reduces food intake. They administered histamine into the lateral ventricle of cats and observed a long-term suppression of food intake. Itowi et al. [13] administered histamine continuously into the suprachiasmatic nucleus of the hypothalamus in rats. Likewise, they found that histamine reduced food intake. Also, an acute injection of histamine into the lateral ventricle of rats has been shown to reduce food intake [14]. In addition, intraperitoneal injection of L-histidine, the histamine precursor, has been shown to have the same effect as histamine, reducing food intake [15–17]. This is possibly the result of an increase in brain histamine, after L-histidine has been transported to the brain and converted to histamine by the HDC enzyme. Metoprine is an inhibitor of N-methyltransferase, the enzyme that breaks down histamine to N-methylhistamine. This compound therefore has the ability to increase the endogenous histamine concentration. Administration of metoprine, both by intraperitoneal injection and by intracerebroventricular infusion, suppresses food intake in rats [18, 19]. ␣-Fluoromethylhistidine (␣-FMH) is a specific and irreversible inhibitor of the HDC enzyme, and has the ability to deplete histamine from the brain neurons. Intracerebroventricular administration of ␣-FMH significantly increases food intake in rats [20–24]. Together, these findings provide consistent evidence for histamine being an anorexigenic agent. Histamine Receptors and Appetite To study the involvement of histamine receptors in appetite regulation, several pharmacological approaches Neuroendocrinology 2007;86:210–214

211

have been used. Injection of an H1 receptor agonist into the lateral ventricle decreased food intake in rats [14]. In contrast, intracerebroventricular administration of an H1 receptor antagonist increased food intake in rats [25– 29]. Furthermore, administration of an H1 receptor antagonist attenuated the histamine-induced suppression of food intake [13, 19]. In contrast, the H2 receptor is not believed to be involved in the regulation of appetite, since neither H2 receptor agonists nor H2 receptor antagonists have any effect on food intake [13, 14, 29]. Also, the administration of an H2 receptor antagonist did not abolish the histamine-induced suppression of food intake [14]. The involvement of the H3 receptors in the regulation of appetite is more complex. The presumed mechanism would be that stimulation of the receptor leads to inhibition of histamine release and thereby less stimulation of postsynaptic H1 receptors, resulting in an increase in appetite. Conversely, blocking the receptor would lead to enhanced release of histamine from the histaminergic neurons which would stimulate the H1 receptors and thereby lead to a decrease in appetite. Activation of H3 receptors by the agonist R-␣-methylhistamine has previously failed to induce an effect on food intake in rats [14, 30]. However, we recently found that R-␣-methylhistamine did indeed increase food intake when administered intraperitonally to mice [31]. Experiments with the H3 receptor antagonist, thioperamide, have also shown mixed results. In some studies it was reported to decrease food intake [14, 23], while in other studies thioperamide was reported to have no significant effect on food intake in neither sated nor fasted rats [32, 33]. These somewhat inconsistent findings might be due to the fact that the H3 receptor, as mentioned above, is a heteroreceptor, and therefore affects the release of substances other than histamine.

develop earlier as indicated by downregulation of leptin receptor gene expression [38]. In addition to the knockout approach, the involvement of the histaminergic system in mediation of the anorexigenic effect of leptin has also been demonstrated pharmacologically. Both blockade of histamine synthesis and blockade of the H1 receptors attenuated the response to leptin [35, 39, 40]. Also, leptin facilitated histamine release from the hypothalamus [41]. In further support of histamine as a mediator of leptin actions, it has been shown that genetically obese animals with defects in the leptin system (ob/ob and db/db mice, fa/fa rats) display lowered levels of hypothalamic histamine [26, 40, 42]. Together, these observations provide clear evidence of histamine as a mediator of leptin actions.

Histamine and Lipolysis

The effect of histamine on regulation of body weight may not be exerted through effects on appetite alone, but direct effects on metabolism are also apparent. Most important are the observations of Bugajski and Janusz [43] who were the first to demonstrate an involvement of histamine in lipolysis. They reported an increase in serum free fatty acid levels as a response to central administration of histamine. Later, neuronal histamine was shown to accelerate lipolysis in white adipose tissue by centrally activating the sympathetic nervous system [44, 45]. In this way, histamine appears to participate in maintaining energy homeostasis by affecting peripheral energy expenditure. Consistent with this, both histamine, histamine H1 receptor and histamine H3 receptor knockout mice have been shown to develop obesity with increasing age [37, 46, 47].

Histamine as a Target for Antiobesity Therapy? Histamine and Leptin

Leptin, which is secreted primarily by adipose tissue, reduces food intake and increases energy expenditure through actions in the hypothalamus [34]. Morimoto et al. [35] were the first to show an involvement of the histaminergic system in the mediation of the anorexigenic effect of leptin. They observed a complete absence of an anorexigenic effect of leptin in H1 receptor knockout mice. This observation has subsequently been confirmed [36, 37]. Recently, we showed that when histamine knockout mice are fed a high-fat diet, leptin resistance seems to 212

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Based on the current evidence of the involvement of histamine in the regulation of body weight, pharmacological manipulation of the histaminergic system seems to be an obvious target for antiobesity drugs. In theory, both central H1 and H3 receptors could be potential targets for the treatment of obesity. However, as the drugs must be administered peripherally, an H1 receptor agonist would also affect the peripheral mast cells and thereby generate an unwanted allergic response as a result of the mast cell-derived histamine release. This is likely to limit the use of H1 receptor agonists and the H1 receptor Jørgensen /Knigge /Warberg /Kjær

as a drug target. In contrast, the H3 receptors may be a promising drug target. Indeed, the use of H3 receptor antagonists to stimulate the histaminergic system may provide a pharmacological means of antiobesity therapy. Before such drugs can be developed, however, several issues

need to be resolved, such as activity of orally-delivered agents, stability and ability to cross the blood-brain barrier. Further work is required before manipulation of the histaminergic system might be considered as a pharmacological target for the control of obesity.

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Histamine and Body Weight

14 Lecklin A, Etu-Seppala P, Stark H, Tuomisto L: Effects of intracerebroventricularly infused histamine and selective H1, H2 and H3 agonists on food and water intake and urine flow in Wistar rats. Brain Res 1998;793:279– 288. 15 Orthen-Gambill N: Antihistaminic drugs increase feeding, while histidine suppresses feeding in rats. Pharmacol Biochem Behav 1988;31:81–86. 16 Sheiner JB, Morris P, Anderson GH: Food intake suppression by histidine. Pharmacol Biochem Behav 1985;23:721–726. 17 Vaziri P, Dang K, Anderson GH: Evidence for histamine involvement in the effect of histidine loads on food and water intake in rats. J Nutr 1997;127:1519–1526. 18 Lecklin A, Tuomisto L, MacDonald E: Metoprine, an inhibitor of histamine N-methyltransferase but not catechol-O -methyltransferase, suppresses feeding in sated and in food deprived rats. Methods Find Exp Clin Pharmacol 1995;17:47–52. 19 Lecklin A, Tuomisto L: The blockade of H1 receptors attenuates the suppression of feeding and diuresis induced by inhibition of histamine catabolism. Pharmacol Biochem Behav 1998;59:753–758. 20 Doi T, Sakata T, Yoshimatsu H, Machidori H, Kurokawa M, Jayasekara LA, et al: Hypothalamic neuronal histamine regulates feeding circadian rhythm in rats. Brain Res 1994; 641:311–318. 21 Ookuma K, Sakata T, Fukagawa K, Yoshimatsu H, Kurokawa M, Machidori H, et al: Neuronal histamine in the hypothalamus suppresses food intake in rats. Brain Res 1993;628:235–242. 22 Orthen-Gambill N, Salomon M: FMH-induced decrease in central histamine levels produces increased feeding and body weight in rats. Physiol Behav 1992;51:891–893. 23 Sakata T, Fukagawa K, Ookuma K, Fujimoto K, Yoshimatsu H, Yamatodani A, et al: Hypothalamic neuronal histamine modulates ad libitum feeding by rats. Brain Res 1990; 537:303–306. 24 Tuomisto L, Yamatodani A, Jolkkonen J, Sainio EL, Airaksinen MM: Inhibition of brain histamine synthesis increases food intake and attenuates vasopressin response to salt loading in rats. Methods Find Exp Clin Pharmacol 1994;16:355–359.

25 Fukagawa K, Sakata T, Shiraishi T, Yoshimatsu H, Fujimoto K, Ookuma K, et al: Neuronal histamine modulates feeding behavior through H1-receptor in rat hypothalamus. Am J Physiol 1989;256:R605–R611. 26 Machidori H, Sakata T, Yoshimatsu H, Ookuma K, Fujimoto K, Kurokawa M, et al: Zucker obese rats: defect in brain histamine control of feeding. Brain Res 1992; 590: 180– 186. 27 Mercer LP, Kelley DS, Humphries LL, Dunn JD: Manipulation of central nervous system histamine or histaminergic receptors (H1) affects food intake in rats. J Nutr 1994; 124: 1029–1036. 28 Ookuma K, Yoshimatsu H, Sakata T, Fujimoto K, Fukagawa F: Hypothalamic sites of neuronal histamine action on food intake by rats. Brain Res 1989;490:268–275. 29 Sakata T, Ookuma K, Fukagawa K, Fujimoto K, Yoshimatsu H, Shiraishi T, et al: Blockade of the histamine H1-receptor in the rat ventromedial hypothalamus and feeding elicitation. Brain Res 1988;441:403–407. 30 Merali Z, Banks K: Does the histaminergic system mediate bombesin/GRP-induced suppression of food intake? Am J Physiol 1994;267:R1589–R1595. 31 Jørgensen EA, Knigge U, Watanabe T, Warberg J, Kjær A: Histaminergic neurons are involved in the orexigenic effect of orexin-A. Neuroendocrinology 2006; 82: 70–77. 32 Itoh E, Fujimiya M, Inui A: Thioperamide, a histamine H3 receptor antagonist, suppresses NPY-but not dynorphin A-induced feeding in rats. Regul Pept 1998; 75–76:373–376. 33 Itoh E, Fujimiya M, Inui A: Thioperamide, a histamine H3 receptor antagonist, powerfully suppresses peptide YY-induced food intake in rats. Biol Psychiatry 1999; 45: 475– 481. 34 Schwartz MW, Woods SC, Porte D Jr, Seeley RJ, Baskin DG: Central nervous system control of food intake. Nature 2000; 404: 661– 671. 35 Morimoto T, Yamamoto Y, Mobarakeh JI, Yanai K, Watanabe T, Watanabe T, et al: Involvement of the histaminergic system in leptin-induced suppression of food intake. Physiol Behav 1999;67:679–683.

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36 Masaki T, Yoshimatsu H, Chiba S, Watanabe T, Sakata T: Targeted disruption of histamine H1-receptor attenuates regulatory effects of leptin on feeding, adiposity, and UCP family in mice. Diabetes 2001;50:385–391. 37 Masaki T, Chiba S, Yasuda T, Noguchi H, Kakuma T, Watanabe T, et al: Involvement of hypothalamic histamine H1 receptor in the regulation of feeding rhythm and obesity. Diabetes 2004;53:2250–2260. 38 Jørgensen EA, Vogelsang TW, Knigge U, Watanabe T, Warberg J, Kjær A: Increased susceptibility to diet-induced obesity in histamine-deficient mice. Neuroendocrinology 2006;83:289–294. 39 Toftegaard CL, Knigge U, Kjær A, Warberg J: The role of hypothalamic histamine in leptin-induced suppression of short-term food intake in fasted rats. Regul Pept 2003; 111:83–90.

