Neuro-endocrinology of Growth and Energy Balance

Neuroendocrinology of Growth and Energy Balance Guest Editors Claude Kordon, Paris, France Iain Clarke, Monash, Vic.,...

Author: Claude Kordon | Iain Clarke

51 downloads 998 Views 3MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

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

Basel • Freiburg • Paris • London • New York • Bangalore • Bangkok • Singapore • Tokyo • Sydney

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.

S. Karger Medical and Scientific Publishers Basel • Freiburg • Paris • London New York • Bangalore • Bangkok Singapore • Tokyo • Sydney

Disclaimer The statements, options and data contained in this publication are solely those of the individual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in the journal is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. Drug Dosage The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.

Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center (see ‘General Information’). © Copyright 2007 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) Printed in Switzerland on acid-free and non-aging paper (ISO 9706) by Reinhardt Druck, Basel ISBN 978–3–8055–8457–9

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

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

Access to full text and tables of contents, including tentative ones for forthcoming issues: www.karger.com/nen_issues

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

© 2007 S. Karger AG, Basel 0028–3835/07/0863–0146$23.50/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/nen

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-



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

Neuroendocrinology 2007;86:147–164

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,

Ghrelin/GHS Receptors


149

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


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



151

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


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].



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


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



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


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).

References 1 Bowers CY, Momany F, Reynolds GA, Chang D, Hong A, Chang K: Structure-activity relationships of a synthetic pentapeptide that specifically releases growth hormone in vitro. Endocrinology 1980;106:663–667. 2 Bowers CY, Momany FA, Reynolds GA, Hong A: On the in vitro and in vivo activity of a new synthetic hexapeptide that acts on the pituitary to specifically release growth hormone. Endocrinology 1984; 114: 1537– 1545. 3 Bowers CY, Reynolds GA, Durham D, Barrera CM, Pezzoli SS, Thorner MO: Growth hormone (GH)-releasing peptide stimulates GH release in normal men and acts synergistically with GH-releasing hormone. J Clin Endocrinol Metabol 1990;70:975–982. 4 Deghenghi R, Cananzi MM, Torsello A, Battisti C, Muller EE, Locatelli V: GH-releasing activity of hexarelin, a new growth hormone-releasing peptide, in infant and adult rats. Life Sci 1994;54:1321–1328. 5 Camanni F, Ghigo E, Arvat E: Growth hormone-releasing peptides and their analogs. Front Neuroendocrinol 1998; 9:47–72. 6 Isidro ML, Cordido F: Growth hormone secretagogues. Comb Chem High Throughput Screen 2006;9:175–180. 7 Smith RG: Development of growth hormone secretagogues. Endocr Rev 2005; 26: 346– 360. 8 Smith RG, Cheng K, Schoen WR, Pong SS, Hickey G, Jacks T, Butler B, Chan WW, Chaung LY, Judith F, et al: A nonpeptidyl growth hormone secretagogue. Science 1993;260:1640–1643. 9 Patchett AA, Nargund RP, Tata JR, Chen MH, Barakat KJ, Johnston DB, Cheng K, Chan WW, Butler B, Hickey G, et al.: Design and biological activities of L-163,191 (MK0677): a potent, orally active growth hormone secretagogue. Proc Natl Acad Sci USA 1995;92:7001–7005.


10 Chapman IM, Bach MA, Van Cauter E, Farmer M, Krupa D, Taylor AM, Schilling LM, Cole KY, Skiles EH, Pezzoli SS, Hartman ML, Veldhuis JD, Gormley GJ, Thorner MO: Stimulation of the growth hormone (GH)-insulin-like growth factor I axis by daily oral administration of a GH secretagogue (MK-677) in healthy elderly subjects. J Clin Endocrinol Metab 1996; 81: 4249– 4257. 11 Smith RG, Sun Y, Betancourt L, Asnicar M: Growth hormone secretagogues: prospects and potential pitfalls. Best Pract Res Clin Endocrinol Metab 2004;18:333–347. 12 Smith RG, Jiang H, Sun Y: Developments in ghrelin biology and potential clinical relevance. Trends Endocrinol Metabol 2005; 16: 436–442. 13 Broglio F, Boutignon F, Benso A, Gottero C, Prodam F, Arvat E, Ghe C, Catapano F, Torsello A, Locatelli V, Muccioli G, Boeglin D, Guerlavais V, Fehrentz JA, Martinez J, Ghigo E, Deghenghi R: EP1572: a novel peptidomimetic GH secretagogue with potent and selective GH-releasing activity in man. J Endocrinol Invest 2002;25:RC26–RC28. 14 Guerlavais V, Boeglin D, Mousseaux D, Oiry C, Heitz A, Deghenghi R, Locatelli V, Torsello A, Ghè C, Catapano F, Muccioli G, Galleyrand JC, Fehrentz JA, Martinez J: New active series of growth hormone secretagogues. J Med Chem 2003;46:1191–1203. 15 Nagamine J, Kawamura T, Tokunaga T, Hume WE, Nagata R, Nakagawa T, Taiji M: Synthesis and pharmacological profile of an orally-active growth hormone secretagogue SM-130686. Comb Chem High Throughput Screen 2006;9:187–196. 16 Codd EE, Yellin T, Walker RF: Binding of growth hormone-releasing hormone and enkephalin-derived growth hormone-releasing peptides to and opioid receptors in forebrain of rat. Neuropharmacology 1988; 27:1019–1025.

17 Sethumadavan K, Veeraragavan K, Bowers CY: Demonstration and characterization of the specific binding of growth hormone-releasing peptide to rat anterior pituitary and hypothalamus. Biochem Biophys Res Commun 1991;178:31–37. 18 Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum CI, Hamelin M, Hreniuk DL, Palyha OC, Anderson J, Paress PS, Diaz C, Chou M, Liu KK, McKee KK, Pong SS, Chaung LY, Elbrecht A, Dashkevicz M, Heavens R, Rigby M, Sirinathsinghji DJ, Dean DC, Melillo DG, Patchett AA, Nargund R, Griffin PR, DeMartino JA, Gupta SK, Schaeffer JM, Smith RG, Van der Ploeg LH: A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 1996;273:974–977. 19 Smith RG, Van der Ploeg LH, Howard AD, Feighner SD, Cheng K, Hickey GJ, Wyvratt MJ Jr, Fisher MH, Nargund RP, Patchett AA: Peptidomimetic regulation of growth hormone secretion. Endocr Rev 1997; 18: 621– 645. 20 Muccioli G, Ghè C, Ghigo MC, Papotti M, Arvat E, Boghen MF, Nilsson MH, Deghenghi R, Ong H, Ghigo E: Specific receptors for synthetic GH secretagogues in the human brain and pituitary gland. J Endocrinol 1998; 157:99–106. 21 Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth hormone-releasing acylated peptide from stomach. Nature 1999;402:656–660. 22 Tomasetto C, Karam SM, Ribieras S, Masson R, Lefebvre O, Staub A, Alexander G, Chenard MP, Rio MC: Identification and characterization of a novel gastric peptide hormone: the motilin-related peptide. Gastroenterology 2000;119:395–405. 23 Coulie BJ, Miller LJ: Identification of motilin-related peptide. Gastroenterology 2001; 120:588–589.


157

24 Masuda Y, Tanaka T, Inomata N, Ohnuma N, Tanaka S, Itoh Z, Hosoda H, Kojima M, Kangawa K: Ghrelin stimulates gastric acid secretion and motility in rats. Biochem Biophys Res Commun 2000;276:905–908. 25 Asakawa A, Inui A, Kaga T, Yuzuriha H, Nagata T, Ueno N, Makino S, Fujimiya M, Niijima A, Fujino MA, Kasuga M: Ghrelin is an appetite-stimulatory signal from stomach with structural resemblance to motilin. Gastroenterology 2001;120:337–345. 26 Mozid AM, Tringali G, Forsling ML, Hendricks MS, Ajodha S, Edwards R, Navarra P, Grossman AB, Korbonits M: Ghrelin is released from rat hypothalamic explants and stimulates corticotrophin-releasing hormone and arginine-vasopressin. Horm Metab Res 2003;35:455–459. 27 Schmid DA, Held K, Ising M, Weikel JC, Steiger A: Ghrelin stimulates appetite, imagination of food, GH, ACTH, and cortisol, but does not affect leptin in normal controls. Neuropsychopharmacology 2005; 30: 1187– 1192. 28 Fernandez-Fernandez R, Tena-Sempere M, Navarro VM, Barreiro ML, Castellano JM, Aguilar E, Pinilla L: Effects of ghrelin upon gonadotropin-releasing hormone and gonadotropin secretion in adult female rats: in vivo and in vitro studies. Neuroendocrinology 2006;82:245–255. 29 Nakazato M, Murakami N, Date Y Kojima M, Matsuo H, Kangawa K, Matsukura S: A role for ghrelin in the central regulation of feeding. Nature 2001;409:194–198. 30 Wren AM, Seal LJ, Cohen MA, Brynes AE, Frost GS, Murphy KG, Dhillo WS, Ghatei MA, Bloom SR: Ghrelin enhances appetite and food intake in humans. J Clin Endocrinol Metab 2001; 86:5992–5995. 31 Date Y, Nakazato M, Murakami N, Kojima M, Kangawa K, Matsukura S: Ghrelin acts in the central nervous system to stimulate gastric acid secretion. Biochem Biophys Res Commun 2001;280:904–907. 32 Inui A, Asakawa A, Bowers CY Mantovani G, Laviano A, Meguid MM, Fujimiya M: Ghrelin, appetite, and gastric motility: the emerging role of stomach as an endocrine organ. FASEB J 2004;18:439–456. 33 Date Y, Shimbara T, Koda S, Toshinai K, Ida T, Murakami N, Miyazato M, Kokame K, Ishizuka Y, Ishida Y, Kageyama H, Shioda S, Kangawa K, Nakazato M: Peripheral ghrelin transmits orexigenic signals through the noradrenergic pathway from the hindbrain to the hypothalamus. Cell Metab 2006; 4: 323–331. 34 Cumming DE: Ghrelin and the short- and long-term regulation of appetite and body weight. Physiol Behav 2006;89:71–84. 35 Tschöp M, Smiley DL, Heiman ML: Ghrelin induces adiposity in rodents. Nature 2000; 407:908–913.

158

36 Broglio F, Arvat E, Benso A, Gottero C, Muccioli G, Papotti M, van der Lely AJ, Deghenghi R, Ghigo E: Ghrelin, a natural GH secretagogue produced by the stomach, induces hyperglycemia and reduces insulin secretion in humans. J Clin Endocrinol Metab 2001; 86:5083–5086. 37 Murata M, Okimura Y, Iida K, Matsumoto M, Sowa H, Kaji H, Kojima M, Kangawa K, Chihara K: Ghrelin modulates the downstream molecules of insulin signaling in hepatoma cells. J Biol Chem 2002;277:5667– 5674. 38 Poykko SM, Kellokoski E, Horkko S, Kauma H, Kesaniemi YA, Ukkola O: Low plasma ghrelin is associated with insulin resistance, hypertension, and the prevalence of type 2 diabetes. Diabetes 2003;52:2546–2553. 39 Thompson NM, Gill DA, Davies R, Loveridge N, Houston PA, Robinson IC, Wells T: Ghrelin and des-octanoyl ghrelin promote adipogenesis directly in vivo by a mechanism independent of the type 1a growth hormone secretagogue receptor. Endocrinology 2004;145:234–242. 40 Gauna C, Delhanty PJ, Hofland L, Janssen JA, Broglio F, Ross RJ, Ghigo E, van der Lely AJ: Ghrelin stimulates, whereas des-octanoyl ghrelin inhibits, glucose output by primary hepatocytes. J Clin Endocrinol Metab 2005;90:1055–1060. 41 Heijboer AC, van den Hoek AM, Parlevliet ET, Havekes LM, Romijn JA, Pijl H, Corssmit EP: Ghrelin differentially affects hepatic and peripheral insulin sensitivity in mice. Diabetologia 2006;49:732–738. 42 Dezaki K, Sone H, Koizumi M, Nakata M, Kakei M, Nagai H, Hosoda H, Kangawa K, Yada T: Blockade of pancreatic islet-derived ghrelin enhances insulin secretion to prevent high-fat diet-induced glucose intolerance. Diabetes 2006;55:3486–3493. 43 Sun Y, Asnicar M, Saha PK, Chan L, Smith RG: Ablation of ghrelin improves the diabetic but not obese phenotype of ob/ob mice. Cell Metab 2006;3:379–386. 44 Gluck EF, Stephens N, Swoap SJ: Peripheral ghrelin deepens torpor bouts in mice through the arcuate nucleus neuropeptide Y signaling pathway. Am J Physiol 2006;291:R1303– R1309. 45 Steiger A: Ghrelin and sleep-wake regulation. Am J Physiol 2007;292:R573–R574. 46 Asakawa A, Inui A, Kaga T, Yuzuriha H, Nagata T, Fujimiya M, Katsuura G, Makino S, Fujino MA, Kasuga M: A role of ghrelin in neuroendocrine and behavioral responses to stress in mice. Neuroendocrinology 2001;74: 143–147. 47 Carlini VP, Varas MM, Cragnolini AB, Schioth HB, Scimonelli TN, de Barioglio SR: Differential role of the hippocampus, amygdala, and dorsal raphe nucleus in regulating feeding, memory, and anxiety-like behavioral responses to ghrelin. Biochem Biophys Res Commun 2004;313:635–641.


48 Kanehisa M, Akiyoshi J, Kitaichi T, Matsushita H, Tanaka E, Kodama K, Hanada H, Isogawa K: Administration of antisense DNA for ghrelin causes an antidepressant and anxiolytic response in rats. Prog Neuropsychopharmacol Biol Psychiatry 2006; 30: 1403–1407. 49 Diano S, Farr SA, Benoit SC, McNay EC, da Silva I, Horvath B, Gaskin FS, Nonaka N, Jaeger LB, Banks WA, Morley JE, Pinto S, Sherwin RS, Xu L, Yamada KA, Sleeman MW, Tschöp MH, Horvath TL: Ghrelin controls hippocampal spine synapse density and memory performance. Nat Neurosci 2006;9: 381–388. 50 Bedendi I, Alloatti G, Marcantoni A Malan D, Catapano F, Ghè C, Deghenghi R, Ghigo E, Muccioli G: Cardiac effect of ghrelin and its endogenous derivatives des-octanoyl ghrelin and des-Gln14-ghrelin. Eur J Pharmacol 2003;476:87–95. 51 Nagaya N, Uematsu M, Kojima M Ikeda Y, Yoshihara F, Shimizu W, Hosoda H, Hirota Y, Ishida H, Mori H, Kangawa K: Chronic administration of ghrelin improves left ventricular dysfunction and attenuates development of cardiac cachexia in rats with heart failure. Circulation 2001;104:1430–1435. 52 Tritos NA, Kissinger KV, Manning WJ, Danias PJ: Association between ghrelin and cardiovascular indexes in healthy obese and lean men. Clin Endocrinol (Oxf) 2004; 60: 60–66. 53 Muccioli G, Broglio F, Tarabra E, Ghigo E: Known and unknown growth hormone secretagogue receptors and their ligands; in Ghigo E (ed): Ghrelin. Boston, Kluwer Academic, 2004, pp 27–46. 54 Broglio F, Prodam F, Me E, Riganti F, Lucatello B, Granata R, Benso A, Muccioli G, Ghigo E Ghrelin: Endocrine, metabolic and cardiovascular actions. J Endocrinol Invest 2005;28:23–25. 55 Cassoni P, Papotti M, Ghè C, Catapano F, Sapino A, Graziani A, Deghenghi R, Reissmann T, Ghigo E, Muccioli G: Identification, characterization and biological activity of specific receptors for natural (ghrelin) and synthetic growth hormone secretagogues in human breast carcinomas and cell lines. J Clin Endocrinol Metab 2001;86:1738–1745. 56 Baldanzi G, Filigheddu N, Cutrupi S, Catapano F, Bonissoni S, Fubini A, Malan D, Baj G, Granata R, Broglio F, Papotti M, Surico N, Bussolino F, Isgaard J, Deghenghi R, Sinigaglia F, Prat M, Muccioli G, Ghigo E, Graziani A: Ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and PI3-kinase/AKT. J Cell Biol 2002; 159:1029–1037. 57 Papotti M, Ghè C, Volante M, Muccioli G: Ghrelin and tumors; in Ghigo E (ed): Ghrelin. Boston, Kluwer Academic, 2004, pp 143– 164.


58 Cassoni P, Ghè C, Marrocco T, Tarabra E, Allia E, Catapano F, Deghenghi R, Ghigo E, Papotti M, Muccioli G: Expression of ghrelin and biological activity of specific receptors for ghrelin and des-acyl ghrelin in human prostate neoplasms and related cell lines. Eur J Endocrinol 2004;150:173–184. 59 Nakahara K, Nakagawa M, Baba Y, Sato M, Toshinai K, Date Y, Nakazato M, Kojima M, Miyazato M, Kaiya H, Hosoda H, Kangawa K, Murakami N: Maternal ghrelin plays an important role in rat fetal development during pregnancy. Endocrinology 2006; 147: 1333–1342. 60 Granata R, Settanni F, Biancone L, Trovato L, Nano R, Bertuzzi F, Destefanis S, Annunziata M, Martinetti M, Catapano F, Ghè C, Isgaard J, Papotti M, Ghigo E, Muccioli G: Acylated and unacylated ghrelin promote proliferation and inhibit apoptosis of pancreatic cells and human islets. Involvement of cAMP/PKA, ERK1/2 and PI3K/AKT signalling. Endocrinology 2007;148:512–529. 61 Delhanty PJ, van der Eerden BC, van der Velde M, Gauna C, Pols HA, Jahr H, Chiba H, van der Lely AJ, van Leeuwen JP: Ghrelin and unacylated ghrelin stimulate human osteoblast growth via mitogen-activated protein kinase/phosphoinositide 3-kinase pathways in the absence of GHS-R1a. J Endocrinol 2006;188:37–47. 62 Dixit VD, Taub DD: Ghrelin and immunity: a young player in an old field. Exp Gerontol 2005;40:900–910. 63 Van der Lely AJ, Tschöp M, Heiman ML, Ghigo E: Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin. Endocr Rev 2004;25:426–457. 64 Kojima M, Kangawa K: Ghrelin: structure and function. Physiol Rev 2005;85:495–522. 65 Ghigo E, Broglio F, Arvat E, Maccario M, Papotti M, Muccioli G: Ghrelin: more than a natural GH secretagogue and/or an orexigenic factor. Clin Endocrinol (Oxf) 2005;62: 1–17. 66 Nogueiras R, Perez-Tilve D, Wortley KE, Tschöp M: Growth hormone secretagogue (ghrelin) receptors – a complex drug target for the regulation of body weight. CNS Neurol Disord Drug Targets 2006;5:335–343. 67 Gualillo O, Lago F, Casanueva FF, Dieguez C: One ancestor, several peptides post-translational modifications of preproghrelin generate several peptides with antithetical effects. Mol Cell Endocrinol 2006;256:1–8. 68 Zhu X, Cao Y, Voodg K, Steiner DF: On the processing of proghrelin to ghrelin. J Biol Chem 2006;281:38867–38870. 69 Bednarek MA, Feighner SD, Pong SS, McKee KK, Hreniuk DL, Silva MV, Warren VA, Howard AD, Van Der Ploeg LH, Heck JV: Structure-functions studies on the new growth hormone-releasing peptide, ghrelin: minimal sequence of ghrelin necessary for activation of growth hormone secretagogue receptor 1a. J Med Chem 2000; 43: 4370– 4376.


70 Torsello A, Ghè C, Bresciani E, Catapano F, Ghigo E, Deghenghi R, Locatelli V, Muccioli G: Short ghrelin peptides neither displace ghrelin binding in vitro nor stimulate GH release in vivo. Endocrinology 2002;143:1968– 1971. 71 Muccioli G, Tschöp M, Papotti M, Deghenghi R, Heiman M, Ghigo E: Neuroendocrine and peripheral activities of ghrelin: implications in metabolism and obesity. Eur J Pharmacol 2002;440:235–254. 72 Broglio F, Prodam F, Riganti E, Muccioli G, Ghigo E: Ghrelin: From somatotrope secretion to new perspectives in the regulation of peripheral metabolic functions. Front Horm Res 2006;35:102–114. 73 Hosoda H, Kojima M, Kangawa K: Biological, physiological, and pharmacological aspects of ghrelin. J Pharmacol Sci 2006; 100: 398–410. 74 Banks WA, Tschöp M, Robinson SM, Heiman ML: Extent and direction of ghrelin transport across the blood-brain barrier is determined by its unique primary structure. J Pharmacol Exp Ther 2002;302:822–827. 75 Matsumoto M, Hosoda H, Kitajima Y, Morozumi N, Minamitake Y, Tanaka S, Matsuo H, Kojima M, Hayashi Y, Kangawa K: Structure-activity relationship of ghrelin: pharmacological study of ghrelin peptides. Biochem Biophys Res Commun 2001; 287: 142–146. 76 Matsumoto M, Kitajima Y, Iwanami T, Hayashi Y, Tanaka S, Minamitake Y, Hosoda H, Kojima M, Matsuo H, Kangawa K: Structural similarity of ghrelin derivatives to peptidyl growth hormone secretagogues. Biochem Biophys Res Commun 2001; 284: 655–659. 77 Tolle V, Zizzari P, Tomasetto C, Rio MC, Epelbaum J, Bluet-Pajot MT: In vivo and in vitro effects of ghrelin/motilin-related peptide on growth hormone secretion in the rat. Neuroendocrinology 2001;73:54–61. 78 Muccioli G, Pons N, Ghè C, Catapano F, Granata R, Ghigo E: Ghrelin and des-acyl ghrelin both inhibit isoproterenol-induced lipolysis in rat adipocytes via a non-type 1a growth hormone secretagogue receptor. Eur J Pharmacol 2004;498:27–35. 79 Hosoda H, Kojima M, Mizushima Y, Shimizu S, Kangawa K: Structural divergence of human ghrelin. Identification of multiple ghrelin-derived molecules produced by posttranslational processing. J Biol Chem 2003; 278:64–70. 80 Hosoda H, Kojima M, Matsuo H, Kangawa K. Ghrelin and des-octanoyl ghrelin: two major forms of rat ghrelin peptide in gastrointestinal tissue. Biochem Biophys Res Commun 2000;279:909–913.