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Neuroendocrinology 2007;86:215–228 DOI: 10.1159/000109094

Received: November 13, 2006 Accepted after revision: November 27, 2006 Published online: September 26, 2007

Central and Peripheral Roles of Ghrelin on Glucose Homeostasis Yuxiang Sun a, b Mark Asnicar a, b Roy G. Smith a–c a

Huffington Center on Aging, b Department of Molecular and Cellular Biology, and c Department of Medicine, Baylor College of Medicine, Houston, Tex., USA

Key Words Ghrelin ⴢ GHS-R ⴢ Pancreas ⴢ Diabetes ⴢ Glucose homeostasis ⴢ Insulin resistance ⴢ Leptin

Abstract Ghrelin, an acylated 28-amino-acid peptide, is an endogenous ligand of the growth hormone secretagogue type 1a (GHS-R1a). Ghrelin is best known for its hypothalamic actions on growth hormone-releasing hormone neurons and neuropeptide Y/agouti-related peptide neurons; however, ghrelin affects multiple organ systems and the complexity of its functions is only now being realized. Although ghrelin is mainly produced in the stomach, it is also produced in low levels by the hypothalamus and by most peripheral tissues. GHS-R1a is expressed predominantly in the anterior pituitary gland, at lower levels in the brain including hypothalamic neurons that regulate feeding behavior and glucose sensing, and at even lower levels in the pancreas. A reciprocal relationship exists between ghrelin and insulin, suggesting that ghrelin regulates glucose homeostasis. Ablation of ghrelin in mice increases glucose-induced insulin secretion, and improves peripheral insulin sensitivity. This review focuses on the newly emerging role of ghrelin in glucose homeostasis and exploration of whether ghrelin is a potential therapeutic target for diabetes. Copyright © 2007 S. Karger AG, Basel

© 2007 S. Karger AG, Basel 0028–3835/07/0863–0215$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/nen

Introduction

GHS-R1a was initially cloned and identified as an orphan G-protein-coupled receptor (GPCR) for a family of synthetic ligands that restore the age-related decline in pulse amplitude of growth hormone (GH) release by activating hypothalamic neurons [1–3]. Subsequently, ghrelin was identified as an endogenous ligand of GHS-R1a [4]. Since its discovery in 1999, nearly 1,800 papers have been published (almost one paper per day) describing ghrelin’s actions. Plasma levels of ghrelin are influenced by nutritional status implicating a role in regulating energy balance. Pharmacologically, ghrelin increases GH release and appetite in humans and rodents, and induces fat deposition in rodents [4–9]. GHS-R1a expression is predominant in the anterior pituitary gland and in specific regions of the brain. Ghrelin mimetics stimulate GH-releasing hormone (GHRH) release by modifying the membrane potential of GHRH neurons and antagonizing somatostatin release from the hypothalamus [10]. Besides central functions, ghrelin has a wide spectrum of peripheral activities which impact endocrine, metabolic, immune, bone, and cardiovascular systems [11, 12]. Ghsr–/– mice are refractory to the orexigenic and GH-releasing properties of ghrelin [13], indicating that the GHS-R1a is a biologically relevant ghrelin receptor that mediates ghrelin action in the central nervous system (CNS). However, adult ghrelin–/– mice are neither reYuxiang Sun Huffington Center on Aging, Baylor College of Medicine One Baylor Plaza, M320, Houston, TX 77030 (USA) Tel. +1 713 798 3837, Fax +1 713 798 1610 E-Mail [email protected]

sistant to diet-induced obesity, nor do they exhibit a dwarf phenotype [14]. The full physiological effects of ghrelin on energy homeostasis remain to be elucidated. Three post-translationally processed peptides are encoded by the ghrelin gene: ghrelin (28-amino-acid ghrelin peptide, octanoylated at serine 3), desacyl ghrelin, and obestatin [4, 15]. Acylation is indispensable for the binding of ghrelin to the GHS-R1a. Since desacyl ghrelin does not bind to GHS-R1a, it was initially thought to be biologically inactive. However, several recent studies have suggested that desacyl ghrelin might also be a functional hormone, with functions mediated through a receptor other than GHS-R1a [16–19]. Recently, obestatin was identified as a product of prepro-ghrelin. Intriguingly, obestatin inhibits food intake and appears to be a natural antagonist of ghrelin, but its anorexic properties are mediated through a distinct G-protein-coupled receptor; GPR39 [15].

Role of Ghrelin on Central Glucose Sensing

Maintaining glucose homeostasis requires glucose sensing by the CNS and by peripheral tissues. The glucose sensors regulate activity of the autonomic nervous system, hormone secretion, glucose production, glucose uptake and utilization. Glucosensing neurons, located in the hypothalamus and the brainstem, exhibit specific excitatory or inhibitory electrical responses to changes in extracellular levels of glucose and are involved in the monitoring of glucose status and the regulation of feeding [20]. In the hypothalamic arcuate nucleus (ARC), excitatory actions of glucose on anorexigenic proopiomelanocortin neurons have been reported, while the appetitepromoting neuropeptide Y (NPY) neurons may be directly inhibited by glucose [21]. It has been proposed that glucose sensing in neurons requires the expression of proteins such as glucose transporter 2 (GLUT2), glucokinase and the ATP-dependent potassium channel [22]. Inactivation of GLUT2 in mice leads to a loss of glucose sensing, and impaired insulin secretion [23]. Recently, it has been reported that the central glucose sensors require GLUT2 expression in glial cells [24]. Ghrelin and GHS-R1a expression increase upon fasting [25], suggesting that ghrelin signaling is involved in sensing of low blood glucose. NPY and agouti-related protein (AGRP) neurons in the ARC are primary targets of ghrelin. Glucose-responding neurons in the lateral hypothalamic area (LHA), the ventromedial hypothalamic nucleus (VMH), and the parvocellular area of the para216

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ventricular nucleus (PVN) are also involved in the orexigenic actions of ghrelin in hypothalamic circuits [26]. We showed that ghrelin mRNA is produced in the brain [14]. In collaboration with Cowley and Horvath, we demonstrated that ghrelin production is localized to a previously uncharacterized group of neurons adjacent to the third ventricle between the dorsal, ventral, paraventricular, and arcuate hypothalamic nuclei [27]. The ghrelinexpressing neurons control key hypothalamic circuits that include those producing NPY, AGRP, proopiomelanocortin, and corticotrophin-releasing hormone [27]. Furthermore, using biotinylated ghrelin, it was demonstrated that ghrelin-binding sites are present in the ARC, lateral anterior hypothalamus, and PVN. Electrophysiological recordings showed that ghrelin stimulates activity of arcuate NPY neurons and mimics the effect of NPY in the PVN of the hypothalamus [27]. The involvement of NPY/AGRP neurons in ghrelin signaling was confirmed by showing that like Ghsr–/– mice, agrp/npy-double knockout mice are refractory to the orexigenic effects of ghrelin [13, 28]. Early studies by Bailey et al. [29] using c-Fos expression as a marker of neuronal activation showed that peripheral administration of a ghrelin mimetic, in addition to activating arcuate neurons, increases Fos protein in the nucleus tractus solitarii (NTS). The NTS provides a direct noradrenergic projection to the hypothalamus which is believed to be important for neural regulation of GH secretion. However, the brainstem cells activated by the GHS-R1a agonists do not include noradrenergic cells, because the induced Fos protein expression did not co-localize with tyrosine hydroxylase [29]. Furthermore, extensive depletion of central noradrenaline by administration of the specific neurotoxin, 5-ADMP, did not modify either the amount or distribution of c-Fos induction in the ARC [29]. The activation of brainstem neurons is not secondary to GH secretion induced by the ghrelin mimetics, because GH release induced by GHRH did not increase c-Fos expression in the brainstem [29]; moreover, Sandostatin, a somatostatin analogue, blocks GH secretion and failed to inhibit brainstem activation in response to the ghrelin mimetics [29]. It was also concluded that activation of brainstem neurons is not secondary to activation of hypothalamic arcuate neurons, since activation of ARC neurons is suppressed by Sandostatin and somatostatin [30, 31]. Hypoglycemia induced by insulin administration also increases c-Fos expression in the NTS, which is accompanied by increased food intake. These responses are inhibited by treatment with anti-ghrelin antibodies, sugSun /Asnicar /Smith

gesting that the NTS facilitates the action of ghrelin on orexigenic signaling as a response to hypoglycemia [32]. In addition to ghrelin causing the release of the orexigenic peptides NPY and AGRP, ghrelin appears to play an important role as a sensor of hypoglycemia. This evidence suggests that ghrelin is part of the regulatory circuit which controls energy homeostasis. In summary, while ghrelin’s effect on appetite and food intake is clearly mediated through the hypothalamic neurons, further study is needed to reveal the role that ghrelin plays in central glucose sensing.

Does Ghrelin Have a Role in Diabetes?

A reciprocal relationship exists between ghrelin and insulin, and ghrelin levels are negatively correlated with the prevalence of type 2 diabetes [33, 34]. It is anticipated that ghrelin may play a role in modifying pancreatic ␤cell function. The pancreatic islets consist of at least four different endocrine cell types: insulin-secreting ␤ cells (65–80% of the total islet cell population), glucagon-secreting ␣ cells (10–15%), somatostatin-producing ␦ cells (5%), and the pancreatic polypeptide (PP)-containing cells (10–15%). In situ hybridization and immunohistochemistry show that ghrelin is localized to pancreatic ␣ cells and ␤ cells [35, 36]. However, it has also been reported that ghrelin is expressed in a novel, developmentallyregulated endocrine islet cell type that shares lineage with glucagon-secreting cells [37]. Targeted disruption of the homeodomain transcription factor Nkx2.2 gene in mice causes a complete lack of insulin-producing ␤ cells, and reduced numbers of ␣ and PP cells. Intriguingly, islets from Nkx2.2–/– mice contain a large population of endocrine cells that do not produce any of the four major islet hormones, but produce ghrelin instead [38]. GHSR1a mRNA was detected in the pancreas using RNA protection assay and RT-PCR analysis [36, 39, 40]. Recently, GHS-R-like immunoreactivity in rat pancreatic islets was co-localized with glucagon-like immunoreactivity and to some extent with insulin-like activity in ␤ cells [41]. In summary, the identity of ghrelin-expressing cell types in pancreatic islets is controversial; nevertheless, ghrelin and its receptor are indeed expressed in cells of the pancreatic islets. Ghrelin may affect pancreatic ␤ cells, by both endocrine and non-endocrine means, to regulate insulin secretion. Ghrelin derived from the stomach, and ghrelin produced locally in pancreatic islets, may affect ␤ cells by endocrine, paracrine and/or autocrine mechanisms.