81 Hosoda H, Kojima M, Matsuo H, Kangawa K: Purification and characterization of rat des-Gln14-ghrelin, a second endogenous ligand for the growth hormone secretagogue receptor. J Biol Chem 2000; 275: 21995– 22000. 82 Jeffery PL, Murray RE, Yeh AH, McNamara JF, Duncan RP, Francis GD, Herington AC, Chopin LK: Expression and function of the ghrelin axis, including a novel preproghrelin isoform, in human breast cancer tissues and cell lines. Endocr Relat Cancer 2005;12:839– 850. 83 Yeh AH, Jeffery PL, Duncan RP, Herington AC, Chopin LK: Ghrelin and a novel preproghrelin isoform are highly expressed in prostate cancer and ghrelin activates mitogen-activated protein kinase in prostate cancer. Clin Cancer Res 2005;11:8295–8303. 84 Zhang JV, Ren PG, Avsian-Kretchmer O, Luo CW, Rauch R, Klein C, Hsueh AJ: Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin’s effects on food intake. Science 2005;310:996–999. 85 Dun SL, Brailoiu GC, Brailoiu E, Yang J, Chang JK, Dun NJ: Distribution and biological activity of obestatin in the rat. J Endocrinol 2006;191:481–489. 86 Samson WK, White MM, Price C, Ferguson AV: Obestatin acts in brain to inhibit thirst. Am J Physiol 2007;292:R637–R643. 87 Holst B, Egerod KL, Schild E, Vickers SP, Cheetham S, Gerlach LO, Storjohann L, Stidsen CE, Jones R, Beck-Sickinger AG, Schwartz TW: GPR39 signaling is stimulated by zinc ions but not by obestatin. Endocrinology 2007;148:13–20. 88 McKee KK, Palyha OC, Feighner SD, Hreniuk DL, Tan CP, Phillips MS, Smith RG, Van der Ploeg LH, Howard AD: Molecular analysis of rat pituitary and hypothalamic growth hormone secretagogue receptors. Mol Endocrinol 1997;11:415–423. 89 Hayashida T, Nakahara K, Mondal MS, Date Y, Nakazato M, Kojima M, Kangawa K, Murakami N: Ghrelin in neonatal rats: distribution in stomach and its possible role. J Endocrinol 2002;173:239–245. 90 Gnanapavan S, Kola B, Bustin SA, Morris DG, McGee P, Fairclough P, Bhattacharya S, Carpenter R, Grossman AB, Korbonits M: The tissue distribution of the mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans. J Clin Endocrinol Metab 2002; 87: 2988–2991. 91 Guan XM, Yu H, Palyha OC, McKee KK, Feighner SD, Sirinathsinghji DJ, Smith RG, van der Ploeg LH, Howard AD: Distribution of mRNA encoding the growth hormone secretagogue receptor in brain and peripheral tissues. Brain Res Mol Brain Res 1997;48:23– 29. 92 Sun Y, Garcia JM, Smith RG: Ghrelin and growth hormone secretagogue receptor expression in mice during aging. Endocrinology 2007;148:1323–1329.


159

93 Zigman JM, Jones JE, Lee CE, Saper CB, Elmquist JK: Expression of ghrelin receptor mRNA in the rat and the mouse brain. J Comp Neurol 2006;494:528–548. 94 Jaszberenyi M, Bujdoso E, Bagosi Z, Telegdy G: Mediation of the behavioral, endocrine and thermoregulatory actions of ghrelin. Horm Behav 2006;50:266–273. 95 Jerlhag E, Egecioglu E, Dickson SL, Andersson M, Svensson L, Engel JA: Ghrelin stimulates locomotor activity and accumbal dopamine-overflow via central cholinergic systems in mice: implications for its involvement in brain reward. Addict Biol 2006;11:45–54. 96 Abizaid A, Liu ZW, Andrews ZB, Shanabrough M, Borok E, Elsworth JD, Roth RH, Sleeman MW, Picciotto MR, Tschöp MH, Gao XB, Horvath TL: Ghrelin modulates the activity and synaptic input organization of midbrain dopamine neurons while promoting appetite. J Clin Invest 2006;116: 3229–3239. 97 Arvat E, Ramunni J, Bellone J, Di Vito L, Baffoni C, Broglio F, Deghenghi R, Bartolotta E, Ghigo E: The GH, prolactin, ACTH and cortisol responses to hexarelin, a synthetic hexapeptide, undergo different agerelated variations. Eur J Endocrinol 1997; 137:635–642. 98 Ghigo E, Arvat E, Camanni F: Growth hormone secretagogues as corticotrophin-releasing factors. Growth Horm IGF Res 1998;8(suppl B):145–148. 99 Ghigo E, Arvat E, Giordano R, Broglio F, Gianotti L, Maccario M, Bisi G, Graziani A, Papotti M, Muccioli G, Deghenghi R, Camanni F: Biologic activities of growth hormone secretagogues in humans. Endocrine 2001;14:87–93. 100 Popovic V, Miljic D, Micic D, Damjanovic S, Arvat E, Ghigo E, Dieguez C, Casanueva FF: Ghrelin main action on the regulation of growth hormone release is exerted at hypothalamic level. J Clin Endocrinol Metab 2003;88:3450–3453. 101 Enomoto M, Nagaya N, Uematsu M, Okumura H, Nakagawa E, Ono F, Hosoda H, Oya H, Kojima M, Kanmatsuse K, Kangawa K: Cardiovascular and hormonal effects of subcutaneous administration of ghrelin, a novel growth hormone-releasing peptide, in healthy humans. Clin Sci (Lond) 2003; 105:431–435. 102 Rubinfeld H, Hadani M, Taylor JE, Dong JZ, Comstock J, Shen Y, DeOliveira D, Datta R, Culler MD, Shimon I: Novel ghrelin analogs with improved affinity for the GH secretagogue receptor stimulate GH and prolactin release from human pituitary cells. Eur J Endocrinol 2004;151:787–795.

160

103 Cowley MA, Smith RG, Diano S, Tschöp M, Pronchuk N, Grove KL, Strasburger CJ, Bidlingmaier M, Esterman M, Heiman ML, Garcia-Segura LM, Nillni EA, Mendez P, Low MJ, Sotonyi P, Friedman JM, Liu H, Pinto S, Colmers WF, Cone RD, Horvath TL: The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis. Neuron 2003; 37: 649– 661. 104 Chen HY, Trumbauer ME, Chen AS, Weingarth DT, Adams JR, Frazier EG, Shen Z, Marsh DJ, Feighner SD, Guan XM, Ye Z, Nargund RP, Smith RG, Van der Ploeg LH, Howard AD, MacNeil DJ, Qian S: Orexigenic action of peripheral ghrelin is mediated by neuropeptide Y and agouti-related protein. Endocrinology 2004; 145: 2607– 2612. 105 Sun Y, Wang P, Zheng H, Smith RG: Ghrelin stimulation of growth hormone release and appetite is mediated through the growth hormone secretagogue receptor. Proc Natl Acad Sci USA 2004; 101: 4679– 4684. 106 Murphy KG, Dhillo WS, Bloom SR: Gut peptides in the regulation of food intake and energy homeostasis. Endocr Rev 2006; 27:719–727. 107 Sibilia V, Muccioli G, Deghenghi R, Pagani F, De Luca V, Rapetti D, Locatelli V, Netti C: Evidence for a role of the GHS-R1a receptors in ghrelin inhibition of gastric acid secretion in the rat. J Neuroendocrinol 2006;18:122–128. 108 Kawczynska-Drozdz A, Olszanecki R, Jawien J, Brzozowski T, Pawlik WW, Korbut R, Guzik TJ: Ghrelin inhibits vascular superoxide production in spontaneously hypertensive rats. Am J Hypertens 2006;19: 764–767. 109 Suematsu M, Katsuki A, Sumida Y, Gabazza EC, Murashima S, Matsumoto K, Kitagawa N, Akatsuka H, Hori Y, Nakatani K, Togashi K, Yano Y, Adachi Y: Decreased circulating levels of active ghrelin are associated with increased oxidative stress in obese subjects. Eur J Endocrinol 2005; 153: 403–407. 110 Koo GC, Huang C, Camacho R, Trainor C, Blake JT, Sirotina-Meisher A, Schleim KD, Wu TJ, Cheng K, Nargund R, McKissick G: Immune enhancing effect of a growth hormone secretagogue. J Immunol 2001; 166: 4195–4201. 111 Dixit VD, Schaffer EM, Pyle RS, Collins GD, Sakthivel SK, Palaniappan R, Lillard JW Jr, Taub DD: Ghrelin inhibits leptinand activation-induced proinflammatory cytokine expression by human monocytes and T cells. J Clin Invest 2004;114:57–66. 112 Camina JP: Cell biology of the ghrelin receptor. J Neuroendocrinol 2006; 18:65–76. 113 Petersenn S: Structure and regulation of the growth hormone secretagogue receptor. Minerva Endocrinol 2002;27:243–256.


114 Bockaert J, Pin JP: Molecular tinkering of G-protein-coupled receptors: an evolutionary success. EMBO J 1999;18:1723–1729. 115 McKee KK, Tan CP, Palyha OC, Liu J, Feighner SD, Hreniuk DL, Smith RG, Howard AD, Van der Ploeg LH: Cloning and characterization of two human G-proteincoupled receptor genes (GPR38 and GPR39) related to the growth hormone secretagogue and neurotensin receptors. Genomics 1997;46:426–434. 116 Tan CP, McKee KK, Liu Q, Palyha OC, Feighner SD, Hreniuk DL, Smith RG, Howard AD: Cloning and characterization of a human and murine T-cell orphan G-protein-coupled receptor similar to the growth hormone secretagogue and neurotensin receptors. Genomics 1998;52:223–229. 117 Palyha OC, Feighner SD, Tan CP, et al: Ligand activation domain of human orphan growth hormone secretagogue receptor conserved from pufferfish to humans. Mol Endocrinol 2000;14:160–169. 118 Muccioli G, Papotti M, Locatelli V, Ghigo E: Binding of 125I-labeled ghrelin to membranes from human hypothalamus and pituitary gland. J Endocrinol Invest 2001;24: RC7–RC9. 119 Tullin S, Hansen BS, Ankersen M, Moller J, Von Cappelen KA, Thim L: Adenosine is an agonist of the growth hormone secretagogue receptor. Endocrinology 2000; 141: 3397–3402. 120 Smith RG, Leonard R, Bailey AR, Palyha O, Feighner S, Tan C, Mckee KK, Pong SS, Griffin P, Howard A: Growth hormone secretagogue receptor family members and ligands. Endocrine 2001;14:9–14. 121 Carreira MC, Camina JP, Smith RG, Casanueva FF: Agonist-specific coupling of growth hormone secretagogue receptor type 1a to different intracellular signaling systems. Role of adenosine. Neuroendocrinology 2004;79:13–25. 122 Johansson S, Fredholm BB, Hjort C, Morein T, Kull B, Hu PS: Evidence against adenosine analogues being agonists at the growth hormone secretagogue receptor. Biochem Pharmacol 2005;70:598–605. 123 Carreira MC, Camina JP, Diaz-Rodriguez E, Alvear-Perez R, Llorens-Cortes C, Casanueva FF: Adenosine does not bind to the growth hormone secretagogue receptor type-1a. J Endocrinol 2006;191:147–157. 124 Deghenghi R, Avallone R, Torsello A, Muccioli G, Ghigo E, Locatelli V: Growth hormone-inhibiting activity of cortistatin in the rat. J Endocrinol Invest 2001;24:RC31– RC33. 125 Deghenghi R, Papotti M, Ghigo E, Muccioli G, Locatelli V: Somatostatin octapeptides (lanreotide, octreotide, vapreotide, and their analogs) share the growth hormonereleasing peptide receptor in the human pituitary gland. Endocrine 2001;14:29–33.


126 Luque RM, Peinado JR, Gracia-Navarro F, Broglio F, Ghigo E, Kineman RD, Malagon MM, Castano JP: Cortistatin mimics somatostatin by inducing a dose-dependent stimulatory and inhibitory effect on growth hormone secretion in somatotrope. J Mol Endocrinol 2006;36:547–555. 127 Ma JN, Schiffer HH, Knapp AE, Wang J, Wong KK, Currier EA, Owens M, Nash NR, Gardell LR, Brann MR, Olsson R, Brurstein ES. Identification of the atypical L-type Ca 2+ channel blocker diltiazem and its metabolites as ghrelin receptor agonists; Mol Pharmacol 2007; [Epub ahead of print]. 128 Broglio F, van Koetsveld P, Benso A, Gottero C, Prodam F, Papotti M, Muccioli G, Gauna C, Hofland L, Deghenghi R, Arvat E, Van Der Lely AJ, Ghigo E: Ghrelin secretion is inhibited by either somatostatin or cortistatin in humans. J Clin Endocrinol Metab 2002;87:4829–4832. 129 Woll PJ, Rozengurt E: [D -Arg1,D -Phe5,D Trp7,9,Leu11]substance P, a potent bombesin antagonist in murine Swiss 3T3 cells, inhibits the growth of human small cell lung cancer cells in vitro. Proc Natl Acad Sci USA 1988;85:1859–1863. 130 Holst B, Cygankiewicz A, Jensen TH, Ankersen M, Schwartz TW: High constitutive signaling of the ghrelin receptor – identification of a potent inverse agonist. Mol Endocrinol 2003;17:2201–2210. 131 Holst B, Lang M, Brandt E, Bach A, Howard A, Frimurer TM, Beck-Sickinger A, Schwartz TW: Ghrelin receptor inverse agonists: identification of an active peptide core and its interaction epitopes on the receptor. Mol Pharmacol 2006;70:936–946. 132 Xin Z, Zhao H, Serby MD, Liu B, Schaefer VG, Falls DH, Kaszubska W, Colins CA, Sham HL, Liu G: Synthesis and structureactivity relationships of isoxazole carboxamides as growth hormone secretagogue receptor antagonists. Bioorg Med Chem Lett 2005;15:1201–1204. 133 Serby MD, Zhao H, Szczepankiewicz BG, Kosogof C, Xin Z, Liu B, Liu M, Nelson LT, Kaszubska W, Falls HD, Schaefer V, Bush EN, Shapiro R, Droz BA, Knourek-Segel VE, Fey TA, Brune ME, Beno DW, Turner TM, Collins CA, Jacobson PB, Sham HL, Liu G: 2,4-Diaminopyrimidine derivatives as potent growth hormone secretagogue receptor antagonists. J Med Chem 2006; 49: 2568–2578. 134 Demange L, Boeglin D, Moulin A, Mousseaux D, Ryan J, Berge G, Gagne D, Heitz A, Perrissoud D, Locatelli V, Torsello A, Galleyrand JC, Fehrentz JA, Martinez J: Synthesis and pharmacological in vitro and in vivo evaluation of novel triazole derivatives as ligands of the ghrelin receptor. 1. J Med Chem 2007;50:1939–1957. 135 Hornby PJ: Designing Spiegelmers to antagonise ghrelin. Gut 2006;55:754–755.


136 Helmling S, Maasch C, Eulberg D, Buchner K, Schroder W, Lange C, Vonhoff S, Wlotzka B, Tschöp MH, Rosewicz S, Klussmann S: Inhibition of ghrelin action in vitro and in vivo by an RNA-Spiegelmer. Proc Natl Acad Sci USA 2004;101:13174–13179. 137 Schwartz TW, Frimurer TM, Holst B, Rosenkilde MM, Elling CE: Molecular mechanism of 7TM receptor activation – a global toggle switch model. Annu Rev Pharmacol Toxicol 2006;46:481–519. 138 Pedretti A, Villa M, Pallavicini M, Valoti E, Vistoli G: Construction of human ghrelin receptor (hGHS-R1a) model using a fragmental prediction approach and validation through docking analysis. J Med Chem 2006;49:3077–3085. 139 Feighner SD, Howard AD, Prendergast K, Palyha OC, Hreniuk DL, Nargund R, Underwood D, Tata JR, Dean DC, Tan CP, McKee KK, Woods JW, Patchett AA, Smith RG, Van der Ploeg LH: Structural requirements for the activation of the human growth hormone secretagogue receptor by peptide and non-peptide secretagogues. Mol Endocrinol 1998;12:137–145. 140 Falls HD, Dayton BD, Fry DG, Ogiela CA, Schaefer VG, Brodjian S, Reilly RM, Collins CA, Kaszubska W: Characterization of ghrelin receptor activity in a rat pituitary cell line RC-4B/C. J Mol Endocrinol 2006;37:51–62. 141 Chen C: Growth hormone secretagogue actions on the pituitary gland: multiple receptors for multiple ligands? Clin Exp Pharmacol Physiol 2000;27:323–329. 142 Malagon MM, Luque RM, Ruiz-Guerrero E, Rodriguez-Pacheco F, Garcia-Navarro S, Casanueva FF, Gracia-Navarro F, Castano JP: Intracellular signaling mechanisms mediating ghrelin-stimulated growth hormone release in somatotropes. Endocrinology 2003;144:5372–5380. 143 Glavaski-Joksimovic A, Jeftinija K, Scanes CG, Anderson LL, Jeftinija S: Stimulatory effect of ghrelin on isolated porcine somatotropes. Neuroendocrinology 2003; 77: 367–379. 144 Rodriguez-Pacheco F, Luque RM, GarciaNavarro S, Gracia-Navarro F, Castano JP, Malagon MM: Ghrelin induces growth hormone secretion via nitric oxide/cGMP signaling. Ann NY Acad Sci 2005; 1040: 452–453. 145 Han XF, Zhu YL, Hernandez M, Keating DJ, Chen C: Ghrelin reduces voltage-gated potassium currents in GH3 cells via cyclic GMP pathways. Endocrine 2005; 28: 217– 224. 146 Kato M, Sakuma Y: The effect of GHRP-6 on the intracellular Na+ concentration of rat pituitary cells in primary culture. J Neuroendocrinol 1999;11:795–800.

147 Kohno D, Gao HZ, Muroya S, Kikuyama S, Yada T: Ghrelin directly interacts with neuropeptide-Y-containing neurons in the rat arcuate nucleus: Ca 2+ signaling via protein kinase A and N-type channel-dependent mechanisms and cross-talk with leptin and orexin. Diabetes 2003;52:948–956. 148 Amar S, Mazella J, Checler F, Kitabgi P, Vincent JP: Regulation of cyclic GMP levels by neurotensin in neuroblastoma clone N1E115. Biochem Biophys Res Commun 1985;129:117–125. 149 Bozou JC, Rochet N, Magnaldo I, Vincent JP, Kitabgi P: Neurotensin stimulates inositol trisphosphate-mediated calcium mobilization but not protein kinase C activation in HT29 cells. Involvement of a G-protein. Biochem J 1989;264:871–878. 150 Bozou JC, de Nadai F, Vincent JP, Kitabgi P: Neurotensin, bradykinin and somatostatin inhibit cAMP production in neuroblastoma N1E115 cells via both pertussis toxin sensitive and insensitive mechanisms. Biochem Biophys Res Commun 1989; 161: 1144–1150. 151 Watson MA, Yamada M, Yamada M, Cusack B, Veverka K, Bolden-Watson C, Richelson E: The rat neurotensin receptor expressed in Chinese hamster ovary cells mediates the release of inositol phosphates. J Neurochem 1992;59:1967–1970. 152 Herrington J, Hille B: Growth hormone-releasing hexapeptide elevates intracellular calcium in rat somatotropes by two mechanisms. Endocrinology 1994; 135: 1100– 1108. 153 Adams EF, Lei T, Buchfelder M, Bowers CY, Fahlbusch R: Protein kinase C-dependent growth hormone-releasing peptides stimulate cyclic adenosine 3,5-monophosphate production by human pituitary somatotropinomas expressing gsp oncogenes: evidence for cross-talk between transduction pathways. Mol Endocrinol 1996;10:432–438. 154 Maudsley S, Martin B, Luttrell LM: The origins of diversity and specificity in G-protein-coupled receptor signaling. J Pharmacol Exp Ther 2005;314:485–494. 155 Mousseaux D, Le Gallic L, Ryan J, Oiry C, Gagne D, Fehrentz JA, Galleyrand JC, Martinez J: Regulation of ERK1/2 activity by ghrelin-activated growth hormone secretagogue receptor 1A involves a PLC/PKCvarepsilon pathway. Br J Pharmacol 2006;148: 350–365. 156 Holst B, Holliday ND, Bach A, Elling CE, Cox HM, Schwartz TW: Common structural basis for constitutive activity of the ghrelin receptor family. J Biol Chem 2004; 279:53806–53817. 157 Nanzer AM, Khalaf S, Mozid AM, Fowkes RC, Patel MV, Burrin JM, Grossman AB, Korbonits M: Ghrelin exerts a proliferative effect on a rat pituitary somatotroph cell line via the mitogen-activated protein kinase pathway. Eur J Endocrinol 2004; 151: 233–240.