There is a possible link between ghrelin production and type 1 and type 2 diabetes. In normal subjects, ghrelin secretion is stimulated by fasting and reduced by feeding [42]. In type 1 diabetic children, however, ghrelin response to meal tests is absent [43]. In type 1 diabetes, ghrelin levels are significantly decreased at time of diagnosis, and the negative correlation between ghrelin and glucose is only observed after insulin therapy [44]. Measuring circulating ghrelin levels may serve as a diagnostic marker for onset, and/or an indicator of the effectiveness of insulin treatment in type 1 diabetes. Low ghrelin levels are associated with obesity, insulin resistance, and type 2 diabetes [33, 34, 45]; however, it is unclear whether lowlevel ghrelin is acting as a risk factor or a compensatory response. Since type 2 diabetes is commonly associated with obesity, it is difficult to distinguish whether low ghrelin is correlated with diabetes alone or in conjunction with obesity. It was reported that in type 2 diabetic patients, plasma levels of ghrelin were significantly lower in the obese patients than in the non-obese patients [45]. Insulin is essential for meal-induced plasma ghrelin suppression. In normal subjects, ghrelin levels increase during fasting, and fall upon feeding [42]. Lack of mealinduced ghrelin suppression has been suggested to contribute to the hyperphagia observed in patients with severe insulin-deficient type 1 diabetes [46, 47]. Abnormal ghrelin secretion may affect both energy balance and metabolic balance of diabetic patients. Streptozotocininduced diabetes mellitus (STZ-DM) in rodents is characterized by hyperglycemia, weight loss, and markedly increased food intake [48]. Ghrelin normally decreases on nutrient ingestion, but is paradoxically elevated in STZ-induced hyperphagia in rats [49], which led to the hypothesis that ghrelin signaling contributes to the pathogenesis of diabetic hyperphagia. A recent study showed that STZ-treated ghrelin–/– mice exhibit a delayed and a reduced maximal increase in food intake when compared to controls [50]. The ghrelin–/– mice initially displayed a greater suppression of food intake, but then gradually increased their intake to become hyperphagic, similar to the STZ-DM wild-type mice. This suggests that while ghrelin signaling is required for onset of diabetic hyperphagia, it does not have a long-term effect; this might be due to a highly adaptive and protective nature of pathways that regulate energy homeostasis. Indeed, this is supported by the observation that diet-induced obesity occurs in ghrelin–/– mice only when the high-fat

Ghrelin’s Role in Glucose Homeostasis

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Expression of Ghrelin in Pancreatic Islets

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diet is initiated immediately after weaning, and it does not occur when the high-fat diet is initiated at adult age [14, 51, 52]. Pharmacological studies designed to investigate of the effects of ghrelin on glucose metabolism and insulin secretion are inconclusive, revealing both stimulatory and inhibitory effects on insulin secretion [35, 53, 54]. In two studies, ghrelin increased insulin release under glucose conditions (at 8.3 mmol/l) in isolated rat ␤ cells and in intravenously injected rats [35, 54]. However, the majority of studies showed that ghrelin inhibits insulin secretion in the pancreas of rats, mice and humans [55–58]. In summary, ghrelin seems to have an impact on insulin secretion, but major questions still remain. Does ghrelin function as a stimulator or an inhibitor of insulin release? Is this function a result of a direct or an indirect action on the pancreatic ␤ cell? What are the molecular mechanisms of this ghrelin effect on insulin release and does low level ghrelin act as a risk factor or a compensatory response in diabetes?

Interrelationship between Ghrelin, Insulin and Leptin

In plasma, reciprocal relationships exist between ghrelin and insulin, and between ghrelin and leptin. In humans, ghrelin inhibits insulin secretion, and ghrelin secretion is inhibited by insulin [53, 57]. In contrast to ghrelin, leptin is an anorexigenic hormone. In rats, fasting augments the pulsatile secretion of ghrelin, and diminishes leptin secretion [59]. It was predicted that in the absence of ghrelin, leptin and insulin levels would not be affected by feeding and fasting. However, identical changes in plasma insulin and leptin levels induced by feeding and fasting of wild-type mice were also observed in ghrelin–/– and Ghsr–/– mice [13, 14]. Hence, the secretion of leptin and insulin in response to changes in energy balance is not dependent upon either ghrelin or its receptor. The opposing effects of ghrelin and leptin on appetite are not explained by ghrelin regulation of leptin secretion, but may involve mutual antagonism at the functional level. Ghrelin and leptin regulate energy balance primarily through hypothalamic neurons in the CNS. Ghrelin stimulates the activity of arcuate NPY neurons, and mimics the effect of NPY in the PVN of the hypothalamus. When leptin is overexpressed in the PVN of obesityprone rats by injection of adeno-associated viral vectorencoding leptin, diet-induced obesity and hyperinsu218

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linemia are then blocked, and circulating ghrelin is increased [60]. Ghrelin and leptin are functional antagonists on hypothalamic NPY/AGRP neurons. In pancreatic ␤ cells, insulin release is inhibited by leptin [61], while the effect of ghrelin on insulin release remains controversial. In hepatoma cells, ghrelin modulates the downstream insulin-signaling molecules [62], and in hepatocytes, leptin increases insulin signal transduction resulting in increased insulin and decreased glucose production [63]. However, there is general agreement that ghrelin receptor, GHSR-1a, is not present in the liver; therefore, any direct effect of ghrelin on the liver must be mediated through a receptor other than GHS-R1a. In summary, ghrelin and leptin function as mutual antagonists on hypothalamic neurons that regulate feeding behavior. Leptin regulates glucose homeostasis through the CNS, adipose tissue, pancreas, liver and muscle [64]; ghrelin may antagonize leptin in specific cells present in each of these target organs.

Ghrelin Deletion in Lean Mice Improves Pancreatic ␤-Cell Function and Peripheral Insulin Sensitivity

Glucose homeostasis is controlled by two key processes: insulin secretion in pancreatic ␤ cells, and insulin sensitivity in peripheral tissues. Preserving viable ␤ cells and improving ␤-cell function are crucial for the outcome of both type 1 and type 2 diabetes. Although autoimmunity affects the majority of ␤cells in type 1 diabetes, a small percentage of insulin-producing ␤ cells remain, particularly at early onset. Insulin resistance is a major pathogenic factor for type 2 diabetes; therefore, improving insulin sensitivity in diabetic patients would enable better glucose control with relatively low doses of insulin. We employed reverse genetics to investigate the role of ghrelin in glucose homeostasis by studying ␤-cell function and peripheral insulin sensitivity in ghrelin–/– mice [65]. Under standard laboratory housing conditions, levels of blood glucose and plasma insulin in ghrelin–/– mice are normal under both fed and fasted conditions. Upon glucose challenge (glucose tolerance tests), ghrelin–/– mice showed reduced blood glucose levels (fig. 1a) and increased insulin levels compared to wild-type mice (fig. 1b), indicating that ghrelin ablation improves ␤-cell function by increasing glucose-induced insulin secretion [65]. In agreement with our findings that exogenous ghrelin blunted glucose-induced insulin response in ghrelin–/– mice during glucose tolerance tests [65], Dezaki et al. [66] reported that acute ghrelin administration negSun /Asnicar /Smith

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atively regulates glucose-stimulated insulin secretion in rats. They also demonstrated that in single ␤ cells, ghrelin attenuated the glucose-induced first-phase and second-phase [Ca2+]i response, and that the GHS-R1a antagonist, [D-Lys3]-GHRP-6, can block this effect (fig. 2) consistent with a GHS-R1a-mediated action. Hyperinsulinemia associated with insulin resistance may suppress ghrelin production in type 2 diabetes [45]. Administration of ghrelin reduces insulin sensitivity, whereas a combination of ghrelin and desacyl ghrelin is reported to improve insulin sensitivity [67]. In ghrelin–/– mice a small but significant reduction in glucose levels occurs 30 min after insulin administration, suggesting that insulin sensitivity is improved in ghrelin–/– mice (fig. 3a). Hyperinsulinemic-euglycemic clamp experiments confirmed this observation. The basal hepatic glucose production rate was the same in both genotypes (fig. 3b). However, glucose production was suppressed during the low-dose insulin clamp, with the lowest rate being exhibited by ghrelin–/– mice (fig. 3b). This result is consistent with findings that the livers of ghrelin–/– mice are more sensitive to the inhibitory effects of insulin on gluconeogenesis. Furthermore, there was a 33% increase in glucose infusion rate, and a 20% increase in glucose disposal rate, during the low-dose insulin clamp (fig. 3c, d). These results are consistent with the improved insulin sensitivity seen in insulin tolerance tests of ghrelin–/– mice (fig. 3a). To summarize, our data indicate that deletion of ghrelin not only improves ␤-cell function, but also increases peripheral tissue insulin sensitivity.

Ghrelin treatment stimulates appetite and fat deposition in rodents, whereas leptin has opposite effects. Leptin-deficient ob/ob mice are severely obese/hyperphagic, hyperglycemic, hyperinsulinemic and extremely glucose-intolerant [63]. Consistent with findings that plasma ghrelin is low in obese human subjects, ghrelin is also low in ob/ob mice and is elevated in leptin-transgenic mice [68]. The GHS-R antagonist, [D-Lys3]-GHRP-6, is reported to reduce food intake, to lower body weight gain and improve glycemic control in ob/ob mice [69]. However, it is unclear whether this effect is directly mediated by specific antagonism of ghrelin signaling or by nonspecific effects on appetite. To address this issue, we generated ghrelin–/– ob/ob, in anticipation that studying ghrelin deficiency in the ob/ob mice would provide a model for investigating ghrelin and leptin mutual antagonism. Remarkably, ghrelin ablation did not reduce the characteristic hyperphagia and obesity exhibited by leptin-deficient ob/ob mice [65]; hence, the hyperphagia and increased fat deposition is not explained by ghrelin unopposed by leptin, and is consistent with our observation that adult ghrelin–/– mice are not resistant to diet-induced obesity [14]. Although ghrelin ablation was unable to rescue the obese hyperphagic phenotype of ob/ob mice, surprisingly ghrelin–/– ob/ob mice have markedly reduced blood glucose levels (fig. 4a) and increased insulin levels (fig. 4b).

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Ghrelin Ablation Partially Rescues the Diabetic Phenotype of ob/ob Mice

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increases in individual rat ␤ cells. [Ca 2+]i was measured in single ␤ cells by dual-wavelength fura-2 microfluorimetry with 340/380 nm excitation. a Effects of ghrelin (10 n M) on the first-phase [Ca 2+]i responses to 8.3 m M glucose. b Effects of ghrelin (10 n M) on [Ca 2+]i oscillations during the second-phase responses to 8.3 m M glucose. This attenuation of [Ca 2+]i oscillations was abolished in the presence of [D -Lys3]-GHRP-6 (1 ␮ M) (produced

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with permission [66], © 2004 The American Diabetes Association). Fig. 3. Ghrelin deletion improves peripheral insulin sensitivity. Insulin tolerance test (a) was performed on 8 h fasted conscious mice injected with Humulin 0.75 U/kg. Clamp studies (b–d) were performed on overnight fasted conscious mice. * p ! 0.05, WT vs. ghrelin–/– mice (produced with permission from [65], © 2006 Elsevier).

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conscious mice with an intraperitoneal injection of D -glucose (0.625 g/kg). * p ! 0.05, ** p ! 0.001, ob/ob vs. ghrelin–/– ob/ob (produced with permission [65], © 2006 Elsevier).

C-peptide levels are also elevated indicating that the increase in serum insulin is due to increased insulin secretion [65]. Furthermore, ghrelin–/– ob/ob mice have dramatically improved glucose tolerance when compared to ob/ob mice as illustrated by markedly reduced blood glucose levels at 15, 30 and 60 min following glucose challenge (fig. 4c). Interestingly, insulin secretion is increased at 15 and 60 min post-glucose dose (fig. 4d), which is noteworthy because the increased ␤-cell response to glucose caused by ghrelin ablation is associated with what is considered first-phase insulin secretion (at 15 min). The first-phase insulin response in glucose tolerance tests is

of clinical relevance since it is one of the earliest detectable signs in individuals predicted to develop diabetes [70]. Hence, ablation of ghrelin improves the diabetic phenotype of ob/ob mice. Ghrelin’s role in glucose homeostasis is summarized in figure 5. Our studies in genetic mouse models show that ghrelin deletion augments glucose-dependent insulin secretion in pancreatic ␤ cells, and improves insulin sensitivity in peripheral tissues. We speculate that ghrelin is a central neuromodulator that controls whole-body metabolism by glucose sensing, ␤-cell function and fat mobilization. In summary, it appears that ghrelin antag-

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in glucose homeostasis. Ghrelin regulates glucose homeostasis by reducing insulin secretion in pancreas and increasing glucose production in liver and reducing glucose uptake in muscle and adipose tissues (the figure is modified from Ahima [95]; produced with permission, © 2006 Elsevier).

␤-Cell-function

onists would enhance ␤-cell function by increasing the sensitivity of ␤ cells to circulating glucose levels and by improving peripheral insulin sensitivity.

Potential Mechanisms of Ghrelin’s Role in Glucose-Induced Insulin Secretion

UCP2 regulates ATP production, and the membrane potential of ␤ cells in pancreatic islets is regulated by ATP-sensitive K+ channels (K ATP). Lowering of UCP2 increases the ATP/ADP ratio, causing inactivation of KATP channels and increased depolarization of the ␤ cell; this results in a further increase of intracellular Ca2+ and insulin release in response to glucose challenge [71]. Overexpression of UCP2 reduces insulin secretion in response to blood glucose levels, and interferes with glucose signaling in ␤ cells [72]. Compared to wild-type mice, Ucp2–/– and Ucp2+/– mice release more insulin in response to the same glucose challenge; this is similar to what is observed in ghrelin–/– mice [71]. Remarkably, as in ghrelin–/– ob/ob mice, deletion of Ucp2 in ob/ob mice improves the diabetic phenotype, but not the obese phenotype, of ob/ob mice. Accordingly, as a consequence of ghrelin ablation, Ucp2 mRNA expression in the pancreas of ghre222

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Adipose

Insulin sensitivity

lin–/– and ghrelin–/– ob/ob mice was reduced by 50% when compared to wild-type and ob/ob mice, respectively [65]. The data suggest that the deletion of ghrelin causes a reduced pancreatic expression of UCP2. Ghrelin has been shown to reduce the influx of Ca2+ into pancreatic ␤ cells [66]; therefore, the increased glucose sensitivity of the ␤ cell associated with ghrelin deletion is likely explained by increased depolarization of ␤ cells which causes increased influx of Ca2+ and increased insulin secretion in response to glucose metabolism [65]. UCP2 appears to be regulated by Sirt1, a mammalian homolog of the silent information regulator 2 (Sir2) protein family [73]. Sirt1 regulates insulin secretion by suppressing the UCP2 promoter in ␤ cells; consistently, Sirt1–/– mice show low plasma insulin levels and impaired glucose tolerance [74]. Transgenic mice engineered to overexpress Sirt1 in pancreatic ␤ cells (BESTO mice) show improved glucose tolerance and enhanced secretion of insulin in response to glucose challenge [75]. The overexpression of Sirt1, like ghrelin deletion, attenuates expression of UCP2. These studies suggest that ghrelin may regulate ␤-cell function through Sirt1, which in turn affects UCP2 levels. Reduced UCP2 expression is consistent with increased sensitivity of pancreatic ␤ cells to glucose; however, this Sun /Asnicar /Smith

may not explain the acute inhibition of insulin secretion which was detected in our ghrelin–/– mice and several other studies following intraperitoneal ghrelin injection [55–58, 65]. Using PCR-select subtraction method (comparing gene expression profile), several ghrelin-induced genes were identified; ␤-cell autoantigen IA-2␤ is one of those genes [76]. Ghrelin increased IA-2␤ mRNA levels in mouse brain, pancreas, and insulinoma cell lines (MIN6 and ␤TC3). Administration of ghrelin or overexpression of IA-2␤ was found to inhibit glucose-stimulated insulin secretion in MIN6 cells. These findings strongly suggest that inhibitory effects of ghrelin on glucosestimulated insulin secretion are at least partly due to the increased IA-2␤ expression induced by ghrelin. Other hormones, such as somatostatin, might also be involved in ghrelin regulation of insulin release. Somatostatin inhibits insulin secretion through somatostatin receptor subtype-5 (sst5) expressed in pancreatic ␤ cells [77, 78]. Ghrelin has an inhibitory effect on pancreatic somatostatin [55]. It is anticipated that ghrelin deletion will allow somatostatin action to be unopposed; this may, in turn, decrease insulin secretion. Paradoxically, our data showed that ghrelin deletion increases insulin, which suggests that ghrelin regulation of insulin release is not mediated through pancreatic somatostatin. Ghrelin may attenuate insulin release indirectly through the release of neurotransmitters such as epinephrine and serotonin (5-HT). Epinephrine is a strong inhibitor of insulin release and ghrelin infusion increases epinephrine levels [79, 80]. Hence, in vivo, the inhibitory effect of ghrelin on insulin release could be due to increased epinephrine. In the brain, 5-HT regulates appetite and energy homeostasis, and a negative feedback system between brain 5-HT and plasma ghrelin has been reported [81]. Besides central expression, 5-HT is also expressed in normal and diabetic pancreatic tissues. Intriguingly, 5-HT exhibits differential effects on insulin release in the pancreas from normal and diabetic rats; for example, 5-HT stimulates insulin release in the normal pancreas, but inhibits insulin release and increases glucagon release in the pancreas from diabetic rats [82]. Hence, 5-HT helps maintain euglycemia in normal rats, but exacerbates hyperglycemia in diabetic rats. In summary, while several potential mechanisms are suggested for ghrelin’s role in glucose homeostasis, many more in vitro and in vivo studies are needed to distinguish direct from indirect central effects and to elucidate the molecular mechanisms involved. Ghrelin ablation increases glucose sensitivity of the pancreatic ␤ cell, suggesting that ghrelin antagonists would reduce the sever-

ity of diabetes and improve control of blood glucose levels. It is important to consider that the apparent benefit is associated with a decrease in UCP2 expression in pancreatic ␤ cells and reducing UCP2 potentially increases the susceptibility of the ␤ cell towards oxidative stress. During long-term treatment this mechanism might result in the loss of insulin-secreting ␤ cells.

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Discrepancies and Future Studies

Ghrelin’s stimulatory effects on feeding are mediated through GHS-R1a [13], but it is unclear whether ghrelin’s effect on insulin secretion is mediated through GHS-R1a exclusively. Since desacyl ghrelin does not bind to, or activate GHS-R1a, its efficacy as an active hormone is debatable [4]. However, recent reports show that ghrelin and desacyl ghrelin may have opposing, concerted, or equivalent actions on glucose homeostasis in different organ systems. Administration of ghrelin in normal young humans induced a decrease in insulin levels and an increase in plasma glucose levels; curiously, while desacyl ghrelin administration had no effect on insulin and glucose levels, co-administration of ghrelin and desacyl ghrelin abolished the effect of ghrelin [16]. Both ghrelin and desacyl ghrelin appear to affect glucose metabolism in the liver. Although GHS-R1a is not expressed in hepatocytes, glucose output by primary hepatocytes is stimulated by ghrelin and inhibited by desacyl ghrelin; furthermore, desacyl ghrelin counteracts the stimulatory effect of ghrelin on glucose release [17]. It has also been reported that ghrelin reduces insulin sensitivity, whereas the combination of ghrelin and desacyl ghrelin improves insulin sensitivity [67]. Transgenic mice overexpressing desacyl ghrelin under the control of a widely expressed viral promoter exhibited no detectable differences in glucose and insulin levels compared to control mice [18]. In another transgenic line, where desacyl ghrelin was overexpressed under the control of the rat insulin II promoter (RIP-G Tg) in pancreatic islets, glucose-stimulated insulin secretion was suppressed [83]. A recent report suggests that ghrelin and desacyl ghrelin have anti-apoptotic effects on ␤ cells, which might be mediated through cAMP/PKA, ERK1/2 and PI3K/Akt [84]. These studies indicate that desacyl ghrelin has a role in glucose homeostasis. However, there is no evidence that desacyl ghrelin activates GHS-R1a; therefore, desacyl ghrelin must signal through an unidentified receptor. Alternatively, since we have no understanding of how acylation of the ghrelin peptide is 223

regulated and what controls the equilibrium between desacyl ghrelin and ghrelin in specific cell types, some of the results in animals and in cell culture might be explained by interconversion. Clearly, while a substantial amount of indirect evidence for a desacyl ghrelin receptor continues to accumulate, unambiguous proof of the existence of this receptor requires that the putative receptor be cloned, isolated and characterized. We await this with eager anticipation. Ghrelin enhances feeding via the neuronal pathways of hypothalamic orexigenic peptides NPY, AGRP and orexin [85]. In collaboration with Toshinai et al. [19], we recently showed that although peripheral administration of desacyl ghrelin to rats or mice did not alter feeding behavior, intracerebroventricular administration of desacyl ghrelin stimulated feeding in Ghsr–/– mice, but not orexin-deficient mice. It was further demonstrated that intracerebroventricular administration of desacyl ghrelin induces Fos expression in orexin-expressing neurons of the LHA, but not in NPY-expressing neurons of the ARC; furthermore, desacyl ghrelin increased intracellular calcium concentrations in isolated orexin neurons [19]. These exciting data are consistent with desacyl ghrelinactivating orexin-expressing neurons through a receptor distinct from the GHS-R. Prepro-orexin mRNA, expressed in LHA, is stimulated by fasting-induced low plasma glucose [86]. Orexin A and orexin B are orexigenic hypothalamic neuropeptides. In rats, orexin A is expressed in the pancreas and is a more potent than orexin B in stimulating insulin secretion [87, 88]. Orexin-containing islet cells, like those in the brain and gut, are glucose-sensitive and become activated when blood glucose levels fall [89]. However, the molecular mechanism of orexin’s effect on insulin and glucose regulation is not well understood. Besides stimulating insulin, orexins evoke glucagon release, and modulate epinephrine and 5-HT [90]. In mouse LHA, orexin/hypocretin neurons (which promote wakefulness, locomotor activity and foraging) are glucose-inhibited, whereas melanin-concentrating hormone neurons (which promote sleep and energy conservation) are glucose-excited [21]. These observations stress the fundamental importance of hypothalamic glucose-sensing neurons in orchestrating sleepwake cycles, energy expenditure and feeding behavior, suggesting that orexin may act to mediate the central and peripheral effects of desacyl ghrelin which regulate glucose homeostasis. [D -Lys3]-GHRP-6 has been used by many researchers as a GHS-R antagonist and has been shown to decrease blood glucose and increase insulin levels in glucose tol224