161

158 Granata R, Settanni F, Trovato L, Destefanis S, Gallo D, Martinetti M, Ghigo E, Muccioli G: Unacylated as well as acylated ghrelin promotes cell survival and inhibit apoptosis in HIT-T15 pancreatic -cells. J Endocrinol Invest 2006;29:RC19–RC22. 159 Bond RA, Ijzerman AP: Recent developments in constitutive receptor activity and inverse agonism, and their potential for GPCR drug discovery. Trends Pharmacol Sci 2006;27:92–96. 160 Wang HJ, Geller F, Dempfle A, Schauble N, Friedel S, Lichtner P, Fontenla-Horro F, Wudy S, Hagemann S, Gortner L, Huse K, Remschmidt H, Bettecken T, Meitinger T, Schafer H, Hebebrand J, Hinney A: Ghrelin receptor gene: identification of several sequence variants in extremely obese children and adolescents, healthy normalweight and underweight students, and children with short normal stature. J Clin Endocrinol Metab 2004;89:157–162. 161 Pantel J, Legendre M, Cabrol S, Hilal L, Hajaji Y, Morisset S, Nivot S, Vie-Luton MP, Grouselle D, de Kerdanet M, Kadiri A, Epelbaum J, Le Bouc Y, Amselem S: Loss of constitutive activity of the growth hormone secretagogue receptor in familial short stature. J Clin Invest 2006; 116: 760– 768. 162 Holst B, Schwartz TW: Ghrelin receptor mutations – too little height and too much hunger. J Clin Invest 2006;116:637–641. 163 Holst B, Brandt E, Bach A, Heding A, Schwartz TW: Non-peptide and peptide growth hormone secretagogues act both as ghrelin receptor agonist and as positive or negative allosteric modulators of ghrelin signaling. Mol Endocrinol 2005; 19: 2400– 2411. 164 Binet V, Brajon C, Le Corre L, Acher F, Pin JP, Prezeau L: The heptahelical domain of GABA B2 is activated directly by CGP7930, a positive allosteric modulator of the GABA B receptor. J Biol Chem 2004; 279: 29085–29091. 165 Holst B, Elling CE, Schwartz TW: Metal ion-mediated agonism and agonist enhancement in melanocortin MC1 and MC4 receptors. J Biol Chem 2002; 277: 47662– 47670. 166 Schwartz TW, Holst B: Ago-allosteric modulation and other types of allostery in dimeric 7TM receptors. J Recept Signal Transduct Res 2006;26:107–128. 167 Hataya Y, Akamizu T, Takaya K, Kanamoto N, Ariyasu H, Saijo M, Moriyama K, Shimatsu A, Kojima M, Kangawa K, Nakao K: A low dose of ghrelin stimulates growth hormone (GH) release synergistically with GH-releasing hormone in humans. J Clin Endocrinol Metab 2001;86:4552–4255. 168 Bowers CY, Sartor AO, Reynolds GA, Badger TM: On the actions of the growth hormone-releasing hexapeptide, GHRP. Endocrinology 1991;128:2027–2035.

162

169 Wu D, Chen C, Katoh K, Zhang J, Clarke IJ: The effect of GH-releasing peptide-2 (GHRP-2 or KP-102) on GH secretion from primary cultured ovine pituitary cells can be abolished by a specific GH-releasing factor receptor antagonist. J Endocrinol 1994; 140:R9–R13. 170 Pandya N, DeMott-Friberg R, Bowers CY, Barkan AL, Jaffe CA: Growth hormone (GH)-releasing peptide-6 requires endogenous hypothalamic GH-releasing hormone for maximal GH stimulation. J Clin Endocrinol Metab 1998;83:1186–1189. 171 Bennett PA, Thomas GB, Howard AD, Feighner SD, van der Ploeg LH, Smith RG, Robinson IC: Hypothalamic growth hormone secretagogue-receptor expression is regulated by growth hormone in the rat. Endocrinology 1997;138:4552–4557. 172 Kamegai J, Wakabayashi I, Miyamoto K, Unterman TG, Kineman RD, Frohman LA: Growth hormone-dependent regulation of pituitary GH secretagogue receptor mRNA levels in the spontaneous dwarf Rat. Neuroendocrinology 1998;68:312–318. 173 Kineman RD, Kamegai J, Frohman LA: Growth hormone (GH)-releasing hormone (GHRH) and the GH secretagogue (GHS), L-692,585, differentially modulate rat pituitary GHS receptor and GHRH receptor messenger ribonucleic acid levels. Endocrinology 1999;140:3581–3586. 174 Lall S, Balthasar N, Carmignac D, Magoulas C, Sesay A, Houston P, Mathers K, Robinson I: Physiological studies of transgenic mice overexpressing growth hormone (GH) secretagogue receptor 1A in GH-releasing hormone neurons. Endocrinology 2004;145:1602–1611. 175 Mano-Otagiri A, Nemoto T, Sekino A, Yamauchi N, Shuto Y, Sugihara H, Oikawa S, Shibasaki T: Growth hormone-releasing hormone (GHRH) neurons in the arcuate nucleus (Arc) of the hypothalamus are decreased in transgenic rats whose expression of ghrelin receptor is attenuated: evidence that ghrelin receptor is involved in the upregulation of GHRH expression in the Arc. Endocrinology 2006;147:4093–4103. 176 Maggio R, Novi F, Scarselli M, Corsini GU: The impact of G-protein-coupled receptor hetero-oligomerization on function and pharmacology. FEBS J 2005; 272: 2939– 2946. 177 Cunha SR, Mayo KE: Ghrelin and growth hormone (GH) secretagogues potentiate GH-releasing hormone (GHRH)-induced cyclic adenosine 3,5-monophosphate production in cells expressing transfected GHRH and GH secretagogue receptors. Endocrinology 2002;143:4570–4582. 178 Jiang H, Betancourt L, Smith RG: Ghrelin amplifies dopamine signaling by cross-talk involving formation of growth hormone secretagogue receptor/dopamine receptor subtype-1 heterodimers. Endocrinology 2006;20:1772–1785.


179 Ferguson SS: Evolving concepts in G-protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev 2001;53:1–24. 180 Camina JP, Carreira MC, El Messari S, Llorens-Cortes C, Smith RG, Casanueva FF: Desensitization and endocytosis mechanisms of ghrelin-activated growth hormone secretagogue receptor 1a. Endocrinology 2004;145:930–940. 181 Parnot C, Miserey-Lenkei S, Bardin S, Corvol P, Clauser E: Lessons from constitutively active mutants of G-protein-coupled receptors. Trends Endocrinol Metab 2002;13: 336–343. 182 Wilbanks AM, Laporte SA, Bohn LM, Barak LS, Caron MG: Apparent loss-offunction mutant GPCRs revealed as constitutively desensitized receptors. Biochemistry 2002;41:11981–11989. 183 Miserey-Lenkei S, Parnot C, Bardin S, Corvol P, Clauser E: Constitutive internalization of constitutively active agiotensin II AT(1A) receptor mutants is blocked by inverse agonists. J Biol Chem 2002;277:5891– 5901. 184 Leterrier C, Bonnard D, Carrel D, Rossier J, Lenkei Z: Constitutive endocytic cycle of the CB1 cannabinoid receptor. J Biol Chem 2004;279:36013–36021. 185 Kim MS, Yoon CY, Park KH, Shin CS, Park KS, Kim SY, Cho BY, Lee HK: Changes in ghrelin and ghrelin receptor expression according to feeding status. Neuroreport 2003;14:1317–1320. 186 Tups A, Helwig M, Khorooshi RM, Archer ZA, Klingenspor M, Mercer JG: Circulating ghrelin levels and central ghrelin receptor expression are elevated in response to food deprivation in a seasonal mammal (Phodopus sungorus). J Neuroendocrinol 2004; 16: 922–928. 187 Wu R, Zhou M, Cui X, Simms HH, Wang P: Upregulation of cardiovascular ghrelin receptor occurs in the hyperdynamic phase of sepsis. Am J Physiol 2004; 287:H1296– H1302. 188 Ong H, McNicoll N, Escher E, Collu R, Deghenghi R, Locatelli V, Ghigo E, Muccioli G, Boghen M, Nilsson M: Identification of a pituitary growth hormone-releasing peptide receptor subtype by photoaffinity labeling. Endocrinology 1998;139:432–435. 189 Ong H, Bodart V, McNicoll N, Lamontagne D, Bouchard JF: Binding sites for growth hormone-releasing peptide. Growth Hormone IGF Res 1998;8:137–140. 190 Papotti M, Ghè C, Cassoni P, Catapano F, Deghenghi R, Ghigo E, Muccioli G: Growth hormone secretagogue binding sites in peripheral human tissues. J Clin Endocrinol Metab 2000;85:3803–3807.


191 Muccioli G, Broglio F, Valetto MR, Ghè C, Catapano F, Graziani A, Papotti M, Bisi G, Deghenghi R, Ghigo E: Growth hormonereleasing peptides and the cardiovascular system. Ann Endocrinol (Paris) 2000; 61: 27–31. 192 Bodart V, Bouchard JF, McNicoll N, Escher E, Carriere P, Ghigo E, Sejlitz T, Sirois MG, Lamontagne D, Ong H: Identification and characterization of a new growth hormonereleasing peptide receptor in the heart. Circ Res 1999;85:796–808. 193 Huang J, Zhou H, Mahavadi S, Sriwai W, Lyall V, Murthy KS: Signaling pathways mediating gastrointestinal smooth muscle contraction and MLC20 phosphorylation by motilin receptors. Am J Physiol 2005; 288:G23–G31. 194 Tremblay F, Perreault M, Klaman LD, Tobin JF, Smith E, Gimeno RE: Normal food intake and body weight in mice lacking the G-protein-coupled receptor GPR39. Endocrinology 2007;148:501–506. 195 Lauwers E, Landuyt B, Arckens L, Schoofs L, Luyten W: Obestatin does not activate orphan G-protein-coupled receptor GPR39. Biochem Biophys Res Commun 2006; 351: 21–25. 196 Bresciani E, Rapetti D, Dona F, Bulgarelli I, Tamiazzo L, Locatelli V, Torsello A: Obestatin inhibits feeding but does not modulate GH and corticosterone secretion in the rat. J Endocrinol Invest 2006; 29: RC16–RC18. 197 Seoane LM, Al-Massadi O, Pazos Y, Pagotto U, Casanueva FF: Central obestatin administration does not modify either spontaneous or ghrelin-induced food intake in rats. J Endocrinol Invest 2006; 29:RC13– RC15. 198 Morello JP, Bouvier M: Palmitoylation: a post-translational modification that regulates signalling from G-protein-coupled receptors. Biochem Cell Biol 1996; 74: 449– 457. 199 Brighton PJ, Szekeres PG, Willars GB: Neuromedin U and its receptors: structure, function, and physiological roles. Pharmacol Rev 2004;56:231–248. 200 Jethwa PH, Smith KL, Small CJ, Abbott CR, Darch SJ, Murphy KG, Seth A, Semjonous NM, Patel SR, Todd JF, Ghatei MA, Bloom SR: Neuromedin U partially mediates leptin-induced hypothalamo-pituitary adrenal (HPA) stimulation and has a physiological role in the regulation of the HPA axis in the rat. Endocrinology 2006; 147: 2886–2892. 201 Nogueiras R, Tovar S, Mitchell SE, Barrett P, Rayner DV, Dieguez C, Williams LM: Negative energy balance and leptin regulate neuromedin-U expression in the rat pars tuberalis. J Endocrinol 2006;190:545– 553.


202 Alevizos I, Mahadevappa M, Zhang X, Ohyama H, Kohno Y, Posner M, Gallagher GT, Varvares M, Cohen D, Kim D, Kent R, Donoff RB, Todd R, Yung CM, Warrington JA, Wong DT: Oral cancer in vivo gene expression profiling assisted by laser capture microdissection and microarray analysis. Oncogene 2001;20:6196–6204. 203 Yamashita K, Upadhyay S, Osada M, Hoque MO, Xiao Y, Mori M, Sato F, Meltzer SJ, Sidransky D: Pharmacologic unmasking of epigenetically silenced tumor suppressor genes in esophageal squamous cell carcinoma. Cancer Cell 2002;2:485–495. 204 Takahashi K, Furukawa C, Takano A, Ishikawa N, Kato T, Hayama S, Suzuki C, Yasui W, Inai K, Sone S, Ito T, Nishimura H, Tsuchiya E, Nakamura Y, Daigo Y: The neuromedin U-growth hormone secretagogue receptor 1b/neurotensin receptor 1 oncogenic signaling pathway as a therapeutic target for lung cancer. Cancer Res 2006; 66:9408–9419. 205 Vincent JP, Mazella J, Kitabgi P: Neurotensin and neurotensin receptors. Trends Pharmacol Sci 1999;20:302–309. 206 Poinot-Chazel C, Portier M, Bouaboula M, Vita N, Pecceu F, Gully D, Monroe JG, Maffrand JP, Le Fur G, Casellas P: Activation of mitogen-activated protein kinase couples neurotensin receptor stimulation to induction of the primary response gene Krox-24. Biochem J 1996;320:145–151. 207 Botto JM, Guillemare E, Vincent JP, Mazella J: Effects of SR 48692 on neurotensin-induced calcium-activated chloride currents in the Xenopus oocyte expression system: agonist-like activity on the levocabastinesensitive neurotensin receptor and absence of antagonist effect on the levocabastine insensitive neurotensin receptor. Neurosci Lett 1997;223:193–196. 208 Sarret P, Gendron L, Kilian P, Nguyen HM, Gallo-Payet N, Payet MD, Beaudet A: Pharmacology and functional properties of NTS2 neurotensin receptors in cerebellar granule cells. J Biol Chem 2002;277:36233– 36243. 209 Gendron L, Perron A, Payet MD, GalloPayet N, Sarret P, Beaudet A: Low-affinity neurotensin receptor (NTS2) signaling: internalization-dependent activation of extracellular signal-regulated kinases 1/2. Mol Pharmacol 2004;66:1421–1430. 210 Perron A, Sarret P, Gendron L, Stroh T, Beaudet A: Identification and functional characterization of a 5-transmembrane domain variant isoform of the NTS2 neurotensin receptor in rat central nervous system. J Biol Chem 2005; 280:10219–10227.

211 Chu KM, Chow KB, Leung PK, Lau PN, Chan CB, Cheng CH, Wise H: Overexpression of the truncated ghrelin receptor polypeptide attenuates the constitutive activation of phosphatidylinositol-specific phospholipase C by ghrelin receptors but has no effect on ghrelin-stimulated extracellular signal-regulated kinase 1/2 activity. Int J Biochem Cell Biol 2007; 39:752–764. 212 Jeffery PL, Herington AC, Chopin LK: Expression and action of the growth hormone-releasing peptide ghrelin and its receptor in prostate cancer cell lines. J Endocrinol 2002;172:R7–R11. 213 De Vriese C, Gregoire F, De Neef P, Robberecht P, Delporte C: Ghrelin is produced by the human erythroleukemic HEL cell line and involved in an autocrine pathway leading to cell proliferation. Endocrinology 2005;146:1514–1522. 214 Barzon L, Pacenti M, Masi G, Stefani AL, Fincati K, Palu G: Loss of growth hormone secretagogue receptor 1a and overexpression of type 1b receptor transcripts in human adrenocortical tumors. Oncology 2005;68:414–421. 215 Sato M, Nakahara K, Goto S, Kalya H, Miyazato M, Date Y, Nakazato M, Kangawa K, Murakami N: Effects of ghrelin and desacyl ghrelin on neurogenesis of the rat fetal spinal cord. Biochem Biophys Res Commun 2006;350:598–603. 216 Gauna C, Meyler FM, Janssen JA, Delhanty PJ, Abribat T, van Koetsveld P, Hofland LJ, Broglio F, Ghigo E, van der Lely AJ: Administration of acylated ghrelin reduces insulin sensitivity, whereas the combination of acylated plus unacylated ghrelin strongly improves insulin sensitivity. J Clin Endocrinol Metab 2004;89:5035–5042. 217 Broglio F, Gottero C, Prodam F, Gauna C, Muccioli G, Papotti M, Abribat T, Van Der Lely AJ, Ghigo E: Non-acylated ghrelin counteracts the metabolic but not the neuroendocrine response to acylated ghrelin in humans. J Clin Endocrinol Metab 2004;89: 3062–3065. 218 Gauna C, Delhanty PJ, van Aken MO, Janssen JA, Themmen AP, Hofland LJ, Culler M, Broglio F, Ghigo E, van der Lely AJ: Unacylated ghrelin is active on the INS-1E rat insulinoma cell line independently of the growth hormone secretagogue receptor type 1a and the corticotropin-releasing factor-2 receptor. Mol Cell Endocrinol 2006; 251:103–111. 219 Toshinai K, Yamaguchi H, Sun Y, Smith RG, Yamanaka A, Sakurai T, Date Y, Mondal MS, Shimbara T, Kawagoe T, Murakami N, Miyazato M, Kangawa K, Nakazato M: Des-acyl ghrelin induces food intake by a mechanism independent of the growth hormone secretagogue receptor. Endocrinology 2006;147:2306–2314.


163

220 Matsuda K, Miura T, Kaiya H, Maruyama K, Shimakura S, Uchiyama M, Kangawa K, Shioda S: Regulation of food intake by acyl and des-acyl ghrelins in the goldfish. Peptides 2006;27:2321–2325. 221 Ghè C, Cassoni P, Catapano F, Marrocco T, Deghenghi R, Ghigo E, Muccioli G, Papotti M: The antiproliferative effect of synthetic peptidyl GH secretagogues in human CALU-1 lung carcinoma cells. Endocrinology 2002;143:484–491. 222 Torsello A, Bresciani E, Rossoni G, Avallone R, Tulipano G, Cocchi D, Bulgarelli I, Deghenghi R, Berti F, Locatelli V: Ghrelin plays a minor role in the physiological control of cardiac function in the rat. Endocrinology 2003;136:1146–1152. 223 Deghenghi R: Structural requirements of growth hormone secretagogues; in Bercu BB, Walker RF (eds): Growth Hormone Secretagogues in Clinical Practice. New York, Dekker, 1998, pp 27–33. 224 Febbraio M, Haijar DP, Silverstein RL: CD36: A class B scavenger receptor involved in angiogenesis, atherosclerosis, inflammation, and lipid metabolism. J Clin Invest 2001;108:785–791.

164

225 Bodart V, Febbraio M, Demers A, McNicoll N, Pohankova P, Perreault A, Sejlitz T, Escher E, Silverstein RL, Lamontagne D, Ong H: CD36 mediates the cardiovascular action of growth hormone-releasing peptides in the heart. Circ Res 2002; 90: 844– 849. 226 Pettersson I, Muccioli G, Granata R, Deghenghi R, Ghigo E, Ohlsson C, Isgaard J: Natural (ghrelin) and synthetic (hexarelin) GH secretagogues stimulate H9c2 cardiomyocyte cell proliferation. J Endocrinol 2002;175:201–209. 227 Frascarelli S, Ghelardoni S, Ronca-Testoni S, Zucchi R: Effect of ghrelin and synthetic growth hormone secretagogues in normal and ischemic rat heart. Basic Res Cardiol 2003;98:401–405. 228 Demers A, McNicoll N, Febbraio M, Servant M, Marleau S, Silverstein R, Ong H: Identification of the growth hormone-releasing peptide binding site in CD36: a photoaffinity cross-linking study. Biochem J 2004;382:417–424. 229 Avallone R, Demers A, Rodrigue-Way A, Bujold K, Harb D, Anghel S, Wahli W, Marleau S, Ong H, Tremblay A: A growth hormone-releasing peptide that binds scavenger receptor CD36 and ghrelin receptor upregulates sterol transporters and cholesterol efflux in macrophages through a peroxisome proliferator-activated receptor-dependent pathway. Mol Endocrinol 2006; 20:3165–3178.


230 Marleau S, Harb D, Bujold K, Avallone R, Iken K, Wang Y, Demers A, Sirois MG, Febbraio M, Silverstein RL, Tremblay A, Ong H: EP-80317, a ligand of the CD36 scavenger receptor, protects apolipoprotein E-deficient mice from developing atherosclerotic lesions. FASEB J 2005;19:1869–1871. 231 Cummings DE, Foster-Shubert KE, Overduin J: Ghrelin and energy balance: focus on current controversies. Current Drug Targets 2006;6:153–169. 232 Wortley KE, del Rincon JP, Murray JD, Garcia K, Iida K, Thorner MO, Sleeman MW: Absence of ghrelin protects against early-onset obesity. J Clin Invest 2005; 115: 3573–3578. 233 Zigman JM, Nakano Y, Coppari R, Balthasar N, Marcus JN, Lee CE, Jones JE, Deysher AE, Waxman AR, White RD, Williams TD, Lachey JL, Seeley RJ, Lowell BB, Elmquist JK: Mice lacking ghrelin receptors resist the development of diet-induced obesity. J Clin Invest 2005;115:3564–3572. 234 Bowers CY: Historical milestones; in Ghigo E (ed): Ghrelin. Boston, Kluwer Academic, 2004, pp 1–13.



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



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


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,

GH and Metabolism in Lower Vertebrates


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.

167

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


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



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.

169

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


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.

References

1 Kawauchi H, Suzuki K, Yamazaki T, Moriyama S, Nozaki M, Yamaguchi K, Takahashi A, Youson J, Sower SA: Identification of a growth hormone in the sea lamprey, an extant representative of a group of the most ancient vertebrates. Endocrinology 2002; 143: 4916–4921. 2 Kawauchi H, Sower SA: The dawn and evolution of hormones in the adenohypophysis. Gen Comp Endocrinol 2006;148:3–14. 3 Rousseau K, Dufour: Phylogenetic evolution of the neuroendocrine control of growth hormone: contribution from teleosts. Cybium 2004;28:181–198. 4 Donaldson EM, Fagerlund UHM, Higgs DA, McBride JR: Hormonal enhancement of growth; in Hoar WS, Randall DJ, Brett JR (eds): Bioenergetic and Growth. New York, Academic Press, 1979, vol VIII, pp 455–597.

Rousseau/Dufour

5 McLean E, Donaldson EM: The role of growth hormone in the growth of poikilotherms; in Schreibman MP, Scanes CG, Pank PKT (eds): The Endocrinology of Growth, Development and Metabolism in Vertebrates. San Diego, Academic Press, 1993, pp 43–71. 6 Le Bail P-Y, Perez-Sanchez J, Yao H, Maisse G: Effect of GH treatment on salmonid growth: study of the variability of response; in Lahlou B, Vitiello P (eds): Aquaculture: Fundamental and Applied Research. Washington, American Geophysical Union, 1993, pp 173–197. 7 Devlin RH, Biagi CA, Yesaki TY, Smailus DE, Byatt JC: Growth of domesticated transgenic fish: a growth-hormone transgene boots the size of wild but not domesticated trout. Nature 2001;409:781–782. 8 Le Bail P-Y, Sumpter JP, Carragher JF, Mourot B, Niu PD, Weil C: Development and validation of a highly sensitive radioimmunoassay for Chinook salmon (Oncorhynchus tshawytscha) growth hormone. Gen Comp Endocrinol 1991;83:75–85. 9 Komourdjian MP, Idler DR: Hepatic mediation of hormonal and nutritional factors influencing the in vitro sulphur uptake by rainbow trout bone. Gen Comp Endocrinol 1978;36:33–39. 10 Mori I, Sakamoto T, Hirano T: Growth hormone (GH)-dependent hepatic GH receptors in the Japanese eel, Anguilla japonica : effects of hypophysectomy and GH injection. Gen Comp Endocrinol 1992; 85: 385– 391. 11 Johnston IA: Muscle development and growth: potential implication for flesh quality in fish. Aquaculture 1999;177:99–115. 12 Rousseau K, Huang Y-S, Le Belle N, Vidal B, Marchelidon J, Epelbaum J, Dufour S: Longterm inhibitory effects of somatostatin and insulin-like growth factor 1 on growth hormone release by serum-free primary culture of pituitary cells from European eel (Anguilla anguilla). Neuroendocrinology 1998; 67: 301–309. 13 Rousseau K, Le Belle N, Pichavant K, Marchelidon J, Chow BKC, Boeuf G, Dufour S: Pituitary growth hormone secretion in the turbot, a phylogenetically recent teleost, is regulated by a species-specific pattern of neuropeptides. Neuroendocrinology 2001; 74:375–385. 14 Markert JR, Higgs DA, Dye HM, MacQuarrie DW: Influence of bovine growth hormone on growth rate appetite, and food conversion of yearling coho salmon (Oncorhynchus kisutch) fed two diets of different composition. Can J Zool 1977;55:74–83. 15 Johnsson JJ, Björnsson BT: Growth hormone increases growth rate, appetite and dominance in juvenile rainbow trout, Oncorhynchus mykiss. Anim Behav 1994;48:177–186.


16 Gill JA, Sumpter JP, Donaldson EM, Dye HM, Souza L, Berg T, Wypych J, Langley K: Recombinant chicken and bovine growth hormone accelerates growth in aquacultured juvenile Pacific salmon (Oncorhynchus kisutch). Bio/Technology 1985;3:643. 17 Garber MM, DeYonge KG, Byatt JC, Lellis WA, Honeyfield DC, Bull RC, Schelling GT, Roeder RA: Dose-response effects of recombinant bovine somatotropin (Posilac) on growth performance and body composition of two-year-old rainbow trout (Oncorhynchus mykiss). J Anim Sci 1995; 73: 3216– 3222. 18 Björnsson BT: The biology of salmon growth hormone: from daylight to dominance. Fish Physiol Biochem 1997;17:9–24. 19 McLean E, Donaldson EM, Teskeredzic E, Souza LM: Growth enhancement following dietary delivery of recombinant porcine somatotropin to diploid and triploid of coho salmon (Oncorhynchus kisutch). Fish Physiol Biochem 1993;11:363–369. 20 Silverstein JT, Wolters WR, Shimizu M, Dickhoff WW: Bovine growth hormone treatment of channel catfish: strain and temperature effects on growth, plasma IGF1 levels, feed intake and efficiency and body composition. Aquaculture 2000;190:77–88. 21 Devlin RH, Byatt JC, McLean E, Yesaki TY, Krivi GG, Jaworski EG, Clarke WC, Donaldson EM: Bovine placental lactogen is a potent stimulator of growth and displays strong binding to hepatic receptor sites of coho salmon. Gen Comp Endocrinol 1994;95:31– 41. 22 Abrahams MV, Sutterlin A: The foraging and antipredator behaviour of growth-enhanced transgenic Atlantic salmon. Anim Behav 1999;58:933–942. 23 Jönsson E, Johansson V, Björnsson BT, Winberg S: Central nervous system actions of growth hormone on brain monoamine levels and behavior of juvenile rainbow trout. Horm Behav 2003;43:367–374. 24 Wilson RP, Poe WW, Nemetz TG, MacMillian JR: Effect of recombinant bovine growth hormone administration on growth and body composition of channel catfish. Aquaculture 1988;73:229–236. 25 Dodd MHI, Dodd JM: The biology of metamorphosis; in Lofts B (ed): Physiology of the Amphibia. New York, Academic Press, 1976, vol 3, pp 467–599. 26 Huang H, Brown DD: Overexpression of Xenopus laevis growth hormone stimulates growth of tadpoles and frogs. Proc Natl Acad Sci USA 2000;97:190–194. 27 Licht P, Hoyer H: Somatotropic effects of exogenous prolactin and growth hormone in juvenile lizards (Lacerta s. sicula). Gen Comp Endocrinol 1968;11:338–346. 28 Brown PS, Giuliano R, Hough G: Pituitary regulation of appetite and growth in the turtles Pseudomys scripta elegans and Chelydra serpentina. J Exp Zool 1974;187:205–215.

29 Duan C, Plisetskaya EM: Nutritional regulation of insulin-like growth factor I mRNA expression in salmon tissues. J Endocrinol 1993;139:243–252. 30 Sumpter JP, Le Bail P-Y, Pickering AD, Pottinger TG, Carragher JF: The effect of starvation on growth and plasma growth hormone concentrations of rainbow trout, Oncorhynchus mykiss. Gen Comp Endocrinol 1991;83: 94–102. 31 Pierce AL, Shimizu M, Beckman BR, Baker DM, Dickhoff WW: Time course of the GH/ IGF axis response to fasting and increased ration in Chinook salmon (Oncorhynchus tshawyscha). Gen Comp Endocrinol 2005; 140:192–202. 32 Gabillard J-C, Kamangar BB, Montserrat N: Coordinated regulation of the GH/IGF sytem genes during refeeding in rainbow trout (Oncorhynchus mykiss). J Endocrinol 2006; 191:15–24. 33 Marchelidon J, Schmitz M, Houdebine LM, Vidal B, Le Belle N, Dufour S: Development of a radioimmunoassay for European eel growth hormone and application to the study of silvering and experimental fasting. Gen Comp Endocrinol 1996;102:360–369. 34 Perez-Sanchez J, Marti-Palanca H, Kaushik SJ: Ration size and protein intake affect circulating growth hormone concentrations, hepatic growth hormone binding and plasma insulin-like growth factor-I in a marine teleost, the gilthead sea bream (Sparus aurata). J Nutr 1995;125:546–552. 35 Weber GM, Grau EG: Changes in serum concentrations and pituitary content of the two prolactins and growth hormone during the reproductive cycle in female tilapia, Oreochromis mossambicus, compared with changes during fasting. Comp Biochem Physiol 1999;124C:323–335. 36 Fox BK, Riley LG, Hirano T, Grau EG: Effects of fasting on growth hormone, growth hormone receptor and insulin-like growth factor-I axis in seawater-acclimated tilapia, Oreochromis mossambicus. Gen Comp Endocrinol 2006;148:340–347. 37 Thissen JP, Ketelslegers JM, Underwood LE: Nutritional regulation of the insulin-like growth factors. Endocr Rev 1994;15:80–101. 38 Sheridan MA: Regulation of lipid metabolism in poikilothermic vertebrates. Comp Biochem Physiol 1994;107B:495–508. 39 Leatherland JF, Nuti RN: Effects of bovine growth hormone on plasma free fatty acid concentrations and liver, muscle and carcass lipid content in rainbow trout, Salmo gairdneri, Richardson. J Fish Biol 1981; 19: 487– 498. 40 O’Connor PK, Reich B, Sheridan MA: Growth hormone stimulates hepatic lipid mobilization in rainbow trout, Oncorhynchus mykiss. J Comp Physiol 1993;163B:427– 431.


171

41 Sheridan MA: Effects of thyroxine, cortisol, growth hormone, and prolactin on lipid metabolism of coho salmon, Oncorhynchus kisutch, during smoltification. Gen Comp Endocrinol 1986;64:220–238. 42 McKeown BA, Leatherland JF, John TM: The effect of growth hormone and prolactin on the mobilization of free fatty acids and glucose in the kokanee salmon, Oncorhynchus nerka. Comp Biochem Physiol 1975; 50B: 425–430. 43 Foster AR, Houlihan DF, Gray C, Medale F, Fauconneau B, Kaushik SJ, Le Bail P-Y: The effects of ovine growth hormone on protein turnover in rainbow trout. Gen Comp Endocrinol 1991;82:111–120. 44 Fauconneau B, Mady M-P, Le Bail P-Y: Effect of growth hormone on muscle protein synthesis in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Fish Physiol Biochem 1996;15:49–56. 45 Inui Y, Ishioka H: In vivo and in vitro effects of growth hormone on the incorporation of [14C]leucine into protein of liver and muscle of the eel. Gen Comp Endocrinol 1985; 59: 295–300. 46 Volkoff H, Eykelbosh AJ, Peter RE: Role of leptin in the control of feeding of goldfish Carassius auratus: interactions with cholecystokinin, neuropeptide Y and orexin A, and modulation by fasting. Brain Res 2003; 972:90–109. 47 Nelson LE, Sheridan MA: Gastroenteropancreatic hormones and metabolism in fish. Gen Comp Endocrinol 2006;148:116–124. 48 Very NM, Sheridan MA: The role of somatostatins in the regulation of growth in fish. Fish Physiol Biochem 2004;27:217–226. 49 Sheridan MA, Kittilson JD: The role of somatostatins in the regulation of metabolism in fish. Comp Biochem Physiol 2004; 138B: 323–330. 50 Canosa LF, Cerda-Reverter JM, Peter RE: Brain mapping of three somatostatin encoding genes in the goldfish. J Comp Neurol 2004;474:43–57. 51 Holloway AC, Reddy PK, Sheridan MA, Leatherland JF: Diurnal rhythms of plasma growth hormone, somatostatin, thyroid hormones, cortisol and glucose concentrations in rainbow trout, Oncorhynchus mykiss, during progressive food deprivation. Biol Rhythm Res 1994;25:415–432. 52 Lin X, Volkoff H, Narnaware Y, Bernier N, Peyon P, Peter R: Brain regulation of feeding behaviour and food intake in fish. Comp Biochem Physiol 2000;126A:415–434. 53 Sheridan MA, Bern HA: Both somatostatin and the caudal neuropeptide, urotensin II, stimulate lipid mobilization from coho salmon liver incubated in vitro. Regul Pept 1986;14:333–344.

172

54 Sheridan MA, Plisetskaya EM, Bern HA, Gorbman A: Effects of somatostatin-25 and urotensin II on lipid and carbohydrate metabolism of coho salmon, Oncorhynchus kisutch. Gen Comp Endocrinol 1987; 66: 405–414. 55 Eilertson CD, Sheridan MA: Differential effects of somatostatin-14 and somatostatin-25 on carbohydrate and lipid metabolism in rainbow trout, Oncorhynchus mykiss. Gen Comp Endocrinol 1993;92:62–70. 56 Eilertson CD, Sheridan MA: Effects of somatostatin-25 on lipid mobilization from rainbow trout, Oncorhynchus mykiss, liver and adipose tissue incubated in vitro. Comparison with somatostatin-14. J Comp Physiol 1994;164B:256–260. 57 Kao Y-H, Youson JH, Holmes JA, Sheridan MA: Effects of somatostatins and insulin on blood glucose levels of larvae and metamorphosing landlocked sea lamprey, Petromyzon marinus. Gen Comp Endocrinol 1998; 111:177–185. 58 Plisetskaya EM, Sheridan MA, Mommsen TP: Metabolic changes in coho and Chinook salmon resulting from acute insufficiency in pancreatic hormones. J Exp Zool 1989; 249: 158–164. 59 Sheridan MA, Kao Y-H: Regulation of metamorphosis-associated changes in the lipid metabolism of selected vertebrates. Am Zool 1998;38:350–368. 60 Plisetskaya EM: Fatty acid levels in the blood of cyclostomes and fish. Environ Biol Fisches 1980;5:273–290. 61 Kao Y-H, Youson JH, Holmes JA, Sheridan MA: Effects of somatostatin on lipid metabolism of larvae and metamorphosing landlocked sea lamprey, Petromyzon marinus. Zool Sci 2001;18:1113–1116. 62 Epple A, Nibbio B, Trachtman MS: Effects of exogenous somatostatin and antisomatostatin on serum parameters of the American eel. Comp Biochem Physiol 1983; 74A:671– 675. 63 Plisetskaya EM, Pollock HG, Rouse JB, Hamilton JW, Kimmel JR, Andrews PC, Gorbman A: Characterization of coho salmon (Oncorhynchus kisutch) islet somatostatins. Gen Comp Endocrinol 1986;63:242–263. 64 Nelson LE, Sheridan MA: Insulin and growth hormone stimulate somatostatin receptor (SSTR) expression by inducing transcription of SSTR mRNAs and by up regulating cell surface SSTRs. Am J Physiol 2006b;291: R163–R169. 65 Carneiro NM, Eilertson CD, Sheridan MA: Lipid-stimulated somatostatin secretion in rainbow trout, Oncorhynchus mykiss. Fish Physiol Biochem 1996;15:447–452. 66 Eilertson CD, Sheridan MA: Pancreatic somatostatin-14 and somatostatin-25 release in rainbow trout is stimulated by glucose and arginine. Am J Physiol 1995; 269:R1017– 1023.


67 Ehrman MM, Melroe GT, Kittilson JD, Sheridan MA: The expression of preprosomatostatin II mRNAs in the Brockmann bodies of rainbow trout, Oncorhynchus mykiss, is regulated by glucose. Gen Comp Endocrinol 2000;118:150–160. 68 Melroe GT, Ehrman MM, Kittilson JD, Sheridan MA: Glucose regulates pancreatic preprosomatostatin I expression. FEBS Lett 2000;465:115–118. 69 Bluet-Pajot M, Epelbaum J, Gourdji D, Hammond C, Kordon C: Hypothalamic and hypophyseal regulation of growth hormone secretion. Cell Mol Neurobiol 1998; 18: 101– 123. 70 Vaudry D, Gonzalez BJ, Basille M, Yon L, Fournier A, Vaudry H: Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to functions. Pharmacol Rev 2000;52:269–324. 71 Montero M, Yon L, Kikuyama S, Dufour S, Vaudry H: Molecular evolution of the growth-hormone/pituitary adenylate cyclase-activating polypeptide family: functional implication in the regulation of growth hormone secretion. J Mol Endocrinol 2000; 25:157–168. 72 Montero M, Yon L, Rousseau K, Arimura A, Fournier A, Dufour S, Vaudry H: Distribution, characterization and growth hormone releasing activity of pituitary adenylate cyclase-activating polypeptide in the European eel, Anguilla anguilla. Endocrinology 1998;139:4300–4310. 73 Matsuda K, Maruyama K, Nakamachi T, Miura T, Uchiyama M, Shioda S: Inhibitory effects of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) on food intake in the goldfish, Carassius auratus. Peptides 2005; 26:1611–1616. 74 Morley JE, Horowitz M, Morley PMK, Flood JF: Pituitary adenylate cyclase-activating polypeptide (PACAP) reduces food intake in mice. Peptides 1992;13:1133–1135. 75 Tachibana T, Saito S, Tomonaga S, Takagi T, Saito E, Boswell T, Furuse M: Intracerebroventricular injection of vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibits feeding in the chick. Neurosci Lett 2003;339:203–206. 76 Matsuda K, Maruyama K, Nakamachi T, Miura T, Uchiyama M, Shioda S: Anorexigenic actions of adenylate cyclase-activating polypeptide (PACAP) in the goldfish; feeding-induced changes in the expression of mRNAs for PACAP and its receptors in the brain, and locomotor response to central injection. Neurosci Lett 2005;386:9–13. 77 Norrholm SD, Das M, Legradi G: Behavioral effects of local microinfusion of pituitary adenylate cyclase activating polypeptide (PACAP) into the paraventricular nucleus of the hypothalamus (PVN). Regul Pept 2005;128: 33–41.

Rousseau/Dufour

78 Gray SL, Cummings KJ, Jirik FR, Sherwood NM: Targeted disruption of the pituitary adenylate cyclase-activating polypeptide gene results in early postnatal death associated with dysfunction of lipid and carbohydrate metabolism. Mol Endocrinol 2001;15: 1739– 1747. 79 Pataki I, Adamik A, Jaszberenyi M, Macsai M, Telegdy G: Pituitary adenylate cyclaseactivating polypeptide induces hyperthermia in the rat. Neuropharmacology 2000;39: 1303–1308. 80 Gray SL, Yamaguchi N, Vencova P, Sherwood NM: Temperature-sensitive phenotype in mice lacking pituitary adenylate cyclase-activating polypeptide. Endocrinology 2002;143:3946–3954. 81 Lovejoy DA, Balment RJ: Evolution and physiology of the corticotrophin-releasing factor (CRF) family of neuropeptides in vertebrates. Gen Comp Endocrinol 1999; 115: 1–22. 82 Rousseau K, Le Belle N, Marchelidon J, Dufour S: Evidence that corticotrophin-releasing hormone acts as a growth-hormone releasing factor in a primitive teleost, the European eel (Anguilla anguilla). J Neuroendocrinol 1999;11:385–392. 83 Denver RJ, Licht P: Neuropeptides influencing in vitro pituitary hormone secretion in hatchling turtles. J Exp Zool 1989; 251: 306– 315. 84 Denver RJ, Licht P: Modulation of neuropeptide-stimulated pituitary hormone secretion in hatchling turtles. Gen Comp Endocrinol 1990;77:107–115. 85 Denver RJ, Licht P: Several hypothalamic peptides stimulate thyrotropin and growth hormone secretion by adult turtle pituitary glands. Comp Biochem Physiol 1991; 100A: 603–606. 86 Bernier NJ, Peter RE: The hypothalamic-pituitary-interrenal axis and the control of food intake in teleost fish. Comp Biochem Physiol 2001;129B:639–644. 87 De Pedro N, Alonso-Gomez AL, Gancedo B, Delgado MJ, Alonso-Bedate M: Role of corticotrophin-releasing factor (CRF) as a food intake regulator in goldfish. Physiol Behav 1993;53:517–520. 88 Bernier NJ, Peter RE: Appetite-suppressing effects of urotensin I and corticotrophin-releasing hormone in goldfish (Carassius auratus). Neuroendocrinology 2001;73:248–260. 89 Negri L, Noviello L, Noviello V: Effects of sauvagine, urotensin I and CRF on food intake in rats. Peptides 1985;6:53–57. 90 De Pedro N, Alonso-Gomez AL, Gancedo B, Valenciano AI, Delgado MJ, Alonso-Bedate M: Effect of -helical-crf(9–41) on feeding in goldfish: involvement of cortisol and catecholamines. Behav Neurosci 1997;111:398– 403.


91 Duval H, Rousseau K, Elies G, Le Bail P-Y, Dufour S, Boeuf G, Boujard D: Cloning, characteization and comparative activity of turbot IGF-I and IGF-II. Gen Comp Endocrinol 2002;126:269–278. 92 Duan C: Nutritional and developmental regulation of insulin-like growth factors in fish. J Nutr 1998;128:306S–314S. 93 Niu PD, Perez-Sanchez J, Le Bail PY: Development of a protein binding assay for teleost insulin-like growth factor (IGF)-like: relationship between growth hormone (GH) and IGF-like in the blood of rainbow trout (Oncorhynchus mykiss). Fish Physiol Biochem 1993;11:381–391. 94 Moriyama S, Swanson P, Nishi M, Takahashi A, Kawauchi H, Dickhoff WW, Plisetskaya EM: Development of homologous radioimmunoassay for coho salmon insulin-like growth factor-I. Gen Comp Endocrinol 1994;96:149–161. 95 Uchida K, Kajimura S, Riley LG, Hirano T, Aida K, Grau EG: Effects of fasting on growth hormone/insulin-like growth factor I axis in the tilapia, Oreochromis mossambicus. Comp Biochem Physiol 2003;134: 429–439. 96 Small BC, Peterson BC: Establishment of a time-resolved fluoroimmunoassay for measuring plasma insulin-like factor I (IGF-I) in fish: effect of fasting on plasma concentrations and tissue mRNA expression of IGF-I and growth hormone (GH) in channel catfish (Ictalurus punctatus). Domest Anim Endocrinol 2005; 28: 202– 215. 97 Duan C, Hirano T: Effects of insulin-like growth factor-I and insulin on the in vitro uptake of sulphate by eel branchial cartilage: evidence for the presence of independent hepatic and prancreatic sulfation factors. J Endocrinol 1992;133:211–219. 98 Matthews SJ, Kinhult AK, Hoeben P, Sara VR, Anderson TA: Nutritional regulation of insulin-like growth factor mRNA expression in barramundi, Lates calcarifer. J Mol Endocrinol 1997;18:273–276. 99 Pedroso FL, de Jesus-Ayson EGT, Cortado HH, Hyodo S, Ayson FG: Changes in mRNA expression of grouper (Epinephelus coiodes) growth hormone and insulin-like growth factor I in response to nutritional status. Gen Comp Endocrinol 2006; 145: 237–246. 100 Gray ES, Kelly KM, Law S, Tsai R, Young G, Bern HA: Regulation of hepatic growth hormone receptors in coho salmon (Oncorhynchus kisutch). Gen Comp Endocrinol 1992;88:243–252. 101 Fukada H, Ozaki Y, Pierce PL, Adachi S, Yamauchi K, Hara A, Swanson P, Dickhoff WW: Salmon growth hormone receptor: molecular cloning, ligand specificity, and response to fasting. Gen Comp Endocrinol 2004;139:61–71.

102 Deng L, Zhang WM, Lin HR, Cheng CHK: Effects of food deprivation on expression of growth hormone receptor and proximate composition in liver of black seabream Acanthopagrus schlegeli. Comp Biochem Physiol 2004;137:421–432. 103 Duan C, Plisetskaya EM, Dickhoff WW: Expression of insulin-like growth factor I in normally and abnormally developing coho salmon (Oncorhynchus kisutch). Endocrinology 1995;136:446–452. 104 De Luze A, Leloup J: Fish growth hormone enhances peripheral conversion of thyroxine to triiodothyronine in the eel (Anguilla anguilla L.). Gen Comp Endocrinol 1984; 56:308–312. 105 Rousseau K, Le Belle N, Sbaihi M, Marchelidon J, Schmitz M, Dufour S: Evidence for a negative feedback in the control of eel growth hormone by thyroid hormones. J Endocrinol 2002;175:605–613. 106 Larsen DA, Beckman BR, Dickhoff WW: The effect of temperature and fasting during the winter on metabolic stores and endocrine physiology (insulin, insulin-like growth factor 1 and thyroxine) of coho salmon, Oncorhynchus kisutch. Gen Comp Endocrinol 2001;123:308–323. 107 Leatherland JF, Farbridge KJ: Chronic fasting reduces the response of the thyroid to growth hormone and TSH, and alters the GH-related changes in hepatic 5-monodeiodinase activity in rainbow trout, Oncorhynchus mykiss. Gen Comp Endocrinol 1992;87:342–353. 108 De Pedro N, Delgado MJ, Gancedo B, Alonso-Bedate M: Changes in glucose, glycogen, thyroid activity and hypothalamic catecholamines in tench by starvation and refeeding. J Comp Physiol 2003; 173: 475– 481. 109 Norris DO: Depression of growth following radiothyroidectomy of larval Chinook salmon and steelhead trout. Trans Am Fish Soc 1969;98:104–106. 110 Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K: Ghrelin is a growthhormone-releasing acylated peptide from stomach. Nature 1999;402:656–660. 111 Palyha OC, Feighner SD, Tan CP, McKee KK, Hreniuk DL, Gao YD, Schleim KD, Yang L, Morriello GJ, Nargund R, Patchett AA, Howard AD, Smith RG: Ligand activation domain of human orphan growth hormone (GH) secretagogue receptor (GHS-R) conserved from pufferfish to humans. Mol Endocrinol 2000;14:160–169. 112 Chan CB, Cheng CH: Identification and functional characterization of two alternatively spliced growth hormone secretagogue receptor transcripts from the pituitary of black seabream, Acanthopagrus schlegeli. Mol Cell Endocrinol 2004;214:81– 95.