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erance tests [66]. However, repeated injections of [D Lys3]-GHRP-6 in ob/ob mice reduced glucose, but did not increase plasma insulin levels [69]. In the INS-1 (832/13) insulin-producing ␤-cell line, ghrelin was reported to inhibit insulin secretion [37], but recently it was reported that both ghrelin and desacyl ghrelin stimulate insulin release in INS-1E cells [91]. Interestingly, in INS-1E cells, [D -Lys3]-GHRP-6 antagonized the ghrelininduced insulin release, while [D -Lys3]-GHRP-6 did not block the stimulatory effect of desacyl ghrelin [91]. These data again suggest that ghrelin and desacyl ghrelin may affect insulin secretion through different signal pathways. A recent report suggested that [D -Lys3]-GHRP-6 activates the 5-HT receptor, 5-HT2B, in smooth muscle [92]. It has also been shown that insulin induces internalization of the 5-HT2A receptor [93]. From these studies, it is not clear whether the effects of [D -Lys3]-GHRP6 represent antagonism of ghrelin activation on GHSR1a, or a stimulatory effect on 5-HT signaling; therefore, we will need to re-evaluate conclusions drawn from experiments with [D -Lys3]-GHRP-6 in order to clarify the meaning of these results. Our studies in mouse models suggested that ghrelin deletion has a beneficial effect on diabetic conditions; thus we anticipate that ghrelin may worsen STZ-induced diabetes. However, a recent article showed that ghrelin prevented the development of diabetes when STZ-treated newborn rats mature to adulthood, which suggests that early administration of ghrelin may help prevent the development of diabetes in disease-prone subjects [94]. It is possible that ghrelin’s effects on STZ-induced diabetes may vary according to the amount of ghrelin administered, and to the developmental stage (e.g., infancy, youth, elderly) in which the ghrelin is administered. Furthermore, in the ghrelin–/– mouse model, both ghrelin and desacyl ghrelin are absent; in the aforementioned STZ-induced diabetes rat experiment, only ghrelin was administered. It is clear that ghrelin plays a role in insulin regulation, but we have much to learn regarding ghrelin’s role in glucose homeostasis, and whether ghrelin is a beneficial or a pathogenic factor for diabetes. While the mouse gene knockout models have provided us with valuable information, given the evolving complexity of ghrelin actions, it is clear that further studies are needed to clarify the independent roles of ghrelin, desacyl ghrelin, and obestatin. We must address whether ghrelin is the sole ligand of GHS-R1a, and whether GHS-R1a is the sole receptor mediating the functions of ghrelin and ghrelin-related peptides both centrally and peripherally. To address the discrepancy between the Sun /Asnicar /Smith

pharmacological effects of ghrelin/desacyl ghrelin and the phenotype of global genetic knockout mouse models, conditional knockouts or inducible over expression of ghrelin or GHS-R1a in pancreatic ␤ cells may be more effective approaches to address ghrelin’s role in pancreatic islets. In summary, while we are assembling more data on ghrelin’s role in glucose homeostasis, the signaling pathways involved remain largely debatable. The effects of ghrelin and desacyl ghrelin on glucose homeostasis may be mediated by a receptor other than GHS-R1a, and the acute and chronic effects of ghrelin and desacyl ghrelin may also be mediated through different pathways.

reports suggest that ghrelin has an inhibitory effect on glucose-induced insulin secretion [55–58, 65, 66]. Even though endogenous ghrelin does not appear to play an essential regulatory role in energy homeostasis in adult mice, ghrelin may play a significant role in glucose homeostasis as a neuromodulator. As more publications on ghrelin appear, we learn that the functions of ghrelin and GHS-R1a are diverse, and interactions between central and peripheral effects are complex. To have full understanding of the biological functions of ghrelin and its related proteins, we must employ all possible approaches (pharmacological and physiological, loss of function and gain of function, in vitro and in vivo, etc.) under different physiological/pathological conditions and developmental stages.

Conclusions Acknowledgements

Ghrelin’s central effects are mainly reflected in its stimulatory effects on GH release and appetite. Whether ghrelin plays a role in central glucose sensing in the brain is largely unknown. Our data and a majority of the published

This review was supported by the National Institutes of Health grants for Roy G. Smith. We thank Michael R. Honig and Edith A. Gibson for editorial assistance.

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Neuroendocrinology 2007;86:229–241 DOI: 10.1159/000108410

Received: November 14, 2006 Accepted after revision: November 27, 2006 Published online: September 12, 2007

Roles of Ghrelin and Leptin in the Control of Reproductive Function Manuel Tena-Sempere Physiology Section, Department of Cell Biology, Physiology and Immunology, University of Córdoba, Córdoba, Spain

Key Words Leptin ⴢ Ghrelin ⴢ GH-secretagogue receptor ⴢ Gonadotropins ⴢ GnRH ⴢ KiSS-1 ⴢ Energy balance ⴢ Testis ⴢ Ovary

Abstract Reproductive function in mammals, defined as the capacity to generate viable male and female gametes, and to support pregnancy and lactation selectively in the female, is sensitive to the metabolic state of the organism. This contention, long assumed on the basis of intuitive knowledge, became formulated on a scientific basis only recently, with the identification of a number of neuroendocrine signals which crucially participate in the joint control of energy balance and reproduction. A paradigmatic example in this context is the adipocyte-derived hormone, leptin; a satiety factor which signals the amount of body energy (fat) stores not only to the circuits controlling food intake but also to a number of neuroendocrine axes, including the reproductive system. More recently, the reproductive dimension of another metabolic hormone, namely the orexigenic stomach-secreted peptide, ghrelin, has been disclosed by observations on its putative roles in the control of gonadal function and gonadotropin secretion. Of note, leptin and ghrelin have been proposed to act as reciprocal regulators of energy homeostasis. However, their potential interplay in the control of reproduction remains largely unexplored. Based on the comparison of the biological actions of leptin and ghrelin at different levels of the hypothalamic-pituitary-gonadal axis, reviewed in detail herein, we propose that, through concurrent or antagonistic

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actions, the leptin-ghrelin pair is likely to operate also as modulator of different reproductive functions, thereby contributing to the physiological integration of reproduction and energy balance. Copyright © 2007 S. Karger AG, Basel

Introduction: Reproduction Is Sensitive to the Metabolic State

The metabolic status of an organism, defined by the availability of energy and nutrients to the tissues, is a pivotal modulator of a myriad of biological functions. Among those, it has been known for ages that the reproductive capacity, characterized in mammals by the ability to produce viable gametes and to support pregnancy and lactation, is sensitive to changes in energy reserves; situations of persistent energy deficit, such as starvation or extreme physical exercise, being invariantly coupled to impaired reproductive function. This contention is especially evident in the female, where pregnancy and lactation are linked to a considerable energetic drain, needed for the nurture of embryos and newborns [1]. The physiologic basis for such a joint regulation of energy balance and reproduction has begun to be unveiled only recently, in a phenomenon that involves multiple common regulatory signals, acting at different levels of the reproductive system. From a neuroendocrine perspective, reproduction critically depends on the concerted development and function of the elements of the so-called hypothalamicManuel Tena-Sempere Physiology Section, Department of Cell Biology, Physiology and Immunology Faculty of Medicine, University of Córdoba Avda. Menéndez Pidal s/n, ES–14004 Córdoba (Spain) Tel. +34 957 218 280, Fax +34 957 218 288, E-Mail [email protected]

pituitary-gonadal (HPG) or gonadotropic axis. Three major groups of signals can be highlighted in this system: the hypothalamic decapeptide gonadotropin-releasing hormone (GnRH), the pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and the hormonal products of the gonads (mainly, sex steroids) [2, 3]. These elements participate in positive and negative feed-forward and feed-back loops, allowing the dynamic regulation of gonadal function along the lifespan. In addition, a large number of central and peripheral signals, of stimulatory and inhibitory nature, participate in the regulation of the gonadotropic axis. Considering the central position of GnRH in the hierarchical control of the HPG axis, many of those regulators are known to target hypothalamic GnRH neurons to regulate fertility. Indeed, based on inferential data, it had been assumed that the major mechanism whereby the metabolic status impacts reproductive function involves modulation of the GnRH neuronal network at the hypothalamus. However, the ultimate elements responsible for such a modulatory action have remained largely unknown until recently.

Endocrine Control of Energy Homeostasis – Leptin as Prototypic Regulator

Energy balance equation is defined by the equilibrium between food intake and energy expenditure – energy homeostasis being one of the most tightly regulated functions of the organism [1]. In order to keep constant body energy stores, it was long anticipated that a state of energy abundance (e.g., by excess of food intake) should activate a series of homeostatic events leading to maintenance of energy balance (e.g., decrease in food consumption and/or increase in energy expenditure), or vice versa. Yet, although indirect evidence strongly suggested the involvement of endocrine factors in this phenomenon, the hormonal effectors for such a key function remained unknown. However, our knowledge of the endocrine mechanisms for the control of body weight significantly expanded in mid-1990s, mostly by the identification of the adipocyte-derived hormone, leptin [1, 4–6]; a finding that virtually opened up a new era in the understanding of the neuroendocrine control of energy homeostasis, and its close relationship with other hormonal systems, including the reproductive axis. Leptin is a 16-kDa adipokine identified in 1994 by positional cloning in the mouse and human [7]. Since its identification, thousands of studies, published on this 230

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topic during the last decade, have defined the structure, regulation, biological effects and major mechanisms of action of leptin in the long-term control of food intake and energy homeostasis [as examples, see reviews 1, 4–6, 8, 9]. As hallmark features of its metabolic role, it is well known that leptin is secreted by the white adipose tissue in proportion to the amount of body energy (fat) stores and functions as satiety factor in the regulation of body weight [1, 4–6]. Most of the effects of leptin in the control of food intake are conducted within the brain, predominantly in the hypothalamus, where leptin is known to modulate both orexigenic neurons, mostly those co-expressing neuropeptide Y and agouti-related peptide, as well as anorexigenic neurons, mostly those co-expressing proopiomelanocortin and cocaine- and amphetamineregulated transcript [reviewed in 9, 10]. In addition to leptin, these orexigenic and anorexigenic pathways are also regulated by a number of modulators of food intake and energy balance that either cooperate or antagonize the effects of leptin on energy homeostasis [10]. Of note, the actions of leptin on these and related circuitries help also to explain its ability to operate as pleiotropic modulator of a wide array of endocrine functions, which are regulated mainly at the hypothalamus [8]. Thus, leptin might be considered as genuine neuroendocrine integrator, linking the control of different essential biological functions to the state of energy reserves of the organism.