173

113 Unniappan S, Lin X, Cervini L, Rivier J, Kaiya H, Kangawa K, Peter RE: Goldfish ghrelin: molecular characterization of the complementary deoxyribonucleic acid, partial gene structure, and evidence for its stimulatory role in food intake. Endocrinology 2002;143:4143–4146. 114 Kaiya H, Kojima M, Hosoda H, Riley LG, Hirano T, Grau EG, Kangawa K: Amidated fish ghrelin: purification, cDNA cloning in the Japanese eel and its biological activity. J Endocrinol 2003;176:415–423. 115 Kaiya H, Kojima M, Hosoda H, Riley LG, Hirano T, Grau EG, Kangawa K: Identification of tilapia ghrelin and its effects on growth hormone and prolactin release in the tilapia, Oreochromis mossambicus. Comp Biochem Physiol B 2003;135: 421– 429. 116 Parhar IS, Sato H, Sakuma Y: Ghrelin gene in cichlid fish is modulated by sex and development. Biochem Biophys Res Commun 2003;305:169–175. 117 Kaiya H, Kojima M, Hosoda H, Moriyama S, Takahashi A, Kawauchi H, Kangawa K: Peptide purification, complementary deoxyribonucleic acid (DNA) and genomic DNA cloning, and functional characterization of ghrelin in rainbow trout. Endocrinology 2003;144:5215–5226. 118 Kaiya H, Small BC, Bilodeau AL, Shepherd BS, Kojima M, Hosoda H, Kangawa K: Purification, cDNA cloning, and characterization of ghrelin in channel catfish, Ictalurus punctatus. Gen Comp Endocrinol 2005;143:201–210. 119 Unniappan S, Canosa LF, Peter RE: Orexigenic actions of ghrelin in goldfish: feeding-induced changes in brain and gut mRNA expression and serum levels, and responses to central and peripheral injections. Neuroendocrinology 2004; 79: 100– 108. 120 Matsuda K, Miura T, Kaiya H, Maruyama K, Uchiyama M, Kangawa K, Shioda S: Stimulatory effect of n-octanoylated ghrelin on locomotor activity in the goldfish, Carassius auratus. Peptides 2006; 27: 1335– 1340. 121 Nieminen P, Mustonen AM, Hyvarinen H: Fasting reduces plasma leptin- and ghrelinimmunoreactive peptide concentrations of the burbot (Lota lota) at 2 degrees C but not at 10 degrees C. Zool Sci 2003; 20: 1109– 1115.

174

122 Kaiya H, Tsukada T, Yuge S, Mondo H, Kangawa K, Takei Y: Identification of eel ghrelin in plasma and stomach by radioimmunoassay and histochemistry. Gen Comp Endocrinol 2006;148:375–382. 123 Hosoda H, Kojima M, Matsuo H, Kangawa K: Ghrelin and des-acyl ghrelin: two major forms of rat ghrelin peptide in gastrointestinal tissue. Biochem Biophys Res Commun 2000;279:909–913. 124 Date Y, Kojima M, Hosoda H, Sawaguchi A, Mondal MS, Suganuma T, Matsukura S, Kangawa K, Nakazato M: Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology 2000; 141: 4255–4261. 125 Riley LG, Hirano T, Grau EG: Rat ghrelin stimulates growth hormone and prolactin release in the tilapia, Oreochromis mossambicus. Zool Sci 2002;19:797–800. 126 Unniappan S, Peter RE: In vitro and in vivo effects of ghrelin on luteinizing hormone and growth hormone release in goldfish. Am J Physiol 2004;286:R1093–R1101. 127 Unniappan S, Peter RE: Structure, distribution and physiological functions of ghrelin in fish. Comp Biochem Physiol 2005; 140A:396–408. 128 Kaiya H, Kojima M, Hosoda H, Koda A, Yamamoto K, Kitajima Y, Matsumoto M, Minamitake Y, Kikuyama S, Kangawa K: Bullfrog ghrelin is modified by n-octanoic acid at its third threonine residue. J Biol Chem 2001;276:40441–40448. 129 Kaiya H, Sakata I, Kojima M, Hosoda H, Sakai T, Kangawa K: Structural determination and histochemical localization of ghrelin in the red-eared slider turtle, Trachemys scripta elegans. Gen Comp Endocrinol 2004;138:50–57. 130 Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM: Positional cloning of the mouse ob gene and its human homologue. Nature 1994;372:425–432. 131 Johnson RM, Johnson TM, Londraville RL: Evidence for leptin expression in fishes. J Exp Zool 2000;286:718–724. 132 Muruzabal FJ, Fruhbeck G, Gomez-Ambrosi J, Archanco M, Burrell MA: Immunocytochemical detection of leptin in nonmammalian vertebrate stomach. Gen Comp Endocrinol 2002;128:149–152. 133 Mustonen AM, Nieminen P, Hyvarinen H: Leptin, ghrelin and energy metabolism of the spawning burbot (Lota lota, L.). J Exp Zool 2002;293:119–126.


134 Ganga R, Bell JG, Montero D, Robaina L, Caballero MJ, Izquierdo MS: Effect of dietary lipids on plasma fatty acid profiles and prostaglandin and leptin production in gilthead seabream (Sparus aurata). Comp Biochem Physiol [B] 2005;142:410–418. 135 Vegusdal A, Sundvold H, Gjoen T, Ruyter B: An in vitro method for studying the proliferation and differentiation of Atlantic salmon preadipocytes. Lipids 2003;38:289– 296. 136 Kurokawa T, Uji S, Suzuki T: Identification of cDNA coding for a homologue to mammalian leptin from pufferfish, Takifugu rubripes. Peptides 2005;26:745–750. 137 Huising MO, Geven EJW, Kruiswijk CP, Nabuurs SB, Stolte EH, Spanings FAT, Verburg-van Kemenade BML, Flik G: Increased leptin expression in common carp (Cyprinus carpio) after food intake but not after fasting or feeding to satiation. Endocrinology 2006;147:5786–5797. 138 Paolucci M, Rocco M, Varrichio E: Leptin presence in plasma, liver and fat bodies in the lizard Podarcis sicula : fluctuations throughout the reproductive cycle. Life Sci 2001;69:2399–2408. 139 Silverstein JT, Plisetskaya EM: The effects of NPY and insulin on food intake regulation in fish. Am Zool 2000;40:296–308. 140 Baker DM, Larsen DA, Swanson P, Dickhoff WW: Long-term peripheral treatment of immature coho salmon (Oncorhynchus kisutch) with human leptin has no clear physiologic effect. Gen Comp Endocrinol 2000;118:134–138. 141 Londraville RL, Duvall CS: Murine leptin injections increase intracellular fatty-acid binding protein in green sunfish (Lepomis cyanellus). Gen Comp Endocrinol 2002; 129:56–62. 142 De Pedro N, Martinez-Alvarez R, Delgado MJ: Acute and chronic leptin reduces food intake and body weight in goldfish (Carassius auratus). J Endocrinol 2006; 188: 513– 520. 143 Niewiarowski PH, Balk ML, Londraville RL: Phenotypic effects of leptin in an ectotherm: a new tool to study the evolution of life histories and endothermy? J Exp Biol 2000;203:295–300. 144 Paolucci M, Buono S, Sciarrillo R, Putti R: Effects of leptin administration on the endocrine pancreas and liver in the lizard, Podarcis sicula. J Exp Zool 2006; 305A:383– 395.

Rousseau/Dufour


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



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


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].


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


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].

References 1 Bagdade JD, Subbaiah P, Albers JJ: Effects of serum from oral contraceptive users on the metabolism of low density lipoprotein. Am J Med Sci 1987;294:225–230. 2 Bronson F, Marsteller F: Effect of short-term food deprivation on reproduction in female mice. Biol Reprod 1985; 33:660–667. 3 Tsukamura H, Maeda K: Non-metabolic and metabolic factors causing lactational anestrus: rat models uncovering the neuroendocrine mechanism underlying the sucklinginduced changes in the mother. Prog Brain Res 2001;133:187–205.

Neuropeptide Signaling in Metabolism and Reproduction

4 Foster D, Ebling FJ, Micka A, Vannerson L, Bucholtz D, Wood R, Suttie J, Fenner DE: Metabolic interfaces between growth and reproduction. I. Nutritional modulation of gonadotropin, prolactin, and growth hormone secretion in the growth-limited female lamb. Endocrinology 1989; 125:342–350. 5 Schneider J: Energy balance and reproduction. Physiol Behav 2004;81:289–317. 6 Krasnow SM, Steiner RA: Physiological mechanisms integrating metabolism and reproduction; in Neill JD (ed): Knobil and Neill’s Physiology of Reproduction, ed 3. St Louis, Elsevier, 2006, pp 2553–2625.

7 Cheung C, Clifton D, Steiner R: Perspectives on leptin’s role as a metabolic signal for the onset of puberty. Front Horm Res 2000; 26: 87–105. 8 Cunningham M, Clifton DK, Steiner RA: Leptin’s actions on the reproductive axis: perspectives and mechanisms. Biol Reprod 1999;60:216–222. 9 Henry B, Goding J, Tilbrook A, Dunshea FR, Clarke IJ: Intracerebroventricular infusion of leptin elevates the secretion of luteinising hormone without affecting food intake in long-term food-restricted sheep, but increases growth hormone irrespective of body weight. J Endocrinol 2001;168:67–77.


179

10 Ingalls AM, Dickie M, Snell G: Obese, a new mutation in the house mouse. J Hered 1950; 41:317–318. 11 Swerdloff R: Physiological control of puberty. Med Clin North Am 1978;62:351–366. 12 Swerdloff R, Batt R, Bray G: Reproductive hormonal function in the genetically obese (ob/ob) mouse. Endocrinology 1976; 98: 1359–1364. 13 Barash I, Cheung CC, Weigle DS, Ren H, Kabigting E, Kuijper J, Clifton D, Steiner RA: Leptin is a metabolic signal to the reproductive system. Endocrinology 1996; 137: 3144– 3147. 14 Chehab F, Lim M, Lu R: Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nat Genet 1996;12:318–320. 15 Mounzih K, Lu R, Chehab FF: Leptin treatment rescues the sterility of genetically obese ob/ob males. Endocrinology 1997;138:1190– 1193. 16 Finn P, Cunningham M, Pau K, Spies H, Clifton D, Steiner R: The stimulatory effect of leptin on the neuroendocrine reproductive axis of the monkey. Endocrinology 1998;139: 4652–4662. 17 Hakansson M, Brown H, Ghilardi N, Skoda R, Meister B: Leptin receptor immunoreactivity in chemically defined target neurons of the hypothalamus. J Neurosci 1998; 18: 559–572. 18 Hileman S, Schillo KK, Hall J: Effects of acute, intracerebroventricular administration of insulin on serum concentrations of luteinizing hormone, insulin, and glucose in ovariectomized lambs during restricted and ad libitum feed intake. Biol Reprod 1993; 48: 117–124. 19 Miller D, Blache D, Martin GB: The role of intracerebral insulin in the effect of nutrition on gonadotrophin secretion in mature male sheep. J Endocrinol 1995; 147:321–329. 20 Bruning J, Gautam D, Burks D, Gillette J, Schubert M, Orban P, Klein R, Krone W, Muller-Wieland D, Kahn C: Role of brain insulin receptor in control of body weight and reproduction. Science 2000;289:2122–2125. 21 Blades RA, Bryant K, Whitehead S: Feedback effects of steroids and gonadotrophin control in adult rats with streptozotocin-induced diabetes mellitus. Diabetologia 1985; 28:348–354. 22 Gentry R, Wade G, Blaustein J: Binding of [3H]estradiol by brain cell nuclei and female rat sexual behavior: inhibition by experimental diabetes. Brain Res 1977; 135: 135– 146. 23 Karkanias G, Morales J, Li C: Deficits in reproductive behavior in diabetic female rats are due to hypoinsulinemia rather than hyperglycemia. Horm Behav 1997;32:19–29. 24 Katayama S, Brownscheidle C, Wootten V, Lee J, Shimaoka K: Absent or delayed preovulatory luteinizing hormone surge in experimental diabetes mellitus. Diabetes 1984; 33:324–327.

180

25 Kirchick H, Keyes P, Frye B: Etiology of anovulation in the immature alloxan-diabetic rat treated with pregnant mare’s serum gonadotropin: absence of the preovulatory luteinizing hormone surge. Endocrinology 1978;102:1867–1873. 26 Kovacs P, Parlow A, Karkanias G: Effect of centrally administered insulin on gonadotropin-releasing hormone neuron activity and luteinizing hormone surge in the diabetic female rat. Neuroendocrinology 2002; 76: 357–365. 27 Lawrence A, Contopoulos A: Reproductive performance in the alloxan diabetic female rat. Acta Endocrinol (Copenh) 1960;33:175– 184. 28 Bucholtz D, Chiesa A, Pappano WN, Nagatani S, Tsukamura H, Maeda K, Foster D: Regulation of pulsatile luteinizing hormone secretion by insulin in the diabetic male lamb. Biol Reprod 2000; 62:1248–1255. 29 Dong Q, Lazarus R, Wong L, Vellios M, Handelsman D: Pulsatile LH secretion in streptozotocin-induced diabetes in the rat. J Endocrinol 1991;131:49–55. 30 Steger R, Kienast SG: Effect of continuous versus delayed insulin replacement on sex behavior and neuroendocrine function in diabetic male rats. Diabetes 1990; 39: 942– 948. 31 Dellovade T, Zhu Y, Krey L, Pfaff D: Thyroid hormone and estrogen interact to regulate behavior. Proc Natl Acad Sci USA 1996; 93: 12581–12586. 32 Morgan MA, Dellovade T, Pfaff D: Effect of thyroid hormone administration on estrogen-induced sex behavior in female mice. Horm Behav 2000;37:15–22. 33 Ortega E, Rodriguez E, Ruiz E, Osorio C: Activity of the hypothalamo-pituitary ovarian axis in hypothyroid rats with or without triiodothyronine replacement. Life Sci 1990;46: 391–395. 34 Chandrasekhar Y, Holland MK, D’Occhio M, Setchell B: Spermatogenesis, seminal characteristics and reproductive hormone levels in mature rams with induced hypothyroidism and hyperthyroidism. J Endocrinol 1985;105:39–46. 35 Vriend J, Bertalanffy F, Ralcewicz T: The effects of melatonin and hypothyroidism on estradiol and gonadotropin levels in female Syrian hamsters. Biol Reprod 1987; 36: 719– 728. 36 Singh U, Krishna A, Bhatnagar K: Seasonal changes in thyroid activity in the female sheath-tailed bat, Taphozous longimanus (Chiroptera: Emballonuridae). Acta Biol Hung 2002;53:267–278. 37 Rodriguez-Pacheco F, Martinez-Fuentes AJ, Tovar S, Pinilla L, Tena-Sempere M, Dieguez C, Castaño JP, Malagon MM: Regulation of pituitary cell function by adiponectin. Endocrinology 2007; 148:401–410.


38 Navarro V, Fernandez-Fernandez R, Nogueiras R, Vigo E, Tovar S, Chartrel N, Le Marec O, Leprince J, Aguilar E, Pinilla L, Dieguez C, Vaudry H, Tena-Sempere M: Novel role of 26RFa, a hypothalamic RFamide orexigenic peptide, as putative regulator of the gonadotropic axis. J Physiol 2006;573:237–249. 39 Yang Y, Zhou L, Liu S, Tang J, Li F, Li R, Song H, Chen M: Expression of feeding-related peptide receptors mRNA in GT1–7 cell line and roles of leptin and orexins in control of GnRH secretion. Acta Pharmacol Sin 2005; 26:976–981. 40 I’Anson H, Sundling L, Roland S, Ritter S: Immunotoxic destruction of distinct catecholaminergic neuron populations disrupts the reproductive response to glucoprivation in female rats. Endocrinology 2003; 144: 4325–4331. 41 Fraley G, Scarlett J, Shimada I, Teklemichael DN, Acohido B, Clifton DK, Steiner R: Effects of diabetes and insulin on the expression of galanin-like peptide in the hypothalamus of the rat. Diabetes 2004; 53: 1237–1242. 42 Jureus A, Cunningham M, Li D, Johnson LL, Krasnow SM, Teklemichael DN, Clifton DK, Steiner RA: Distribution and regulation of galanin-like peptide in the hypothalamus of the mouse. Endocrinology 2001; 142: 5140– 5144. 43 Kumano S, Matsumoto H, Takatsu Y, Noguchi J, Kitada C, Ohtaki T: Changes in hypothalamic expression levels of galanin-like peptide in rat and mouse models support that it is a leptin-target peptide. Endocrinology 2003;144:2634–2643. 44 Shen J, Gundlach A: Galanin-like peptide mRNA alterations in arcuate nucleus and neural lobe of streptozotocin-diabetic and obese Zucker rats. Further evidence for leptin-dependent and independent regulation. Neuroendocrinology 2004; 79:327–337. 45 Gottsch M, Clifton D, Steiner R: Galaninlike peptide as a link in the integration of metabolism and reproduction. Trends Endocrinol Metab 2004;15:215–221. 46 Kauffman A, Buenzle J, Fraley G, Rissman EF: Effects of galanin-like peptide on locomotion, reproduction, and body weight in female and male mice. Horm Behav 2005;48: 141–151. 47 Krasnow S, Fraley G, Schuh S, Baumgartner J, Clifton DK, Steiner R: A role for galaninlike peptide in the integration of feeding, body weight regulation, and reproduction in the mouse. Endocrinology 2003; 144: 813– 822. 48 Matsumoto H, Noguchi J, Takatsu Y, Horikoshi Y, Kumano S, Ohtaki T, Kitada C, Itoh T, Onda H, Nishimura O, Fujino M: Stimulation effect of galanin-like peptide on luteinizing hormone-releasing hormone-mediated luteinizing hormone secretion in male rats. Endocrinology 2001; 142:3693–3696.


49 Morton G, Schwartz M: The NPY/AgRP neuron and energy homeostasis. Int J Obes Relat Metab Disord 2001; 25(suppl 5):S56– S62. 50 Akabayashi A, Wahlestedt C, Alexander J, Leibowitz S: Specific inhibition of endogenous neuropeptide Y synthesis in arcuate nucleus by antisense oligonucleotides suppresses feeding behavior and insulin secretion. Brain Res Mol Brain Res 1994; 21: 55– 61. 51 Marin-Bivens CL, Kalra S, Olster D: Intraventricular injection of neuropeptide Y antisera curbs weight gain and feeding, and increases the display of sexual behaviors in obese Zucker female rats. Regul Pept 1998; 75/76:327–334. 52 Ahima R, Prabakaran D, Mantzoros C, Qu D, Lowell B, Maratos-Flier E, Flier J: Role of leptin in the neuroendocrine response to fasting. Nature 1996;382:250–252. 53 Schwartz M, Baskin D, Bukowski T, Kuijper J, Foster D, Lasser G, Prunkard D, Porte DJ, Woods S, Seeley R, Weigle D: Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice. Diabetes 1996; 45: 531–535. 54 Mercer J, Hoggard N, Williams L, Lawrence C, Hannah L, Morgan P, Trayhurn P: Coexpression of leptin receptor and preproneuropeptide Y mRNA in arcuate nucleus of mouse hypothalamus. J Neuroendocrinol 1996; 8: 733–735. 55 Erickson J, Ahima R, Hollopeter G, Flier J, Palmiter R: Endocrine function of neuropeptide Y knockout mice. Regul Pept 1997; 70:199–202. 56 Erickson J, Hollopeter G, Palmiter R: Attenuation of the obesity syndrome of ob/ob mice by the loss of neuropeptide Y. Science 1996; 274:1704–1707. 57 Schwartz M, Sipols A, Marks J, Sanacora G, White J, Scheurink A, Kahn S, Baskin D, Woods S, Figlewicz D, Et A: Inhibition of hypothalamic neuropeptide Y gene expression by insulin. Endocrinology 1992; 130: 3608– 3616. 58 Sipols A, Baskin D, Schwartz M: Effect of intracerebroventricular insulin infusion on diabetic hyperphagia and hypothalamic neuropeptide gene expression. Diabetes 1995;44:147–151. 59 Kalra S, Crowley W: Norepinephrine-like effects of neuropeptide Y on LH release in the rat. Life Sci 1984;35:1173–1176. 60 Allen L, Kalra P, Crowley WR, Kalra S: Comparison of the effects of neuropeptide Y and adrenergic transmitters on LH release and food intake in male rats. Life Sci 1985; 37: 617–623. 61 Bauer-Dantoin AC, Urban J, Levine J: Neuropeptide Y gene expression in the arcuate nucleus is increased during preovulatory luteinizing hormone surges. Endocrinology 1992;131:2953–2958.