Roles of Leptin in the Control of the HPG Axis

The ability of leptin to regulate reproductive function became evident soon after its identification in the context of the ob/ob mouse [5]. This model was well-known to harbor serious reproductive defects long before leptin deficiency was found responsible for in its morbid obesity. Thus, absence of leptin was also hypothesized as causative for its reproductive impairment, defined by lack of puberty onset, variable degrees of hypogonadotropic hypogonadism and infertility [5, 6]. Accordingly, leptin administration was able to rescue puberty onset and prevented sterility in ob/ob female mice [11], and leptin restored reproductive capacity in ob/ob males [12]. Of note, while ob/ob females were found invariantly infertile, a small but detectable proportion of ob/ob males have been reported to have normal reproductive development and fertility [6]. This observation indirectly suggested a more prominent role of leptin in the control of female reproduction, in line with the contention that metabolic gating Tena-Sempere

of reproductive function must be much more restrictive in the female, due to the energetic drain of pregnancy and lactation. In fact, the importance of leptin in the control of female reproduction was soon confirmed in women carrying inactivating mutations of leptin or leptin receptor genes, who showed primary amenorrhea and severe hypogonadism [6]. Nonetheless, data from male (human and mouse) models of leptin deficiency clearly evidence that leptin is also an important regulator of male fertility. Hypothalamic Actions of Leptin: Roles in Puberty and Gonadotropin Secretion One of the functions of leptin in the context of reproduction that initially drew more attention was its role in puberty onset. Based on the reproductive phenotypes of human and animal models of leptin insufficiency, it was hypothesized that leptin plays a role in pubertal maturation. Yet, the precise nature of this action was the matter of intense debate initially, due to contradictory reports on the ability of leptin to actually advance puberty onset in rodents [5, 6, 11, 13, 14]. The present consensus indicates that leptin is predominantly a permissive signal for puberty onset, as threshold leptin levels appear mandatory for normal puberty to proceed, especially in the female [5, 6, 13]. This permissive role (i.e., adequate leptin levels are absolutely essential for but probably not sufficient to trigger puberty onset) is indirectly supported by human studies showing that leptin levels progressively increase in girls along pubertal maturation [15]. In contrast, similar analyses in healthy boys revealed that serum leptin concentrations increased between 5 and 10 years of age, but consistently declined thereafter [15]. Such dissociation probably reflects partial differences in the role of leptin in male and female puberty, and points out the divergent regulatory actions of sex steroids (androgen and estrogen) on leptin secretion (see below: Gonadal Steroids as Modulators of Leptin Secretion). In addition to its role in puberty, the capacity of leptin to regulate gonadotropin secretion has been also studied in detail. In rats, leptin was shown to enhance gonadotropin levels in the female, while blockade of endogenous leptin disrupted pulsatile LH secretion and estrous cyclicity [11, 16]. Likewise, systemic administration of leptin or its active fragment, leptin116–130 amide, elicited FSH and LH secretion in male mice and rats, respectively [13, 17]. In good agreement, variations of LH levels were found tightly coupled to the pulsatile release of leptin in cycling women [18], whereas leptin administration clearly ameliorated the suppressed LH secretion in women under Control of Reproduction by Ghrelin and Leptin

acute caloric deprivation [19]. Moreover, treatment with recombinant leptin significantly improved defective reproductive function in hypoleptinemic patients with hypothalamic amenorrhea [20]. Altogether, these observations strongly suggest a positive role of leptin in the control of the gonadotropic axis. In close parallelism with its hypothalamic actions in the control of energy balance, leptin was soon assumed to conduct its stimulatory/permissive actions upon the HPG axis mainly through effects on the GnRH neuronal system at the hypothalamus. This hypothesis was fully confirmed by studies showing the ability of intrahypothalamic infusion of leptin to elicit GnRH release in vivo [21], and the normalization of pulsatile GnRH/LH secretion after central leptin administration to fasted rats [22]. However, whether this action is conducted directly upon GnRH neurons or indirectly (via intermediate pathways) remained unknown for years. In fact, it was initially reported that leptin receptors are expressed in the immortalized GnRH-secreting cell line, GT1–7, thus suggesting direct actions of leptin on GnRH neurons [23]. However, it is now accepted that, under physiological conditions, GnRH neurons do not express leptin receptors; a phenomenon that suggests the involvement of intermediate circuits and signals [18, 21]. Although the nature of such mediators has been partially disclosed [as examples, see 18, 21], a major breakthrough in our understanding of the neuroendocrine networks responsible for relaying leptin effects onto GnRH neurons has just taken place, with the demonstration that kisspeptin neurons are direct targets for leptin within the hypothalamus [24]; kisspeptin being one of the most potent stimulators of the GnRH/LH axis known so far [25]. Interestingly, hypothalamic Kiss1 gene expression is decreased in conditions of negative energy balance, whereas administration of kisspeptin appears sufficient to rescue defective pubertal activation of the reproductive axis negative energy balance conditions [26], yet it is possible that KiSS-1 neurons are not the only elements that transmit leptin effects onto the GnRH system. An excellent review of the major features of the KiSS-1 system as mediator of leptin effects on the HPG axis can be found in the accompanying paper by Steiner and colleagues in this issue of Neuroendocrinology. Pituitary Effects of Leptin in the Control of the Reproductive Axis Although the hypothalamus is undisputedly considered as the major site for its positive effects on the gonadotropic system, a body of evidence strongly suggests that the sites and mechanisms for the actions of leptin on the Neuroendocrinology 2007;86:229–241

231

reproductive axis are more diverse, and probably involve stimulatory and inhibitory effects, at different levels of the HPG axis. Based on the presence of leptin receptors at the pituitary, these might include direct pituitary actions of leptin in the control of gonadotrope function [18]. In fact, leptin was initially reported to slightly but significantly increase basal and GnRH-stimulated LH secretion by hemipituitaries of normally-fed male rats [27], yet absence of stimulatory effects has also been described [28]. Of note, the net effects of leptin on gonadotrope function might depend on the prevailing metabolic status and are apparently defined by specific domains of the leptin molecule, as direct inhibitory LH responses have been reported in food-restricted animals and after in vitro challenge with the leptin fragment, leptin116–130 amide [28, 29]. The physiologic importance of leptin actions directly at the pituitary level in the control of the gonadotropic axis is yet to be to be fully elucidated. Direct Gonadal Effects of Leptin Additional extrahypothalamic actions of leptin in the control of the HPG axis include direct effects of this adipokine on the gonads, where expression of leptin receptors was described [30]. Concerning the ovary, a large number of studies in different species, including the rat and human, have documented the ability of leptin to modulate ovarian steroidogenesis. Surprisingly enough, most of those studies demonstrated inhibitory actions of leptin on ovarian steroid synthesis [31–35], in apparent contrast with the proven stimulatory/permissive reproductive roles of leptin at the hypothalamus, yet stimulatory effects of leptin on aromatase activity and/or estrogen secretion from the human and mouse ovary have also been reported [36, 37]. The ability of leptin to directly regulate ovarian steroidogenesis is in good agreement with the expression of leptin receptor (Ob-R) mRNA in theca and granulosa cells of the human ovary [38], as well as with the presence of the transcripts encoding the long (OB-Rb) and short (Ob-Ra) forms of leptin receptor in the rat ovary [39]. Not only the receptor, but leptin itself appears to be expressed in the human and rat ovary [40–43], where its levels fluctuate according to functional state of the cycle. Another putative function of leptin in the ovary is its potential implication in the direct control of folliculogenesis and ovulation. However, there is no clear consensus on the predominant function of leptin in this particular facet of ovarian physiology, since leptin has been demonstrated either to impair early follicular development and ovulation [44, 45] or to promote oocyte maturation [46]. 232

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Moreover, follicular responsiveness to gonadotropin priming appears to be decreased in leptin-deficient mice [47], and leptin has been reported to directly induce ovulation in a LH-independent manner [48], although the physiological relevance of the latter is yet to be defined. Overall, it is tempting to speculate that, as has been suggested for the testis (see below), such a combination of inhibitory and stimulatory effects might reflect the differential impact of low or high leptin levels on the ovary. Thus, it has been proposed that massive hyperleptinemia causes direct inhibitory effects on ovulation that might contribute to the adverse impact of morbid obesity on fertility [49]. Expression of leptin receptors was also reported in the testis [30], suggesting potential direct testicular actions of this adipokine. In detail, studies on the developmental and hormonal regulation of Ob-R gene expression in rodent (mostly rat) testis revealed a scattered pattern of distribution within adult testicular tissue, with specific signals in Leydig and Sertoli cells [50, 51]. In addition, Ob-R expression has been also identified in mouse germ cells [52]. Expression of Ob-R gene was persistently detected throughout postnatal testicular development, at rather constant relative levels [32]. Moreover, testicular expression of the Ob-R gene consisted of an array of alternatively spliced isoforms, with abundant levels of Ob-Rb mRNA (i.e., the transcript encoding the long functional variant of leptin receptor) as well as detectable expression of Ob-Ra, Ob-Rf, Ob-Re and Ob-Rc transcripts [50], whose functional role in the tuning of leptin signaling in the testis remains to be elucidated. In keeping with the observed expression of Ob-R, leptin was able to inhibit basal and stimulated testosterone secretion by adult rat testes ex vivo [29]; an effect which is analogous to the inhibition of ovarian steroidogenesis by leptin (see above). This inhibitory action was independently confirmed using primary cultures of rat Leydig cells and the murine mLTC-1 clone from Leydig cell origin [53, 54]. The molecular basis for the inhibitory effects of leptin on testosterone secretion likely involves the decreased expression levels of the mRNAs encoding key steroidogenic factors, such as steroidogenic factor-1 (SF-1), steroid acute regulatory (StAR) protein, and P450 side-chain cleavage (P450scc) enzyme, but not of those of 17␤-hydroxyl steroid dehydrogenase (17␤-HSD) type III [55]. Overall, it is tempting to hypothesize that, while subthreshold leptin concentrations (as signal for energy insufficiency) can induce the suppression of reproductive function at the brain, massively elevated leptin levels may induce direct inhibitory actions on the testis, which could Tena-Sempere

explain some forms of hypogonadism linked to morbid obesity [56, 57]. In good agreement, decreased testosterone concentrations have been recently described in nongenetic (rat) models of hyperleptinemia [57]. Gonadal Steroids as Modulators of Leptin Secretion An additional facet of the interactions between leptin and the gonads is the ability of gonadal hormones to modulate leptin secretion by the white adipose tissue. In this sense, leptin levels are invariantly higher in women than in men, even after correction for body mass index or fat mass [8]. This likely reflects the divergent effects of sex steroids of leptin expression and secretion, as estrogens mainly stimulate leptin release by adipocytes, whereas androgens decrease leptin gene expression and secretion from white adipose tissue [8]. On the latter, a negative correlation between leptin and testosterone levels has been described in men and boys [58], which might derive from direct effect of androgen upon the adipose secretion of leptin [14], as well as the direct inhibitory actions of leptin upon testicular testosterone secretion [29, 53].

Ghrelin: A Ubiquitous Factor with Key Roles in Food Intake and Energy Balance

Ghrelin was identified in late 1999 as the natural ligand of the growth hormone (GH) secretagogue receptor [59], GH secretagogues (GHS) being a large family of peptidyl and non-peptidyl synthetic compounds with ability to elicit GH release in different species, including humans [60]. The mature ghrelin peptide, which is cleaved out from a precursor of 117 amino acids, is composed of 28 amino acids, where the residue Ser3 contains a n-octanoyl group (acylation). Such a post-translational modification was the first of this kind described in a secreted molecule, and appears essential for ghrelin to bind to its receptor (the type 1a GHS-R) and to elicit GH secretion [59, 61–63]. However, although the unacylated form of ghrelin (UAG) was initially considered totally inert, growing evidence indicates that UAG is provided with some biological activities, which are either similar or distinct to those of acylated ghrelin in different physiologic systems [61, and references therein]. This contention, which has emerged very recently, has attracted considerable attention during the last 2 years [as examples, see 64–66], especially considering that UAG is the predominant circulating form of ghrelin in plasma. From a functional perspective, ghrelin was initially catalogued as the endogenous counterpart of GHS, with Control of Reproduction by Ghrelin and Leptin

ability to stimulate GH secretion [59]. However, soon after its cloning, it became evident that the biological effects of ghrelin are much more diverse than those originally described, including both endocrine and nonendocrine effects. Such a pleiotropism justifies the extraordinary attention drawn by this molecule, which has resulted in over 1,800 publications on this topic in the last 7 years. These studies have defined the multifaceted nature of ghrelin, characterized not only by its multiple effects but also by its diverse mechanisms of action and sites of expression [61–63]. For the purpose of this review, it is relevant to highlight that, in addition to its GH-releasing effects, it was soon demonstrated that ghrelin is provided with a relevant metabolic dimension. Thus, ghrelin was proven as a potent orexigenic signal, acting at the hypothalamus [61–63]. Moreover, ghrelin is mainly produced by the stomach (actually it was isolated from this source using a reverse-pharmacology approach [see 59]), its gene expression is enhanced after food deprivation, and its plasma levels increase during the preprandrial state and are (in most cases) negatively correlated with the body mass index. On this basis, ghrelin has been recently proposed as circulating signal for energy insufficiency (the only known circulating orexigen in mammals), which may play a major role in the short- and longterm control of body weight [10, 67].

Ghrelin as Novel Pleiotropic Regulator of Reproductive Function

Considering the prototypic example of leptin and the emergent biological profile of ghrelin (see above), we, as well as others, hypothesized that ghrelin might contribute, in conjunction with other metabolic signals, to the physiologic coupling of reproduction and energy reserves. This possibility has been now supported by a number of experimental observations, demonstrating biological actions of ghrelin at different levels of the reproductive system. These are systematically reviewed in the following sections and summarized in table 1. Central Effects of Ghrelin in the Control of Reproduction In the context of reproduction, one of the functions of ghrelin analyzed in different species was its potential role in the control of gonadotropin secretion. In this sense, studies conducted in the rat, rhesus monkey and sheep have demonstrated that central administration of ghrelin is able to suppress pulsatile LH secretion [68–71]. In good Neuroendocrinology 2007;86:229–241

233

Table 1. Reported experimental evidence supporting the role of ghrelin as putative regulator of the HPG axis

Tissue/system

Evidence

Reference

Gonadotropic axis

Inhibition of LH secretion in the rat by ghrelin in vivo Inhibition of LH secretion in the monkey by ghrelin in vivo Inhibition of LH secretion in the sheep by ghrelin in vivo Inhibition of LH secretion in the human by ghrelin in vivo Delay of puberty onset in the male rat by ghrelin in vivo Inhibition of LH secretion and delay of puberty by UAGa in vivo Ghrelin inhibition of GnRH-induced LH secretion in vitro Ghrelin stimulation of basal LH and FSH secretion in vitro

68, 70, 73, 74 69 71 72 74 66 70, 73 70, 73

Testis

Expression of GGDTb in mouse testis Expression of ghrelin gene/protein in rat testis Expression of ghrelin gene/protein in human testis Expression of GHS-R gene/protein in rat testis Expression of GHS-R gene/protein in human testis Ghrelin-induced inhibition of testosterone secretion Ghrelin-induced inhibition of testicular mRNA levels of StAR, p450scc and other steroidogenic factors Ghrelin modulation of testicular GHS-R levels Ghrelin-induced inhibition of SCFc gene expression Ghrelin-induced inhibition of Leydig cell proliferation

77 78, 80 79, 81, 86 78, 82 79, 81 78, 86

Expression of ghrelin gene/protein in rat ovary Expression of ghrelin protein in human ovary Expression of GHS-R protein in human ovary Expression of ghrelin and GHS-R in chicken ovary

83 84 85 87

Ovary

78 82 86 86

Major findings and original references are included. Unacylated ghrelin. b Ghrelin gene-derived transcript. c Stem cell factor.

a

agreement, ghrelin administration was recently shown to suppress LH pulsatility in healthy humans [72]. Such a predominantly inhibitory effect of ghrelin has been characterized in detail by our group in the rat, where intracerebral administration of ghrelin inhibited LH secretion in prepubertal male rats, adult males and cyclic females, as well as in gonadectomized animals [70, 73]. Interestingly, the inhibitory effects of ghrelin on LH secretion are mimicked by the unacylated form of the molecule in different experimental paradigms [66], suggesting the contribution of mechanisms independent of the classical type 1a GHS receptor. In addition, ghrelin decreased GnRH release by hypothalamic explants ex vivo [73], reinforcing the contention of a major central (hypothalamic) site of action for its inhibitory effects on the gonadotropic axis. As it might be expected for a signal involved in the control of energy homeostasis, ghrelin is likely to conduct also long-term effects on the reproductive system. This is clearly illustrated by the effects of repeated injections of 234

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ghrelin in male and female rats at puberty [74]. A protocol of ghrelin administration, at a dose of 0.5 nmol/12 h for 7 days during puberty transition, significantly decreased serum LH and testosterone levels, and delayed the normal occurrence of balanopreputial separation (as external index of puberty onset). On the contrary, a similar protocol of repeated injections of ghrelin to peripubertal females failed to induce major changes in serum levels of gonadotropins or estradiol, nor did it significantly alter the timing of puberty, as estimated by the ages of vaginal opening and first estrus [74]. On the basis of these observations, we hypothesize that persistently elevated ghrelin levels, as putative signal for energy insufficiency, may operate not only to acutely inhibit LH secretion but also to impair the normal timing of puberty. Curiously enough, in contrast to leptin (see above: Hypothalamic Actions of Leptin: Roles in Puberty and Gonadotropin Secretion), the female rat is apparently less sensitive than the male to the effects of ghrelin at puberty; a contention which is supported by our recent data showing that twice daily adTena-Sempere

ministration of a higher dose (1 nmol) of ghrelin was able to delay vaginal opening in pubertal female rats [manuscript in preparation]. Finally, as was the case for its acute actions on LH secretion, UAG mimicked the effects of acylated ghrelin in terms of induction of a partial delay in puberty onset, suggesting again the potential involvement of GHS-R1a-independent mechanisms in the effects of ghrelin on the gonadotropic axis [66]. Direct Pituitary Effects of Ghrelin in the Control of Gonadotropin Secretion Besides central (likely hypothalamic) effects, another potential site of action for the effects of ghrelin upon the gonadotropic axis is the pituitary. Indeed, the GH-releasing effects of ghrelin are mostly conducted directly at the pituitary level, where its functional receptor is abundantly expressed [61–63], yet the distribution of this receptor in pituitary cell populations other than somatotropes has not been fully characterized. Studies using rat pituitary explants as experimental setting evidenced a dual mode of action of ghrelin at the pituitary in the direct control of gonadotropin secretion. Thus, ghrelin was able to inhibit GnRH-induced LH release by pituitaries from prepubertal animals and adult cyclic female rats, regardless of the stage of the estrous cycle [70, 73]. Conversely, ghrelin evoked clear-cut stimulatory responses in terms of LH and FSH secretion ex vivo, by pituitaries from prepubertal and adult male and female rats [70, 73, and pers. unpubl. data]. This antithetical model of action of ghrelin in the direct control of gonadotrope function might reflect the differential role of systemically derived and locally produced ghrelin in the regulation of gonadotropin secretion. In this sense, in the rat, the stimulatory effects of ghrelin on pituitary LH release become evident at high concentrations (␮M range), but ghrelin expression has been reported at the pituitary [75], making it possible that high local levels of ghrelin are achieved at certain conditions. Of note, direct stimulatory effects of ghrelin on LH release have been also described in non-mammalian species such as goldfish, but effective doses in this case are much lower (pM range) [76]. Thus, it is tempting to speculate that, during evolution, the reproductive roles of ghrelin might have switched from stimulatory effects solely at the pituitary towards a predominant, central inhibitory action in mammals, coincident with maximal specialization of the hypothalamus in the hierarchical control of reproductive axis. Ghrelin and the Gonads: Expression and Actions In the multifaceted mode of action of ghrelin in the control of the HPG axis, compelling evidence demonControl of Reproduction by Ghrelin and Leptin

strates that ghrelin is expressed and conducts specific biological effects directly at the gonadal level. Chronologically, the first (indirect) evidence for the potential involvement of ghrelin in the control of gonadal function came from the demonstration of expression of a testisspecific ghrelin gene-derived transcript in the mouse, although the functional relevance of this transcript is yet to be fully clarified [77]. Immediately afterwards, expression of ghrelin gene was reported in the rat and human testis [78, 79]. On this basis, our group undertook the detailed characterization of testicular ghrelin expression (at the mRNA and peptide levels) in both rat and human species [78, 80]. In the rat, testicular expression of ghrelin gene was persistently detected throughout post-natal development, with ghrelin peptide being selectively detected in Leydig cells at advanced stages of maturation, regardless of their fetal or adult origin [78, 80]. In good agreement, ghrelin mRNA levels appeared regulated mainly (if not exclusively) by pituitary LH; LH receptors being solely expressed in Leydig cells within the testis [80]. Expression of ghrelin mRNA was also detected in the human testis [79, 81], where immunohistochemical assays demonstrated that, as in the rat, ghrelin peptide is abundantly present in interstitial mature Leydig cells. However, a specific feature of testicular expression of ghrelin in the human is the presence of this peptide, albeit at low levels, also in Sertoli cells [81]. Not only the ligand, but also its cognate receptor, the GHS-R type 1a, was identified in the rat and human testis. In the rat, abundant testicular expression of GHS-R1a mRNA has been demonstrated from puberty onwards, with a scattered pattern of distribution of GHS-R1a (mRNA and peptide) in the adult characterized by expression in somatic Sertoli and Leydig cells, and likely in germ cells [82]. Similarly, expression of GHS-R1a has been also reported in the human testis [81], with specific GHS-R1a immunostaining being detected in somatic Sertoli and Leydig cells, as well as in germ cells, mainly in pachytene spermatocytes. In the rat, testicular expression of GHS-R gene is under the control of hormonal signals, mainly the cognate ligand, ghrelin, and pituitary FSH [82]. As in the male gonad, ghrelin and its cognate receptor have been also detected in the mammalian (human and rat) ovary. Ghrelin gene is expressed in the adult rat ovary in a cycle-dependent manner, with peak levels during the luteal phase of the estrous cycle that were prevented by blockade of the preovulatory surge of gonadotropins [83]. In good agreement, within the rat ovarian tissue, strong ghrelin immunoreactivity was predominantly deNeuroendocrinology 2007;86:229–241