Neuropeptide Signaling in Metabolism and Reproduction

62 Sahu A, Crowley W, Kalra S: Hypothalamic neuropeptide-Y gene expression increases before the onset of the ovarian steroid-induced luteinizing hormone surge. Endocrinology 1994; 134:1018–1022. 63 Pierroz D, Gruaz N, D’Alieves V, Aubert ML: Chronic administration of neuropeptide Y into the lateral ventricle starting at 30 days of life delays sexual maturation in the female rat. Neuroendocrinology 1995;61:293–300. 64 Toufexis D, Kyriazis D, Woodside B: Chronic neuropeptide Y Y5 receptor stimulation suppresses reproduction in virgin female and lactating rats. J Neuroendocrinol 2002; 14:492–497. 65 Estrada K, Pompolo S, Morris M, Tilbrook A, Clarke I: Neuropeptide Y (NPY) delays the oestrogen-induced luteinizing hormone (LH) surge in the ovariectomized ewe: further evidence that NPY has a predominant negative effect on LH secretion in the ewe. J Neuroendocrinol 2003;15:1011–1020. 66 Xu M, Hill J, Levine JE: Attenuation of luteinizing hormone surges in neuropeptide Y knockout mice. Neuroendocrinology 2000; 72: 263–271. 67 Clark J, Kalra P, Kalra S: Neuropeptide Y stimulates feeding but inhibits sexual behavior in rats. Endocrinology 1985; 117: 2435– 2442. 68 Erickson J, Hollopeter G, Palmiter R: Attenuation of the obesity syndrome of ob/ob mice by the loss of neuropeptide Y. Science 1996; 274:1704–1707. 69 Hill J, Levine JE: Abnormal response of the neuropeptide Y-deficient mouse reproductive axis to food deprivation but not lactation. Endocrinology 2003; 144:1780–1786. 70 Bergendahl M, Wiemann J, Clifton DK, Huhtaniemi I, Steiner R: Short-term starvation decreases POMC mRNA but does not alter GnRH mRNA in the brain of adult male rats. Neuroendocrinology 1992; 56:913–920. 71 Koegler F, Grove K, Schiffmacher A, Smith M, Cameron J: Central melanocortin receptors mediate changes in food intake in the rhesus macaque. Endocrinology 2001; 142: 2586–2592. 72 Mizuno T, Kleopoulos S, Bergen H, Roberts JL, Priest C, Mobbs C: Hypothalamic proopiomelanocortin mRNA is reduced by fasting and [corrected] in ob/ob and db/db mice, but is stimulated by leptin. Diabetes 1998;47: 294–297. 73 Schwartz M, Seeley R, Woods S, Weigle D, Campfield L, Burn P, Baskin D: Leptin increases hypothalamic pro-opiomelanocortin mRNA expression in the rostral arcuate nucleus. Diabetes 1997;46:2119–2123. 74 Thornton J, Cheung CC, Clifton D, Steiner RA: Regulation of hypothalamic proopiomelanocortin mRNA by leptin in ob/ob mice. Endocrinology 1997; 138:5063–5066.

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.


181

87 Messager S, Chatzidaki E, Ma D, Hendrick A, Zahn D, Dixon J, Thresher R, Malinge I, Lomet D, Carlton MB, Colledge W, Caraty A, Aparicio S: Kisspeptin directly stimulates gonadotropin-releasing hormone release via G protein-coupled receptor 54. Proc Natl Acad Sci USA 2005;102:1761–1766. 88 Smith J, Cunningham M, Rissman EF, Clifton DK, Steiner RA: Regulation of Kiss1 gene expression in the brain of the female mouse. Endocrinology 2005; 146:3686–3692. 89 Smith J, Dungan H, Stoll E, Gottsch M, Braun R, Eacker SM, Clifton D, Steiner RA: Differential regulation of KiSS-1 mRNA expression by sex steroids in the brain of the male mouse. Endocrinology 2005;146:2976– 2984. 90 Han S, Gottsch ML, Lee K, Popa S, Smith J, Jakawich S, Clifton D, Steiner RA, Herbison A: Activation of gonadotropin-releasing hormone neurons by kisspeptin as a neuroendocrine switch for the onset of puberty. J Neurosci 2005;25:11349–11356.

182

91 Matsui H, Takatsu Y, Kumano S, Matsumoto H, Ohtaki T: Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat. Biochem Biophys Res Commun 2004;320:383–388. 92 Navarro VM, Castellano J, Fernandez-Fernandez R, Barreiro M, Roa J, Sanchez-Criado JE, Aguilar E, Dieguez C, Pinilla L, TenaSempere M: Developmental and hormonally regulated messenger ribonucleic acid expression of KiSS-1 and its putative receptor, GPR54, in rat hypothalamus and potent luteinizing hormone-releasing activity of KiSS-1 peptide. Endocrinology 2004; 145: 4565–4574. 93 Navarro VM, Castellano J, Fernandez-Fernandez R, Tovar S, Roa J, Mayen A, Barreiro M, Casanueva F, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M: Effects of KiSS-1 peptide, the natural ligand of GPR54, on follicle-stimulating hormone secretion in the rat. Endocrinology 2005; 146:1689–1697. 94 Navarro VM, Castellano J, Fernandez-Fernandez R, Tovar S, Roa J, Mayen A, Nogueiras R, Vazquez MJ, Barreiro M, Magni P, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M: Characterization of the potent luteinizing hormone-releasing activity of KiSS-1 peptide, the natural ligand of GPR54. Endocrinology 2005; 146:156–163.


95 Castellano J, Navarro V, Fernandez-Fernandez R, Nogueiras R, Tovar S, Roa J, Vazquez M, Vigo E, Casanueva F, Aguilar E, Pinilla L, Dieguez C, Tena-Sempere M: Changes in hypothalamic KiSS-1 system and restoration of pubertal activation of the reproductive axis by kisspeptin in undernutrition. Endocrinology 2005; 146:3917–3925. 96 Castellano J, Navarro VM, Fernandez-Fernandez R, Roa J, Vigo E, Pineda R, Dieguez C, Aguilar E, Pinilla L, Tena-Sempere M: Expression of hypothalamic KiSS-1 system and rescue of defective gonadotropic responses by kisspeptin in streptozotocin-induced diabetic male rats. Diabetes 2006; 55: 2602– 2610. 97 Smith J, Acohido B, Clifton D, Steiner R: KiSS-1 neurones are direct targets for leptin in the ob/ob mouse. J Neuroendocrinol 2006; 18:298–303.




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


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


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


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.


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).


187

References 1 Morley JE, Thomas DR, Wilson MM: Cachexia: pathophysiology and clinical relevance. Am J Clin Nutr 2006;83:735–743. 2 Plata-Salaman CR: Anorexia during acute and chronic disease. Nutrition 1996; 12: 69– 78. 3 Wong S, Pinkney J: Role of cytokines in regulating feeding behaviour. Curr Drug Targets 2004;5:251–263. 4 Hansen MK, Taishi P, Chen Z, Krueger JM: Cafeteria feeding induces interleukin-1␤ mRNA expression in rat liver and brain. Am J Physiol 1998;274:R1734–R1739. 5 Wisse BE, Ogimoto K, Morton GJ, Wilkinson CW, Frayo RS, Cummings DE, Schwartz MW: Physiological regulation of hypothalamic IL-1␤ gene expression by leptin and glucocorticoids: implications for energy homeostasis. Am J Physiol 2004; 287:E1107– E1113. 6 Martin MU, Falk W: The interleukin-1 receptor complex and interleukin-1 signal transduction. Eur Cytokine Netw 1997; 8: 5– 17. 7 Buchanan JB, Peloso E, Satinoff E: Influence of ambient temperature on peripherally induced interleukin-1␤ fever in young and old rats. Physiol Behav 2006;88:453–458. 8 Nelson KP, Marks NL, Heyen JR, Johnson RW: Behavior of adult and aged mice before and after central injection of interleukin-1␤. Physiol Behav 1999;66:673–679. 9 Bluthe RM, Dantzer R, Kelley KW: Central mediation of the effects of interleukin-1 on social exploration and body weight in mice. Psychoneuroendocrinology 1997;22:1–11. 10 Avital A, Goshen I, Kamsler A, Segal M, Iverfeldt K, Richter-Levin G, Yirmiya R: Impaired interleukin-1 signaling is associated with deficits in hippocampal memory processes and neural plasticity. Hippocampus 2003;13:826–834. 11 Barrientos RM, Higgins EA, Sprunger DB, Watkins LR, Rudy JW, Maier SF: Memory for context is impaired by a post context exposure injection of interleukin-1␤ into dorsal hippocampus. Behav Brain Res 2002; 134: 291–298. 12 Matsuki T, Horai R, Sudo K, Iwakura Y: IL-1 plays an important role in lipid metabolism by regulating insulin levels under physiological conditions. J Exp Med 2003; 198: 877– 888. 13 Butera PC, Briffa CF, Whitaker EE: Devazepide fails to reverse the inhibitory effect of interleukin-1␤ on food intake in female rats. Physiol Behav 2004;82: 777–783. 14 Moldawer LL, Andersson C, Gelin J, Lundholm KG: Regulation of food intake and hepatic protein synthesis by recombinant-derived cytokines. Am J Physiol 1988; 254: G450–G456.

188

15 Reyes TM, Sawchenko PE: Involvement of the arcuate nucleus of the hypothalamus in interleukin-1-induced anorexia. J Neurosci 2002;22:5091–5099. 16 De Souza CT, Araujo EP, Bordin S, Ashimine R, Zollner RL, Boschero AC, Saad MJ, Velloso LA: Consumption of a fat-rich diet activates a proinflammatory response and induces insulin resistance in the hypothalamus. Endocrinology 2005; 146:4192–4199. 17 Sonti G, Ilyin SE, Plata-Salaman CR: Anorexia induced by cytokine interactions at pathophysiological concentrations. Am J Physiol 1996;270:R1394–R1402. 18 Gregoire F, De Broux N, Hauser N, Heremans H, Van Damme J, Remacle C: Interferon-␥ and interleukin-1␤ inhibit adipoconversion in cultured rodent preadipocytes. J Cell Physiol 1992;151:300–309. 19 Memon RA, Feingold KR, Moser AH, Fuller J, Grunfeld C: Regulation of fatty acid transport protein and fatty acid translocase mRNA levels by endotoxin and cytokines. Am J Physiol 1998;274:E210–E217. 20 Suto G, Kiraly A, Plourde V, Tache Y: Intravenous interleukin-1␤-induced inhibition of gastric emptying: involvement of central corticotrophin-releasing factor and prostaglandin pathways in rats. Digestion 1996;57: 135–140. 21 Quan N, Stern EL, Whiteside MB, Herkenham M: Induction of pro-inflammatory cytokine mRNAs in the brain after peripheral injection of subseptic doses of lipopolysaccharide in the rat. J Neuroimmunol 1999;93: 72–80. 22 Ma XC, Chen LT, Oliver J, Horvath E, Phelps CP: Cytokine and adrenal axis responses to endotoxin. Brain Res 2000;861:135–142. 23 Turrin NP, Gayle D, Ilyin SE, Flynn MC, Langhans W, Schwartz GJ, Plata-Salaman CR: Pro-inflammatory and anti-inflammatory cytokine mRNA induction in the periphery and brain following intraperitoneal administration of bacterial lipopolysaccharide. Brain Res Bull 2001;54:443–453. 24 Laye S, Gheusi G, Cremona S, Combe C, Kelley K, Dantzer R, Parnet P: Endogenous brain IL-1 mediates LPS-induced anorexia and hypothalamic cytokine expression. Am J Physiol 2000;279:R93–R98. 25 Swiergiel AH, Dunn AJ: The roles of IL-1, IL6, and TNF- ␣ in the feeding responses to endotoxin and influenza virus infection in mice. Brain Behav Immun 1999; 13: 252– 265. 26 Kozak WZH, Conn CA, Soszynski D, van der Ploeg LH, Kluger MJ: Thermal and behavioral effects of lipopolysaccharide and influenza in interleukin-1␤-deficient mice. Am J Physiol 1995;269:R969–R977.


27 Yao JH, Ye SM, Burgess W, Zachary JF, Kelley KW, Johnson RW: Mice deficient in interleukin-1␤-converting enzyme resist anorexia induced by central lipopolysaccharide. Am J Physiol 1999;277:R1435–R1443. 28 Leon LR, Kozak W, Peschon J, Kluger MJ: Exacerbated febrile responses to LPS, but not turpentine, in TNF double receptor-knockout mice. Am J Physiol 1997; 272:R563– R569. 29 Harden LM, du Plessis I, Poole S, Laburn HP: Interleukin-6 and leptin mediate lipopolysaccharide-induced fever and sickness behavior. Physiol Behav 2006;89:146–155. 30 Plata-Salaman CR, Oomura Y, Kai Y: Tumor necrosis factor and interleukin-1␤: suppression of food intake by direct action in the central nervous system. Brain Res 1988;448: 106–114. 31 Coppack SW: Pro-inflammatory cytokines and adipose tissue. Proc Nutr Soc 2001; 60: 349–356. 32 Langhans W, Hrupka B: Interleukins and tumor necrosis factor as inhibitors of food intake. Neuropeptides 1999;33:415–424. 33 Tollner B, Roth J, Storr B, Martin D, Voigt K, Zeisberger E: The role of tumor necrosis factor in the febrile and metabolic responses of rats to intraperitoneal injection of a high dose of lipopolysaccharide. Pflügers Arch 2000;440:925–932. 34 Porter MH, Hrupka BJ, Altreuther G, Arnold M, Langhans W: Inhibition of TNF- ␣ production contributes to the attenuation of LPS-induced hypophagia by pentoxifylline. Am J Physiol 2000;279:R2113–R2120. 35 Rothwell NJ, Busbridge NJ, Lefeuvre RA, Hardwick AJ, Gauldie J, Hopkins SJ: Interleukin-6 is a centrally acting endogenous pyrogen in the rat. Can J Physiol Pharmacol 1991;69:1465–1469. 36 Wallenius V, Wallenius K, Ahren B, Rudling M, Carlsten H, Dickson SL, Ohlsson C, Jansson JO: Interleukin-6-deficient mice develop mature-onset obesity. Nat Med 2002; 8: 75– 79. 37 Plata-Salaman CR, Sonti G, Borkoski JP, Wilson CD, French-Mullen JM: Anorexia induced by chronic central administration of cytokines at estimated pathophysiological concentrations. Physiol Behav 1996;60: 867– 875. 38 Wallenius K, Wallenius V, Sunter D, Dickson SL, Jansson JO: Intracerebroventricular interleukin-6 treatment decreases body fat in rats. Biochem Biophys Res Commun 2002; 293:560–565. 39 Kozak W, Poli V, Soszynski D, Conn CA, Leon LR, Kluger MJ: Sickness behavior in mice deficient in interleukin-6 during turpentine abscess and influenza pneumonitis. Am J Physiol 1997;272:R621–R630.

Buchanan /Johnson

40 Beretta E, Dhillon H, Kalra PS, Kalra SP: Central LIF gene therapy suppresses food intake, body weight, serum leptin and insulin for extended periods. Peptides 2002;23:975– 984. 41 Janoschek R, Plum L, Koch L, Munzberg H, Diano S, Shanabrough M, Muller W, Horvath TL, Bruning JC: gp130 signaling in proopiomelanocortin neurons mediates the acute anorectic response to centrally applied ciliary neurotrophic factor. Proc Natl Acad Sci USA 2006;103:10707–10712. 42 Shapiro L, Zhang XX, Rupp RG, Wolff SM, Dinarello CA: Ciliary neurotrophic factor is an endogenous pyrogen. Proc Natl Acad Sci USA 1993;90:8614–8618. 43 Espat NJ, Auffenberg T, Rosenberg JJ, Rogy M, Martin D, Fang CH, Hasselgren PO, Copeland EM, Moldawer LL: Ciliary neurotrophic factor is catabolic and shares with IL-6 the capacity to induce an acute phase response. Am J Physiol 1996; 271:R185– R190. 44 Gloaguen I, Costa P, Demartis A, Lazzaro D, Di Marco A, Graziani R, Paonessa G, Chen F, Rosenblum CI, Van der Ploeg LH, Cortese R, Ciliberto G, Laufer R: Ciliary neurotrophic factor corrects obesity and diabetes associated with leptin deficiency and resistance. Proc Natl Acad Sci USA 1997; 94: 6456–6461. 45 Xu B, Dube MG, Kalra PS, Farmerie WG, Kaibara A, Moldawer LL, Martin D, Kalra SP: Anorectic effects of the cytokine, ciliary neurotropic factor, are mediated by hypothalamic neuropeptide Y: comparison with leptin. Endocrinology 1998; 139:466–473. 46 Ettinger MP, Littlejohn TW, Schwartz SL, Weiss SR, McIlwain HH, Heymsfield SB, Bray GA, Roberts WG, Heyman ER, Stambler N, Heshka S, Vicary C, Guler HP: Recombinant variant of ciliary neurotrophic factor for weight loss in obese adults: a randomized, dose-ranging study. JAMA 2003; 289:1826–1832. 47 Lambert PD, Anderson KD, Sleeman MW, Wong V, Tan J, Hijarunguru A, Corcoran TL, Murray JD, Thabet KE, Yancopoulos GD, Wiegand SJ: Ciliary neurotrophic factor activates leptin-like pathways and reduces body fat, without cachexia or rebound weight gain, even in leptin-resistant obesity. Proc Natl Acad Sci USA 2001;98:4652–4657. 48 Kokoeva MV, Yin H, Flier JS: Neurogenesis in the hypothalamus of adult mice: potential role in energy balance. Science 2005; 310: 679–683. 49 Anand BK, Brobeck JR: Hypothalamic control of food intake in rats and cats. Yale J Biol Med 1951;24:123–140. 50 Sahu A: Minireview: A hypothalamic role in energy balance with special emphasis on leptin. Endocrinology 2004; 145:2613–2620. 51 Banks WA, Kastin AJ, Broadwell RD: Passage of cytokines across the blood-brain barrier. Neuroimmunomodulation 1995; 2:241– 248.

Cytokines and Food Intake

52 Nakamori T, Morimoto A, Yamaguchi K, Watanabe T, Murakami N: Interleukin-1␤ production in the rabbit brain during endotoxin-induced fever. J Physiol 1994;476:177– 186. 53 Konsman JP, Kelley K, Dantzer R: Temporal and spatial relationships between lipopolysaccharide-induced expression of Fos, interleukin-1␤ and inducible nitric oxide synthase in rat brain. Neuroscience 1999; 89: 535–548. 54 Goehler LE, Erisir A, Gaykema RP: Neuralimmune interface in the rat area postrema. Neuroscience 2006;140:1415–1434. 55 Harre EM, Roth J, Pehl U, Kueth M, Gerstberger R, Hubschle T: Selected contribution: role of IL-6 in LPS-induced nuclear STAT3 translocation in sensory circumventricular organs during fever in rats. J Appl Physiol 2002;92:2657–2666. 56 Kurosawa M, Bucinskaite V, Miyasaka K, Funakoshi A, Lundeberg T: Effects of systemic injection of interleukin-1␤ on gastric vagal afferent activity in rats lacking type A cholecystokinin receptors. Neurosci Lett 2000;293:9–12. 57 Ek M, Kurosawa M, Lundeberg T, Ericsson A: Activation of vagal afferents after intravenous injection of interleukin-1␤: role of endogenous prostaglandins. J Neurosci 1998; 18:9471–9479. 58 Niijima A: The afferent discharges from sensors for interleukin-1␤ in the hepatoportal system in the anesthetized rat. J Auton Nerv Syst 1996;61:287–291. 59 Hosoi T, Okuma Y, Nomura Y: Electrical stimulation of afferent vagus nerve induces IL-1␤ expression in the brain and activates HPA axis. Am J Physiol 2000; 279:R141– R147. 60 Hansen MK, Krueger JM: Subdiaphragmatic vagotomy blocks the sleep- and fever-promoting effects of interleukin-1␤. Am J Physiol 1997;273:R1246–R1253. 61 Gaykema RP, Goehler LE, Hansen MK, Maier SF, Watkins LR: Subdiaphragmatic vagotomy blocks interleukin-␤-induced fever but does not reduce IL-1␤ levels in the circulation. Auton Neurosci 2000;85: 72–77. 62 Gautron L, Mingam R, Moranis A, Combe C, Laye S: Influence of feeding status on neuronal activity in the hypothalamus during lipopolysaccharide-induced anorexia in rats. Neuroscience 2005;134:933–946. 63 Wan W, Wetmore L, Sorensen CM, Greenberg AH, Nance DM: Neural and biochemical mediators of endotoxin and stress-induced c-fos expression in the rat brain. Brain Res Bull 1994;34:7–14. 64 Bret-Dibat JL, Bluthe RM, Kent S, Kelley KW, Dantzer R: Lipopolysaccharide and interleukin-1 depress food-motivated behavior in mice by a vagal-mediated mechanism. Brain Behav Immun 1995;9:242–246.

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.


189

79 Lugarini F, Hrupka BJ, Schwartz GJ, PlataSalaman CR, Langhans W: Acute and chronic administration of immunomodulators induces anorexia in Zucker rats. Physiol Behav 2005;84:165–173. 80 Nahon JL: The melanocortins and melaninconcentrating hormone in the central regulation of feeding behavior and energy homeostasis. C R Biol 2006; 329: 623–638; discussion 53–55. 81 Ericsson A, Liu C, Hart RP, Sawchenko PE: Type 1 interleukin-1 receptor in the rat brain: distribution, regulation, and relationship to sites of IL-1-induced cellular activation. J Comp Neurol 1995;361:681–698. 82 Kalra SP, Xu B, Dube MG, Moldawer LL, Martin D, Kalra PS: Leptin and ciliary neurotropic factor inhibit fasting-induced suppression of luteinizing hormone release in rats: role of neuropeptide Y. Neurosci Lett 1998;240:45–49. 83 Sergeyev V, Broberger C, Hokfelt T: Effect of LPS administration on the expression of POMC, NPY, galanin, CART and MCH mRNAs in the rat hypothalamus. Brain Res Mol Brain Res 2001;90:93–100. 84 Marks DL, Cone RD: Central melanocortins and the regulation of weight during acute and chronic disease. Recent Prog Horm Res 2001;56:359–375. 85 Marks DL, Ling N, Cone RD: Role of the central melanocortin system in cachexia. Cancer Res 2001;61:1432–1438. 86 DeBoer MD, Marks DL: Cachexia: lessons from melanocortin antagonism. Trends Endocrinol Metab 2006;17:199–204. 87 Lawrence CB, Rothwell NJ: Anorexic but not pyrogenic actions of interleukin-1 are modulated by central melanocortin-3/4 receptors in the rat. J Neuroendocrinol 2001; 13: 490– 495.

190

88 Wisse BE, Frayo RS, Schwartz MW, Cummings DE: Reversal of cancer anorexia by blockade of central melanocortin receptors in rats. Endocrinology 2001; 142:3292–3301. 89 Wisse BE, Schwartz MW, Cummings DE: Melanocortin signaling and anorexia in chronic disease states. Ann NY Acad Sci 2003;994:275–281. 90 Swiergiel AH, Dunn AJ: Feeding, exploratory, anxiety- and depression-related behaviors are not altered in interleukin-6-deficient male mice. Behav Brain Res 2006; 171: 94– 108. 91 Plata-Salaman CR: Anorexia induced by activators of the signal transducer gp130. Neuroreport 1996;7:841–844. 92 Netea MG, Joosten LA, Lewis E, Jensen DR, Voshol PJ, Kullberg BJ, Tack CJ, van Krieken H, Kim SH, Stalenhoef AF, van de Loo FA, Verschueren I, Pulawa L, Akira S, Eckel RH, Dinarello CA, van den Berg W, van der Meer JW: Deficiency of interleukin-18 in mice leads to hyperphagia, obesity and insulin resistance. Nat Med 2006;12:650–656. 93 Darling G, Fraker DL, Jensen JC, Gorschboth CM, Norton JA: Cachectic effects of recombinant human tumor necrosis factor in rats. Cancer Res 1990;50:4008–4013. 94 Reyes-Vazquez C, Prieto-Gomez B, Dafny N: Alpha-interferon suppresses food intake and neuronal activity of the lateral hypothalamus. Neuropharmacology 1994; 33: 1545– 1552. 95 Plata-Salaman CR: Interferons and central regulation of feeding. Am J Physiol 1992;263: R1222–R1227.


96 Martin D, Merkel E, Tucker KK, McManaman JL, Albert D, Relton J, Russell DA: Cachectic effect of ciliary neurotrophic factor on innervated skeletal muscle. Am J Physiol 1996;271:R1422–R1428. 97 Nakagawa T, Ogawa Y, Ebihara K, Yamanaka M, Tsuchida A, Taiji M, Noguchi H, Nakao K: Anti-obesity and anti-diabetic effects of brain-derived neurotrophic factor in rodent models of leptin resistance. Int J Obes Relat Metab Disord 2003;27:557–565. 98 Pelleymounter MA, Cullen MJ, Wellman CL: Characteristics of BDNF-induced weight loss. Exp Neurol 1995;131:229–238. 99 Reed JA, Clegg DJ, Smith KB, Tolod-Richer EG, Matter EK, Picard LS, Seeley RJ: GMCSF action in the CNS decreases food intake and body weight. J Clin Invest 2005; 115:3035–3044. 100 Hanai K, Oomura Y, Kai Y, Nishikawa K, Shimizu N, Morita H, Plata-Salaman CR: Central action of acidic fibroblast growth factor in feeding regulation. Am J Physiol 1989;256:R217–R223. 101 Oomura Y, Sasaki K, Li A: Pathophysiological significance of brain acidic fibroblast growth factor. Patol Fiziol Eksp Ter 1992;4: 71–74. 102 Akiyama Y, Kajimura N, Matsuzaki J, Kikuchi Y, Imai N, Tanigawa M, Yamaguchi K: In vivo effect of recombinant human leukemia inhibitory factor in primates. Jpn J Cancer Res 1997;88:578–583. 103 Agnello D, Wang H, Yang H, Tracey KJ, Ghezzi P: HMGB-1, a DNA-binding protein with cytokine activity, induces brain TNF and IL-6 production, and mediates anorexia and taste aversion. Cytokine 2002; 18:231–236.

Buchanan /Johnson



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



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


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


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


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

Adipokine Expression in Brain


193

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


[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



195

% control

A 300

Sham TBI

200 100 0

B 300 % control

***

Ipsilateral

Contralateral

**

200 100 0

Ipsilateral

Contralateral

Ipsilateral

Contralateral

% control

C 200 150 100 50 0

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


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



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).

197

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


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

B

socs-3 mRNA expression (% of controls)

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.

C

120 100 80 60

*

40 20 0

STH CTL

STH R4

200

*

150 100 50 0

STH CTL

200

STH R4

*

150 100 50 0

STH CTL

STH R4

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



Fasting-Induced Adipose Factor

199

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


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.



201

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


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

203

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

References

204

[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.

1 Krieger DT: Brain peptides: What, where, and why? Science 1983; 222:975–985. 2 Bryant MG, Polak MM, Modlin I, Bloom SR, Albuquerque RH, Pearse AG: Possible dual role for vasoactive intestinal peptide as gastrointestinal hormone and neurotransmitter substance. Lancet 1976;1:991–993. 3 Dockray GJ: The G.L. Brown lecture. Regulatory peptides and the neuroendocrinology of gut-brain relations. Q J Exp Physiol 1988; 73:703–727.


4 Polak JM, Bloom SR: Regulatory peptides of the gastrointestinal and respiratory tracts. Arch Int Pharmacodyn Ther 1986; 280: 16– 49. 5 Sato T, Fukue Y, Teranishi H, Yoshida Y, Kojima M: Molecular forms of hypothalamic ghrelin and its regulation by fasting and 2deoxy-D -glucose administration. Endocrinology 2005; 146:2510–2516.

Wilkinson /Brown /Imran /Ur

6 Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM: Positional cloning of the mouse obese gene and its human homologue. Nature 1994;372:425–432. 7 Trayhurn P, Bing C, Wood IS: Adipose tissue and adipokines – energy regulation from the human perspective. J Nutr 2006;136:1935S– 1939S. 8 Lafontan M: Fat cells: afferent and efferent messages define new approaches to treat obesity. Annu Rev Pharmacol Toxicol 2005; 45:119–146. 9 Koerner A, Kratzsch J, Kiess W: Adipocytokines: Leptin – the classical, resistin – the controversical, adiponectin – the promising, and more to come. Best Pract Res Clin Endocrinol Metab 2005;19:525–546. 10 Steppan CM, Bailey ST, Bhat S, Brown EJ, Banerjee RR, Wright CM, Patel HR, Ahima RS, Lazar MA: The hormone resistin links obesity to diabetes. Nature 2001;409:307–312. 11 Scherer PE, Williams S, Fogliano M, Baldini G, Lodish HF: A novel serum protein similar to C1q, produced exclusively in adipocytes. J Biol Chem 1995; 270:26746–26749. 12 Kersten S, Mandard S, Tan NS, Escher P, Metzger D, Chambon P, Gonzalez FJ, Desvergne B, Wahli W: Characterization of the fasting-induced adipose factor FIAF, a novel peroxisome proliferator-activated receptor target gene. J Biol Chem 2000; 275: 28488– 28493. 13 Fukuhara A, Matsuda M, Nishizawa M, Segawa K, Tanaka M, Kishimoto K, Matsuki Y, Murakami M, Ichisaka T, Murakami H, Watanabe E, Takagi T, Akiyoshi M, Ohtsubo T, Kihara S, Yamashita S, Makishima M, Funahashi T, Yamanaka S, Hiramatsu R, Matsuzawa Y, Shimomura I: Visfatin: A protein secreted by visceral fat that mimics the effects of insulin. Science 2005;307:426–430. 14 Hida K, Wada J, Eguchi J, Zhang H, Baba M, Seida A, Hashimoto I, Okada T, Yasuhara A, Nakatsuka A, Shikata K, Hourai S, Futami J, Watanabe E, Matsuki Y, Hiramatsu R, Akagi S, Makino H, Kanwar YS: Visceral adipose tissue-derived serine protease inhibitor: a unique insulin-sensitizing adipocytokine in obesity. Proc Natl Acad Sci USA 2005; 102: 10610–10615. 15 Bullo M, Peeraully MR, Trayhurn P: Stimulation of ngf expression and secretion in 3T31 adipocytes by prostaglandins PGD2, PGJ2, and ⌬12-PGJ2. Am J Physiol 2005; 289:E62– E67. 16 Kratchmarova I, Kalume DE, Blagoev B, Scherer PE, Podtelejnikov AV, Molina H, Bickel PE, Andersen JS, Fernandez MM, Bunkenborg J, Roepstorff P, Kristiansen K, Lodish HF, Mann M, Pandey A: A proteomic approach for identification of secreted proteins during the differentiation of 3T3-L1 preadipocytes to adipocytes. Mol Cell Proteomics 2002;1:213–222.


17 Yang YS, Song HD, Li RY, Zhou LB, Zhu ZD, Hu RM, Han ZG, Chen JL: The gene expression profiling of human visceral adipose tissue and its secretory functions. Biochem Biophys Res Commun 2003; 300:839–846. 18 Elmquist JK, Bjorbaek C, Ahima RS, Flier JS, Saper CB: Distributions of leptin receptor mRNA isoforms in the rat brain. J Comp Neurol 1998;395:535–547. 19 Funahashi H, Yada T, Suzuki R, Shioda S: Distribution, function, and properties of leptin receptors in the brain. Int Rev Cytol 2003;224:1–27. 20 Burguera B, Couce ME, Long J, Lamsam J, Laakso K, Jensen MD, Parisi JE, Lloyd RV: The long form of the leptin receptor (OB-Rb) is widely expressed in the human brain. Neuroendocrinology 2000; 71:187–195. 21 Jin L, Burguera BG, Couce ME, Scheithauer BW, Lamsan J, Eberhardt NL, Kulig E, Lloyd RV: Leptin and leptin receptor expression in normal and neoplastic human pituitary: evidence of a regulatory role for leptin on pituitary cell proliferation. J Clin Endocrinol Metab 1999;84:2903–2911. 22 Cai A, Hyde JF: The human growth hormone-releasing hormone transgenic mouse as a model of modest obesity: differential changes in leptin receptor (OBR) gene expression in the anterior pituitary and hypothalamus after fasting and obr localization in somatotrophs. Endocrinology 1999; 140: 3609–3614. 23 Morash B, Li A, Murphy PR, Wilkinson M, Ur E: Leptin gene expression in the brain and pituitary gland. Endocrinology 1999; 140: 5995–5998. 24 Wilkinson M, Morash B, Ur E: The brain is a source of leptin. Front Horm Res 2000;26: 106–125. 25 Wiesner G, Vaz M, Collier G, Seals D, Kaye D, Jennings G, Lambert G, Wilkinson D, Esler M: Leptin is released from the human brain: influence of adiposity and gender. J Clin Endocrinol Metab 1999;84:2270–2274. 26 Eikelis N, Esler M: The neurobiology of human obesity. Exp Physiol 2005;90:673–682. 27 Bennett BD, Solar GP, Yuan JQ, Mathias J, Thomas GR, Matthews W: A role for leptin and its cognate receptor in hematopoiesis. Curr Biol 1996;6:1170–1180. 28 Himms-Hagen J: Physiological roles of the leptin endocrine system: differences between mice and humans. Crit Rev Clin Lab Sci 1999;36:575–655. 29 Arner P: Resistin: Yet another adipokine tells us that men are not mice. Diabetologia 2005; 48:2203–2205. 30 Banks WA, Kastin AJ, Huang W, Jaspan JB, Maness LM: Leptin enters the brain by a saturable system independent of insulin. Peptides 1996;17:305–311. 31 Banks WA: The blood-brain barrier as a regulatory interface in the gut-brain axes. Physiol Behav 2006;89:472–476.

32 Zlokovic BV, Jovanovic S, Miao W, Samara S, Verma S, Farrell CL: Differential regulation of leptin transport by the choroid plexus and blood-brain barrier and high affinity transport systems for entry into hypothalamus and across the blood-cerebrospinal fluid barrier. Endocrinology 2000; 141: 1434– 1441. 33 Kurrimbux D, Gaffen Z, Farrell CL, Martin D, Thomas SA: The involvement of the blood-brain and the blood-cerebrospinal fluid barriers in the distribution of leptin into and out of the rat brain. Neuroscience 2004;123:527–536. 34 Ahima RS: Central actions of adipocyte hormones. Trends Endocrinol Metab 2005; 16: 307–313. 35 Rivest S: Does circulating leptin have the ability to cross the blood-brain barrier and target neurons directly? Endocrinology 2002;143:3211–3213. 36 Banks WA, Clever CM, Farrell CL: Partial saturation and regional variation in the blood-to-brain transport of leptin in normal weight mice. Am J Physiol 2000;278:E1158– E1165. 37 Hileman SM, Pierroz DD, Masuzaki H, Bjorbaek C, El-Haschimi K, Banks WA, Flier JS: Characterizaton of short isoforms of the leptin receptor in rat cerebral microvessels and of brain uptake of leptin in mouse models of obesity. Endocrinology 2002; 143:775– 783. 38 Kastin AJ, Pan W: Dynamic regulation of leptin entry into brain by the blood-brain barrier. Regul Pept 2000; 92:37–43. 39 Sinha MK, Opentanova I, Ohannesian JP, Kolaczynski JW, Heiman ML, Hale J, Becker GW, Bowsher RR, Stephens TW, Caro JF: Evidence of free and bound leptin in human circulation. Studies in lean and obese subjects and during short-term fasting. J Clin Invest 1996;98:1277–1282. 40 Zastrow O, Seidel B, Kiess W, Thiery J, Keller E, Bottner A, Kratzsch J: The soluble leptin receptor is crucial for leptin action: evidence from clinical and experimental data. Int J Obes Relat Metab Disord 2003; 27: 1472– 1478. 41 Wong ML, Licinio J, Yildiz BO, Mantzoros CS, Prolo P, Kling M, Gold PW: Simultaneous and continuous 24-hour plasma and cerebrospinal fluid leptin measurements: dissociation of concentrations in central and peripheral compartments. J Clin Endocrinol Metab 2004;89:258–265. 42 Adam CL, Findlay PA, Miller DW: Bloodbrain leptin transport and appetite and reproductive neuroendocrine responses to intracerebroventricular leptin injection in sheep: influence of photoperiod. Endocrinology 2006; 147:4589–4598.


205

43 Uotani S, Bjorbaek C, Tornoe J, Flier JS: Functional properties of leptin receptor isoforms: internalization and degradation of leptin and ligand-induced receptor downregulation. Diabetes 1999;48:279–286. 44 Ahima RS, Qi Y, Singhal NS: Adipokines that link obesity and diabetes to the hypothalamus. Prog Brain Res 2006; 153: 155– 174. 45 Kelly JF, Elias CF, Lee CE, Ahima RS, Seeley RJ, Bjorbaek C, Oka T, Saper CB, Flier JS, Elmquist JK: Ciliary neurotrophic factor and leptin induce distinct patterns of immediate early gene expression in the brain. Diabetes 2004;53:911–920. 46 Hakansson ML, Meister B: Transcription factor STAT-3 in leptin target neurons of the rat hypothalamus. Neuroendocrinology 1998;68:420–427. 47 Vaisse C, Halaas JL, Horvath CM, Darnell JE Jr, Stoffel M, Friedman JM: Leptin activation of STAT-3 in the hypothalamus of wild-type and ob/ob mice but not db/db mice. Nat Genet 1996;14:95–97. 48 Hosoi T, Kawagishi T, Okuma Y, Tanaka J, Nomura Y: Brain stem is a direct target for leptin’s action in the central nervous system. Endocrinology 2002; 143:3498–3504. 49 Polizzotto MN, Bartlett PF, Turnley AM: Expression of ‘suppressor of cytokine signalling’ (SOCS) genes in the developing and adult mouse nervous system. J Comp Neurol 2000;423:348–358. 50 Stromberg H, Svensson SP, Hermanson O: Distribution of the transcription factor signal transducer and activator of transcription 3 in the rat central nervous system and dorsal root ganglia. Brain Res 2000;853:105–114. 51 Pan W, Tu H, Kastin AJ: Differential BBB interactions of three ingestive peptides: obestatin, ghrelin, and adiponectin. Peptides 2006;27:911–916. 52 Spranger J, Verma S, Gohring I, Bobbert T, Seifert J, Sindler AL, Pfeiffer A, Hileman SM, Tschop M, Banks WA: Adiponectin does not cross the blood-brain barrier but modifies cytokine expression of brain endothelial cells. Diabetes 2006;55:141–147. 53 Qi Y, Takahashi N, Hileman SM, Patel HR, Berg AH, Pajvani UB, Scherer PE, Ahima RS: Adiponectin acts in the brain to decrease body weight. Nat Med 2004;10:524–529. 54 Yamauchi T, Kamon J, Ito Y, Tsuchida A, Yokomizo T, Kita S, Sugiyama T, Miyagishi M, Hara K, Tsunoda M, Murakami K, Ohteki T, Uchida S, Takekawa S, Waki H, Tsuno NH, Shibata Y, Terauchi Y, Froguel P, Tobe K, Koyasu S, Taira K, Kitamura T, Shimizu T, Nagai R, Kadowaki T: Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature 2003;423:762–769. 55 Maddineni S, Metzger S, Ocón O, Hendricks G 3rd, Ramachandran R: Adiponectin gene is expressed in multiple tissues in the chicken: food deprivation influences adiponectin messenger ribonucleic acid expression. Endocrinology 2005; 146:4250–4256.

206

56 Kastin AJ, Akerstrom V, Pan W: Validity of multiple-time regression analysis in measurement of tritiated and iodinated leptin crossing the blood-brain barrier: meaningful controls. Peptides 2001; 22:2127–2136. 57 Zelissen PM, Stenlof K, Lean ME, Fogteloo J, Keulen ET, Wilding J, Finer N, Rossner S, Lawrence E, Fletcher C, McCamish M: Effect of three treatment schedules of recombinant methionyl human leptin on body weight in obese adults: a randomized, placebo-controlled trial. Diabetes Obes Metab 2005; 7: 755–761. 58 Bell-Anderson KS, Bryson JM: Leptin as a potential treatment for obesity: progress to date. Treat Endocrinol 2004;3:11–18. 59 Farooqi IS, O’Rahilly S: Monogenic obesity in humans. Annu Rev Med 2005; 56: 443– 458. 60 Licinio J, Caglayan S, Ozata M, Yildiz BO, de Miranda PB, O’Kirwan F, Whitby R, Liang L, Cohen P, Bhasin S, Krauss RM, Veldhuis JD, Wagner AJ, DePaoli AM, McCann SM, Wong ML: Phenotypic effects of leptin replacement on morbid obesity, diabetes mellitus, hypogonadism, and behavior in leptindeficient adults. Proc Natl Acad Sci USA 2004;101:4531–4536. 61 Pan W, Yu Y, Cain CM, Nyberg F, Couraud PO, Kastin AJ: Permeation of growth hormone across the blood-brain barrier. Endocrinology 2005; 146:4898–4904. 62 Donahue CP, Kosik KS, Shors TJ: Growth hormone is produced within the hippocampus where it responds to age, sex, and stress. Proc Natl Acad Sci USA 2006; 103: 6031– 6036. 63 Li XL, Aou S, Oomura Y, Hori N, Fukunaga K, Hori T: Impairment of long-term potentiation and spatial memory in leptin receptor-deficient rodents. Neuroscience 2002; 113:607–615. 64 Harvey J, Solovyova N, Irving A: Leptin and its role in hippocampal synaptic plasticity. Prog Lipid Res 2006;45:369–378. 65 Farr SA, Banks WA, Morley JE: Effects of leptin on memory processing. Peptides 2006; 27:1420–1425. 66 Oomura Y, Hori N, Shiraishi T, Fukunaga K, Takeda H, Tsuji M, Matsumiya T, Ishibashi M, Aou S, Li XL, Kohno D, Uramura K, Sougawa H, Yada T, Wayner MJ, Sasaki K: Leptin facilitates learning and memory performance and enhances hippocampal CA1 long-term potentiation and CaMK II phosphorylation in rats. Peptides 2006; 27: 2738– 2749. 67 Elias MF, Elias PK, Sullivan LM, Wolf PA, D’Agostino RB: Obesity, diabetes and cognitive deficit: The Framingham Heart Study. Neurobiol Aging 2005;26(suppl 1):11–16. 68 Steppan CM, Lazar MA: The current biology of resistin. J Intern Med 2004;255:439–447. 69 Sahu A: Leptin signaling in the hypothalamus: emphasis on energy homeostasis and leptin resistance. Front Neuroendocrinol 2003;24:225–253.


70 Hakansson ML, Brown H, Ghilardi N, Skoda RC, Meister B: Leptin receptor immunoreactivity in chemically defined target neurons of the hypothalamus. J Neurosci 1998; 18: 559–572. 71 Patz S, Wahle P: Developmental changes of neurotrophin mRNA expression in the layers of rat visual cortex. Eur J Neurosci 2006; 24:2453–2460. 72 Ur E, Wilkinson DA, Morash BA, Wilkinson M: Leptin immunoreactivity is localized to neurons in rat brain. Neuroendocrinology 2002;75:264–272. 73 Wilkinson M, Wilkinson D, Wiesner G, Morash B, Ur E: Hypothalamic resistin immunoreactivity is reduced by obesity in the mouse: co-localization with ␣-melanostimulating hormone. Neuroendocrinology 2005;81:19–30. 74 Morash B, Wilkinson D, Murphy P, Ur E, Wilkinson M: Developmental regulation of leptin gene expression in rat brain and pituitary. Mol Cell Endocrinol 2001; 185: 151– 159. 75 Morash BA, Ur E, Wilkinson M: Pituitary leptin gene expression is reduced by neonatal androgenization of female rats. Pituitary 2001;4:63–70. 76 Thompson HJ, Lifshitz J, Marklund N, Grady MS, Graham DI, Hovda DA, McIntosh TK: Lateral fluid percussion brain injury: a 15year review and evaluation. J Neurotrauma 2005;22:42–75. 77 Wilkinson M, Brown R, Thompson H, Imran A, Ur E, Lebold DG: Expression of brainderived leptin and fasting-induced adipose factor following traumatic brain injury. Front Neuroendocrinol 2006;27:93. 78 Morash B, Johnstone J, Leopold C, Li A, Murphy P, Ur E, Wilkinson M: The regulation of leptin gene expression in the C6 glioblastoma cell line. Mol Cell Endocrinol 2000; 165:97–105. 79 Li AW, Morash B, Hollenberg AN, Ur E, Wilkinson M, Murphy PR: Transcriptional regulation of the leptin gene promoter in rat GH3 pituitary and C6 glioma cells. Mol Cell Endocrinol 2001;176:57–65. 80 Slieker LJ, Sloop KW, Surface PL, Kriauciunas A, LaQuier F, Manetta J, Bue-Valleskey J, Stephens TW: Regulation of expression of ob mRNA and protein by glucocorticoids and cAMP. J Biol Chem 1996; 271:5301–5304. 81 Miller VM, Paulson HL, Gonzalez-Alegre P: RNA interference in neuroscience: progress and challenges. Cell Mol Neurobiol 2005;25: 1195–1207. 82 Makimura H, Mizuno TM, Mastaitis JW, Agami R, Mobbs CV: Reducing hypothalamic AGRP by RNA interference increases metabolic rate and decreases body weight without influencing food intake. BMC Neurosci 2002;3:18.