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tected in the corpus luteum. Clear-cut ghrelin immunostaining has been also observed in young and mature corpora lutea of the human ovary, whereas no discernible ghrelin signal was detected in ovarian follicles at any developmental stage [84]. In addition, the presence of ghrelin peptide was also demonstrated in the interstitial gland as well as in ovarian hilus cells, also termed Leydig cells of the ovary based on their remarkable morphological and functional similarities with differentiated testicular Leydig cells [84]. GHS-R1a is also expressed in the human ovary, as revealed by immunohistochemical analyses that demonstrated a scattered pattern of distribution with detectable signals in oocytes as well as in somatic follicular cells, luteal cells, and, to a lower extent, in interstitial hilus cells [84]. Expression of GHS-R1a has been also demonstrated in the ovarian surface epithelium [85]. Overall, the expression of GHS-R1a in several ovarian compartments makes it possible that circulating ghrelin could act directly on the female gonad. In addition, the expression of ghrelin itself strongly suggests the existence of potential autocrine/paracrine roles of ghrelin in the regulation of ovarian function, whose physiologic relevance awaits to be elucidated. From a functional standpoint, identification of expression of ghrelin and its functional receptor in the gonads highlighted the possibility of direct effects of this hormone in the control of gonadal function. This facet of ghrelin physiology has been thoroughly analyzed by our group using the rat testis as model, by measuring different indices of testicular function (as testosterone secretion, seminiferous tubule gene expression and cell proliferation) after ghrelin challenge in different in vivo and ex vivo settings. Ghrelin was able to inhibit, in a dose-dependent manner, stimulated testosterone secretion ex vivo and to decrease the mRNA levels of several key factors in the steroidogenic route, such as StAR, P450scc, 3␤-HSD and testis-specific 17␤-HSD type III [78]. In good agreement, an inverse correlation between ghrelin expression in Leydig cells and serum testosterone concentrations has been very recently reported in humans [86]. In addition, ghrelin is likely to modulate relevant seminiferous tubule functions, as evidenced by its ability to inhibit expression of the Sertoli-cell product stem cell factor (SCF). SCF is a major paracrine stimulator of germ cell development, which operates as key survival factor for spermatogonia, spermatocytes and spermatids in the seminiferous epithelium [87]. SCF has been involved also in Leydig cell development and survival. Using models of intratesticular injection in vivo, we have recently demonstrated that ghrelin decreases the proliferative rate of im236

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mature Leydig cells both during pubertal development and after selective ablation of preexisting mature Leydig cells by administration of the cytotoxic compound EDS [87]. In sum, those functional data illustrate the multifaceted nature of ghrelin actions in the testis, where it might operate as putative regulator of key aspects of testicular physiology, such as steroidogenesis, Leydig cell proliferation and/or seminiferous tubule functions. In contrast to the testis, the role, if any, of ghrelin in the direct control of mammalian ovary remains totally unexplored to date. However, recent studies have demonstrated that functional ghrelin receptors are expressed in the chicken ovary, where expression of ghrelin gene is also detected [88]. Moreover, ghrelin might participate in the modulation of essential ovarian functions in the chick, such as cell proliferation, apoptosis and hormone (steroids and peptides) secretion [88]. Although similar functional analyses in the rat and/or human ovary are yet to be conducted, the ovarian expression of ghrelin and its receptor in birds and mammals is highly suggestive of an evolutionary conserved role of ghrelin in the direct control of female gonadal function. The physiologic relevance of this phenomenon is yet to be determined. Ghrelin Roles in Placental Physiology and Pregnancy The effects of ghrelin on the reproductive system very likely involve also putative functions in pregnancy and placental physiology. Expression of ghrelin has been reported in the rat and human placenta [89], and it is measurable in human fetal circulation [90]. In addition, ghrelin is present in non-pregnant and decidualized endometrium, and it has been proposed as paracrine/autocrine regulator of decidualization of human endometrial stromal cells, with a potential role in the interaction between endometrium and embryo during implantation [91]. Functional studies conducted in rodents have demonstrated that ghrelin levels in the uterine fluid considerably increase during fasting, and ghrelin has been reported to inhibit the development of mouse preimplantation embryos in vitro [92]. In good agreement, in the pregnant rat, repeated ghrelin treatment causing hyperghrelinemia during the first half of pregnancy significantly reduced the litter size, without major adverse effects in terms of sex ratio, pregnancy length and body weight at birth [74]. Altogether, these findings make it tempting to speculate that elevated ghrelin levels, of systemic or uterine origin, might operate as negative modifier for early embryo development in order to avoid the excessive metabolic drain linked to pregnancy and lactation in situations of negative energy balance. Tena-Sempere

+

KiSS-1

??

–

Hypothalamus +

Hypothalamus GnRH

GnRH

+ GnRH

+/–

+ GnRH

+/–

Pituitary

Pituitary Leptin

Ghrelin Stomach

WAT LH FSH

LH FSH

– +

Leptin

+

T Ghrelin

Testis

Testis

a

Fig. 1. Schematic presentation of the reproductive roles of leptin

and ghrelin, and their contribution to the metabolic control of reproductive function. The systemic effects of leptin, produced by the adipose tissue as signal of energy abundance, and ghrelin, secreted by the stomach as signal of energy insufficiency, in the control of the gonadotropic axis appear to be antithetical: leptin operates as indispensable permissive/positive factor for puberty onset, gonadotropin release and fertility (a), whereas ghrelin conducts mostly inhibitory actions (b); for details of leptin and ghrelin actions at different levels of the HPG axis see the text. According to this model, in altered metabolic conditions, such as situa-

Proposed Model: Leptin-Ghrelin Interplay in Energy Balance and Reproduction

Our knowledge of the molecular signals and neuroendocrine mechanisms responsible for the homeostatic control of body weight and energy balance has substantially expanded during the last decade. In this complex physiological network, leptin and ghrelin have been demonstrated as mutual functional antagonists, in terms of their effects on food intake and energy expenditure, acting on partially overlapping circuitries involved in the maintenance of energy homeostasis within the hypothalamus [93]. Indeed, the fact that both signals are hormones whose circulating levels fluctuate in a reciprocal manner and conduct opposite biological actions (leptin as signal for energy abundance with potent anorectic effects; ghrelin as signal of energy insufficiency with significant orexigenic actions) has led to the proposal that these two factors operate in a tightly coupled, reciprocal manner in the long-term control of body weight and energy balance [10]. Control of Reproduction by Ghrelin and Leptin

–

b

–

tions of persistent negative energy balance, reciprocal changes in circulating leptin (decrease) and ghrelin (increase) levels are likely to cooperate in the suppression of HPG axis. In addition, leptin and ghrelin are able to conduct direct actions at the gonadal level, which include (in the case of the testis) inhibitory effects on testosterone (T) secretion, as well as potential modulatory actions at the seminiferous tubules (not depicted). These inhibitory effects (in terms of T secretion) might contribute to the hypogonadal state of cachexia (hyperghrelinemia) or morbid obesity (hyperleptinemia). Taken from references 93 and 94, with modifications.

The functional properties of ghrelin in this binomial are unique, as this is the only known circulating factor with clear-cut orexigenic function in mammals [67]. As reflection of the long-known link between energy balance and reproduction, in the last years we have become aware of the reproductive roles of leptin and, more recently, ghrelin, which participate in the control of several aspects of reproductive function, likely through a multifaceted mode of action with effects at different levels of the HPG axis. In line with the proposed ‘yin-yang’ model for leptin-ghrelin interplay in the long-term control of energy balance [10], the systemic effects of leptin and ghrelin in the control of central elements of the gonadotropic axis appear to be opposite to each other: leptin operates as essential permissive/stimulatory factor for puberty onset and proper gonadotropin secretion, whereas ghrelin conducts mostly inhibitory effects in terms of LH secretion and timing of puberty. These antithetical actions could be regarded as extension of the functional antagonism between leptin and ghrelin in the control of Neuroendocrinology 2007;86:229–241

237

energy balance [10, 93], suggesting that the leptin-ghrelin binomial may conduct similar reciprocal regulatory actions upon the HPG axis. In addition to their systemic effects on the reproductive axis, leptin and ghrelin appear to conduct direct actions at the gonads. Yet, rather than functional antagonists, leptin and ghrelin likely carry out similar biological actions at this level. This is clearly the case in the testis, where both peptides have been reported to inhibit testosterone secretion [94–96]. Such concurrent actions might be mechanistically relevant to define the spectrum of reproductive disturbances linked to alterations in energy balance, such as defective testosterone secretion not only in conditions of energy insufficiency, where excess of ghrelin has been proposed as causative factor [97], but also in situations of extreme obesity, defined by massive hyperleptinemia [56, 57]. In summary, the analysis of the wide array of biological functions of leptin and ghrelin reveals the potential involvement of both peptides in the control of relevant aspects of reproductive physiology, such as puberty onset, gonadotropin secretion and fertility (some of which are schematically depicted in fig. 1). Importantly, the physiologic relevance of leptin and ghrelin in the overall control of reproduction is likely dissimilar, as illustrated by the fact that absence of leptin or leptin receptors is undisputedly associated to disturbed pubertal development and impaired reproductive capacity, while disruption of ghrelin signaling does not apparently induce major reproductive defects [98, 99]. It is plausible, however, that

the absence of ghrelin as signal of energy insufficiency may not be as deleterious in terms of reproductive function as its hypersecretion, which is the expected condition in situations of negative energy balance. Indeed, our recent studies of acute and repeated administration of ghrelin in the rat strongly suggest that hyperghrelinemia might be deleterious for critical functional parameters of the reproductive axis, such as gonadotropin (LH) secretion and puberty onset (see above: Central Effects of Ghrelin in the Control of Reproduction). In any event, although critical aspects of ghrelin function in the control of the HPG axis remain to be fully elucidated, the experimental data so far available make it tempting to propose that, through concurrent and opposite actions, the leptinghrelin pair contributes not only to the long-term regulation of body weight but also to the integrative control of energy balance and reproduction.

Acknowledgments The author is indebted to E. Aguilar and L. Pinilla (University of Cordoba, Spain) and C. Dieguez and F.F. Casanueva (University of Santiago de Compostela, Spain) for continuous support and helpful discussions during preparation of the manuscript. The experimental work conducted in the author’s laboratory was supported by grants BFI 2002-00176 and BFI 2005-07446 from Ministerio de Educación y Ciencia (Spain), funds from Instituto de Salud Carlos III (Project PI042082), and EU research contract EDEN QLK4-CT-2002-00603.

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Subject Index Vol. 86, No. 3, 2007

Adiponectin 175, 191 Appetite, neuroendocrine regulation 210 Arcuate nucleus 175 Blood-brain barrier 191 Cachexia 183 Cephalokines 191 Diabetes 215 Energy balance 229 Fasting-induced adipose factor 191 Feeding 183 Galanin-like peptide 175 Ghrelin 147, 215, 229 GH-secretagogue receptor 229 GHS-R 215 Glucose homeostasis 215 GnRH 229 Gonadotropins 229 Growth hormone 165 – – regulators 165 – – secretagogues 147 Histamine 210 –, body weight regulation 210 Histaminergic system 210

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Hypothalamus 183, 191 Inflammatory cytokines 183 Insulin 175 – resistance 215 Kiss1 175 KiSS-1 229 Kisspeptin 175 Leptin 175, 191, 210, 215, 229 Lower vertebrates 165 Metabolism 165, 175 Neuropeptide 175 – Y 175 Ovary 229 Pancreas 215 Proopiomelanocortin 175 Receptor antagonists 210 – types 147 Reproduction 175 Resistin 191 RNA interference 191 Testis 229 Thyroid hormone 175