83 Brown R, Morash B, Ur E, Wilkinson M: RNAi-mediated silencing of leptin gene expression increases cell death in C6 glioblastoma cells. Brain Res Mol Brain Res 2005; 139:357–360. 84 Russo VC, Metaxas S, Kobayashi K, Harris M, Werther GA: Antiapoptotic effects of leptin in human neuroblastoma cells. Endocrinology 2004; 145:4103–4112. 85 Knerr I, Schuster S, Nomikos P, Buchfelder M, Dotsch J, Schoof E, Fahlbusch R, Rascher W: Gene expression of adrenomedullin, leptin, their receptors and neuropeptide Y in hormone-secreting and non-functioning pituitary adenomas, meningiomas and malignant intracranial tumours in humans. Neuropathol Appl Neurobiol 2001; 27:215–222. 86 Eikelis N, Esler M, Barton D, Dawood T, Wiesner G, Lambert G: Reduced brain leptin in patients with major depressive disorder and in suicide victims. Mol Psychiatry 2006; 11:800–801. 87 Eikelis N, Lambert G, Wiesner G, Kaye D, Schlaich M, Morris M, Hastings J, Socratous F, Esler M: Extra-adipocyte leptin release in human obesity and its relation to sympathoadrenal function. Am J Physiol 2004; 286: E744–E752. 88 Esler M, Vaz M, Collier G, Nestel P, Jennings G, Kaye D, Seals D, Lambert G: Leptin in human plasma is derived in part from the brain, and cleared by the kidneys. Lancet 1998;351: 879. 89 Reichlin S: Is leptin a secretion of the brain? J Clin Endocrinol Metab 1999; 84: 2267– 2269. 90 Korbonits M, Chitnis MM, Gueorguiev M, Norman D, Rosenfelder N, Suliman M, Jones TH, Noonan K, Fabbri A, Besser GM, Burrin JM, Grossman AB: The release of leptin and its effect on hormone release from human pituitary adenomas. Clin Endocrinol (Oxf) 2001;54:781–789. 91 Smith EL, Hainsworth AH: Acute effects of interleukin-1␤ on noradrenaline release from the human neuroblastoma cell line SHSY5Y. Neurosci Lett 1998;251:89–92. 92 Steppan CM, Wang J, Whiteman EL, Birnbaum MJ, Lazar MA: Activation of SOCS-3 by resistin. Mol Cell Biol 2005; 25: 1569– 1575. 93 Jung HS, Park KH, Cho YM, Chung SS, Cho HJ, Cho SY, Kim SJ, Kim SY, Lee HK, Park KS: Resistin is secreted from macrophages in atheromas and promotes atherosclerosis. Cardiovasc Res 2006;69:76–85. 94 Yang RZ, Huang Q, Xu A, McLenithan JC, Eisen JA, Shuldiner AR, Alkan S, Gong DW: Comparative studies of resistin expression and phylogenomics in human and mouse. Biochem Biophys Res Commun 2003; 310: 927–935.


95 Lappas M, Yee K, Permezel M, Rice GE: Release and regulation of leptin, resistin and adiponectin from human placenta, fetal membranes, and maternal adipose tissue and skeletal muscle from normal and gestational diabetes mellitus-complicated pregnancies. J Endocrinol 2005; 186:457–465. 96 Minn AH, Patterson NB, Pack S, Hoffmann SC, Gavrilova O, Vinson C, Harlan DM, Shalev A: Resistin is expressed in pancreatic islets. Biochem Biophys Res Commun 2003;310:641–645. 97 Nogueiras R, Gallego R, Gualillo O, Caminos JE, Garcia-Caballero T, Casanueva FF, Dieguez C: Resistin is expressed in different rat tissues and is regulated in a tissueand gender-specific manner. FEBS Lett 2003;548:21–27. 98 Tsukamoto H: Adipogenic phenotype of hepatic stellate cells. Alcohol Clin Exp Res 2005;29:132S–133S. 99 McTernan PG, Kusminski CM, Kumar S: Resistin. Curr Opin Lipidol 2006; 17: 170– 175. 100 Morash BA, Ur E, Wiesner G, Roy J, Wilkinson M: Pituitary resistin gene expression: effects of age, gender and obesity. Neuroendocrinology 2004; 79:149–156. 101 Morash BA, Wilkinson D, Ur E, Wilkinson M: Resistin expression and regulation in mouse pituitary. FEBS Lett 2002; 526: 26– 30. 102 Savage DB, Sewter CP, Klenk ES, Segal DG, Vidal-Puig A, Considine RV, O’Rahilly S: Resistin/fizz3 expression in relation to obesity and peroxisome proliferator-activated receptor-␥ action in humans. Diabetes 2001;50:2199–2202. 103 Tovar S, Nogueiras R, Tung LY, Castaneda TR, Vazquez MJ, Morris A, Williams LM, Dickson SL, Dieguez C: Central administration of resistin promotes short-term satiety in rats. Eur J Endocrinol 2005;153:R1– R5. 104 Ort T, Arjona AA, MacDougall JR, Nelson PJ, Rothenberg ME, Wu F, Eisen A, Halvorsen YD: Recombinant human fizz3/resistin stimulates lipolysis in cultured human adipocytes, mouse adipose explants, and normal mice. Endocrinology 2005; 146: 2200– 2209. 105 Bouret SG, Simerly RB: Leptin and development of hypothalamic feeding circuits. Endocrinology 2004; 145:2621–2626. 106 Bouret SG, Draper SJ, Simerly RB: Formation of projection pathways from the arcuate nucleus of the hypothalamus to hypothalamic regions implicated in the neural control of feeding behavior in mice. J Neurosci 2004;24:2797–2805. 107 Bouret SG, Draper SJ, Simerly RB: Trophic action of leptin on hypothalamic neurons that regulate feeding. Science 2004; 304: 108–110. 108 Flier JS: Obesity wars: molecular progress confronts an expanding epidemic. Cell 2004;116:337–350.

109 Liu F, Guo XR, Gong HX, Ni YH, Fei L, Pan XQ, Guo M, Chen RH: A resistin binding peptide selected by phage display inhibits 3T3-L1 preadipocyte differentiation. Chin Med J (Engl) 2006;119:496–503. 110 Brunetti L, Orlando G, Recinella L, Michelotto B, Ferrante C, Vacca M: Resistin, but not adiponectin, inhibits dopamine and norepinephrine release in the hypothalamus. Eur J Pharmacol 2004;493:41–44. 111 Belsham DD, Cai F, Cui H, Smukler SR, Salapatek AM, Shkreta L: Generation of a phenotypic array of hypothalamic neuronal cell models to study complex neuroendocrine disorders. Endocrinology 2004; 145:393–400. 112 Brown R, Imran S, Belsham DD, Ur E, Wilkinson M: Valproic acid reduces resistin, but increases SOCS-3, expression in a novel hypothalamic cell line. Front Neuroendocrinol 2006;27:93. 113 Brown R, Imran S, Belsham DD, Ur E, Wilkinson M: Adipokine gene expression in a novel hypothalamic neuronal cell line: resistin-dependent regulation of fastinginduced adipose factor and SOCS-3. Neuroendocrinology 2007; 85: 232–241. 114 Brown RE, Imran A, Belsham DD, Ur E, Wilkinson M: Regulation of brain-derived resistin and FIAF in a novel hypothalamic neuronal cell line. 8th European Congress of Endocrinology, Glasgow 2006. 115 Kim S, Moustaid-Moussa N: Secretory, endocrine and autocrine/paracrine function of the adipocyte. J Nutr 2000; 130: 3110S– 3115S. 116 Kim I, Moon SO, Koh KN, Kim H, Uhm CS, Kwak HJ, Kim NG, Koh GY: Molecular cloning, expression, and characterization of angiopoietin-related protein. Angiopoietin-related protein induces endothelial cell sprouting. J Biol Chem 1999;274:26523– 26528. 117 Wiesner G, Morash BA, Ur E, Wilkinson M: Food restriction regulates adipose-specific cytokines in pituitary gland but not in hypothalamus. J Endocrinol 2004; 180:R1– R6. 118 Le Jan S, Amy C, Cazes A, Monnot C, Lamande N, Favier J, Philippe J, Sibony M, Gasc JM, Corvol P, Germain S: Angiopoietin-like 4 is a proangiogenic factor produced during ischemia and in conventional renal cell carcinoma. Am J Pathol 2003;162: 1521–1528. 119 Lal A, Peters H, St Croix B, Haroon ZA, Dewhirst MW, Strausberg RL, Kaanders JH, van der Kogel AJ, Riggins GJ: Transcriptional response to hypoxia in human tumors. J Natl Cancer Inst 2001; 93: 1337– 1343. 120 Kim I, Kim HG, Kim H, Kim HH, Park SK, Uhm CS, Lee ZH, Koh GY: Hepatic expression, synthesis and secretion of a novel fibrinogen/angiopoietin-related protein that prevents endothelial-cell apoptosis. Biochem J 2000;346:603–610.


207

121 Belanger AJ, Lu H, Date T, Liu LX, Vincent KA, Akita GY, Cheng SH, Gregory RJ, Jiang C: Hypoxia up-regulates expression of peroxisome proliferator-activated receptor-␥ angiopoietin-related gene in cardiomyocytes: role of hypoxia inducible factor 1␣. J Mol Cell Cardiol 2002;34:765–774. 122 Wiesner G, Brown RE, Robertson GS, Imran SA, Ur E, Wilkinson M: Increased expression of the adipokine genes resistin and fasting-induced adipose factor in hypoxic/ ischaemic mouse brain. Neuroreport 2006; 17:1195–1198. 123 Hayashi T, Noshita N, Sugawara T, Chan PH: Temporal profile of angiogenesis and expression of related genes in the brain after ischemia. J Cereb Blood Flow Metab 2003;23:166–180. 124 Nag S: The blood-brain barrier and cerebral angiogenesis: lessons from the cold-injury model. Trends Mol Med 2002;8:38–44. 125 Trayhurn P, Wood IS: Adipokines: Inflammation and the pleiotropic role of white adipose tissue. Br J Nutr 2004;92:347–355. 126 Xu A, Lam MC, Chan KW, Wang Y, Zhang J, Hoo RL, Xu JY, Chen B, Chow WS, Tso AW, Lam KS: Angiopoietin-like protein 4 decreases blood glucose and improves glucose tolerance but induces hyperlipidemia and hepatic steatosis in mice. Proc Natl Acad Sci USA 2005;102:6086–6091. 127 Mobbs CV, Isoda F, Makimura H, Mastaitis J, Mizuno T, Shu IW, Yen K, Yang XJ: Impaired glucose signaling as a cause of obesity and the metabolic syndrome: the glucoadipostatic hypothesis. Physiol Behav 2005;85: 3–23. 128 Jin L, Zhang S, Burguera BG, Couce ME, Osamura RY, Kulig E, Lloyd RV: Leptin and leptin receptor expression in rat and mouse pituitary cells. Endocrinology 2000; 141: 333–339. 129 Korbonits M, Chitnis MM, Gueorguiev M, Jordan S, Norman D, Kaltsas G, Burrin JM, Grossman AB: Leptin in pituitary adenomas – a novel paracrine regulatory system. Pituitary 2001;4:49–55. 130 Smolinska N, Przala J, Kaminski T, Siawrys G, Gajewska A, Kochman K, Okrasa S: Leptin gene expression in the hypothalamus and pituitary of pregnant pigs. Neuro Endocrinol Lett 2004; 25:191–195. 131 Yonekura S, Senoo T, Kobayashi Y, Yonezawa T, Katoh K, Obara Y: Effects of acetate and butyrate on the expression of leptin and short-form leptin receptor in bovine and rat anterior pituitary cells. Gen Comp Endocrinol 2003;133:165–172. 132 Crespi EJ, Denver RJ: Leptin (ob gene) of the South African clawed frog Xenopus laevis. Proc Natl Acad Sci USA 2006; 103: 10092– 10097. 133 Sone M, Nagata H, Takekoshi S, Osamura RY: Expression and localization of leptin receptor in the normal rat pituitary gland. Cell Tissue Res 2001;305:351–356.

208

134 Morash BA, Imran A, Wilkinson D, Ur E, Wilkinson M: Leptin receptors are developmentally regulated in rat pituitary and hypothalamus. Mol Cell Endocrinol 2003; 210:1–8. 135 McDuffie IA, Akhter N, Childs GV: Regulation of leptin mRNA and protein expression in pituitary somatotropes. J Histochem Cytochem 2004;52:263–273. 136 Sone M, Osamura RY: Leptin and the pituitary. Pituitary 2001;4:15–23. 137 Fauquier T, Lacampagne A, Travo P, Bauer K, Mollard P: Hidden face of the anterior pituitary. Trends Endocrinol Metab 2002; 13:304–309. 138 Jin L, Tsumanuma I, Ruebel KH, Bayliss JM, Lloyd RV: Analysis of homogeneous populations of anterior pituitary folliculostellate cells by laser capture microdissection and reverse transcription-polymerase chain reaction. Endocrinology 2001; 142: 1703–1709. 139 Lloyd RV, Jin L, Ruebel KH, Bayliss JM: Analysis of folliculostellate cells by laser capture microdissection and reverse transcription-polymerase chain reaction. Methods Enzymol 2002;356:248–255. 140 Lloyd RV, Jin L, Tsumanuma I, Vidal S, Kovacs K, Horvath E, Scheithauer BW, Couce ME, Burguera B: Leptin and leptin receptor in anterior pituitary function. Pituitary 2001;4:33–47. 141 Vidal S, Cohen SM, Horvath E, Kovacs K, Scheithauer BW, Burguera BG, Lloyd RV: Subcellular localization of leptin in non-tumorous and adenomatous human pituitaries: an immuno-ultrastructural study. J Histochem Cytochem 2000;48:1147–1152. 142 Cammisotto PG, Bukowiecki LJ: Role of calcium in the secretion of leptin from white adipocytes. Am J Physiol 2004; 287: R1380–R1386. 143 Bjorbaek C, Kahn BB: Leptin signaling in the central nervous system and the periphery. Recent Prog Horm Res 2004; 59: 305– 331. 144 Horsch D, Kahn CR: Region-specific mRNA expression of phosphatidylinositol 3-kinase regulatory isoforms in the central nervous system of C57BL/6J mice. J Comp Neurol 1999;415:105–120. 145 Tsumanuma I, Jin L, Zhang S, Bayliss JM, Scheithauer BW, Lloyd RV: Leptin signal transduction in the HP75 human pituitary cell line. Pituitary 2000;3:211–220. 146 Gautron L, Lafon P, Chaigniau M, Tramu G, Laye S: Spatiotemporal analysis of signal transducer and activator of transcription 3 activation in rat brain astrocytes and pituitary following peripheral immune challenge. Neuroscience 2002;112:717–729. 147 Bousquet C, Zatelli MC, Melmed S: Direct regulation of pituitary proopiomelanocortin by STAT-3 provides a novel mechanism for immuno-neuroendocrine interfacing. J Clin Invest 2000;106:1417–1425.


148 Glavaski-Joksimovic A, Rowe EW, Jeftinija K, Scanes CG, Anderson LL, Jeftinija S: Effects of leptin on intracellular calcium concentrations in isolated porcine somatotropes. Neuroendocrinology 2004; 80: 73–82. 149 Casanueva FF, Dieguez C: Neuroendocrine regulation and actions of leptin. Front Neuroendocrinol 1999; 20:317–363. 150 Ahima RS, Saper CB, Flier JS, Elmquist JK: Leptin regulation of neuroendocrine systems. Front Neuroendocrinol 2000;21:263– 307. 151 Kaminski T, Smolinska N, Gajewska A, Siawrys G, Okrasa S, Kochman K, Przala J: Leptin and long form of leptin receptor genes expression in the hypothalamus and pituitary during the luteal phase and early pregnancy in pigs. J Physiol Pharmacol 2006;57:95–108. 152 Szczepankiewicz D, Wojciechowicz T, Kaczmarek P, Nowak KW: Leptin and its receptors in the course of pregnancy in the rat. Int J Mol Med 2006;17:95–99. 153 Bennett PA, Lindell K, Karlsson C, Robinson IC, Carlsson LM, Carlsson B: Differential expression and regulation of leptin receptor isoforms in the rat brain: effects of fasting and oestrogen. Neuroendocrinology 1998;67:29–36. 154 Meli R, Pacilio M, Raso GM, Esposito E, Coppola A, Nasti A, Di Carlo C, Nappi C, Di Carlo R: Estrogen and raloxifene modulate leptin and its receptor in hypothalamus and adipose tissue from ovariectomized rats. Endocrinology 2004; 145:3115–3121. 155 Ramachandran R, Ocón-Grove OM, Metzger SL: Molecular cloning and tissue expression of chicken AdipoR1 and AdipoR2 complementary deoxyribonucleic acids. Domest Anim Endocrinol 2007;33:19– 31. 156 Malagon MM, Rodriguez-Pacheco F, Martinez-Fuentes AJ, Tovar S, Pinilla L, TenaSempere M, Dieguez C, Castano JP: Regulation of pituitary cell function by the adipokine adiponectin. Front Neuroendocrinol 2006;27:35. 157 Lu M, Tang Q, Olefsky JM, Webster NJG, Mellon PL: Adiponectin activates AMPK and increases luteinizing hormone secretion in LbT2 gonadotropes. Endocrine Society. Abstracts 2006, P1–381. 158 Brown R, Wiesner G, Ur E, Wilkinson M: Pituitary resistin gene expression is upregulated in vitro and in vivo by dexamethasone but is unaffected by rosiglitazone. Neuroendocrinology 2005; 81:41–48. 159 Unger JW, Betz M: Insulin receptors and signal transduction proteins in the hypothalamo-hypophyseal system: a review on morphological findings and functional implications. Histol Histopathol 1998;13: 1215–1224.


160 Kawamata Y, Habata Y, Fukusumi S, Hosoya M, Fujii R, Hinuma S, Nishizawa N, Kitada C, Onda H, Nishimura O, Fujino M: Molecular properties of apelin: tissue distribution and receptor binding. Biochim Biophys Acta 2001; 1538:162–171. 161 Platt KA, Min HY, Ross SR, Spiegelman BM: Obesity-linked regulation of the adipsin gene promoter in transgenic mice. Proc Natl Acad Sci USA 1989;86:7490–7494. 162 Zhang HT, Li LY, Zou XL, Song XB, Hu YL, Feng ZT, Wang TT: The immunohistochemical distribution of NGF, BDNF, NT-3, NT-4 in the brains of adult rhesus monkeys. J Histochem Cytochem 2007;55:1–19. 163 Peeraully MR, Jenkins JR, Trayhurn P: NGF gene expression and secretion in white adipose tissue: regulation in 3T3-L1 adipocytes by hormones and inflammatory cytokines. Am J Physiol 2004;287:E331–E339. 164 Gomez-Ambrosi J, Catalan V, Diez-Caballero A, Martinez-Cruz LA, Gil MJ, GarciaFoncillas J, Cienfuegos JA, Salvador J, Mato JM, Fruhbeck G: Gene expression profile of omental adipose tissue in human obesity. FASEB J 2004;18:215–217.


165 Guan H, Arany E, van Beek JP, ChamsonReig A, Thyssen S, Hill DJ, Yang K: Adipose tissue gene expression profiling reveals distinct molecular pathways that define visceral adiposity in offspring of maternal protein-restricted rats. Am J Physiol 2005; 288:E663–E673. 166 Friedberg M, Zoumakis E, Hiroi N, Bader T, Chrousos GP, Hochberg Z: Modulation of 11␤-hydroxysteroid dehydrogenase type 1 in mature human subcutaneous adipocytes by hypothalamic messengers. J Clin Endocrinol Metab 2003;88:385–393. 167 Seres J, Bornstein SR, Seres P, Willenberg HS, Schulte KM, Scherbaum WA, EhrhartBornstein M: Corticotropin-releasing hormone system in human adipose tissue. J Clin Endocrinol Metab 2004;89:965–970.

168 McIntosh TK, Vink R, Noble L, Yamakami I, Fernyak S, Soares H, Faden AL: Traumatic brain injury in the rat: characterization of a lateral fluid-percussion model. Neuroscience 1989;28:233–244. 169 Beretta E, Dube MG, Kalra PS, Kalra SP: Long-term suppression of weight gain, adiposity, and serum insulin by central leptin gene therapy in prepubertal rats: effects on serum ghrelin and appetite-regulating genes. Pediatr Res 2002;52:189–198. 170 Ehrhardt RA, Bell AW, Boisclair YR: Spatial and developmental regulation of leptin in fetal sheep. Am J Physiol 2002; 282: R1628–R1635. 171 Boswell T, Dunn IC, Wilson PW, Joseph N, Burt DW, Sharp PJ: Identification of a nonmammalian leptin-like gene: characterization and expression in the tiger Salamander (Ambystoma tigrinum). Gen Comp Endocrinol 2006;146:157–166. 172 Buono S, Putti R: Leptin, leptin receptors and ACTH immunoreactivities are present in the gastrointestinal tract and the neural tube of tadpoles of the newt Triturus. J Mol Histol 2004;35:103–109.


209



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



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


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.

References 1 Flegal KM: Epidemiologic aspects of overweight and obesity in the United States. Physiol Behav 2005;86:599–602. 2 Wada H, Yamatodani A, Inagaki N, Itowi N, Wang NP, Fukui H: Histaminergic neuron system and its function. Adv Exp Med Biol 1988;236:343–357. 3 Kalucy RS: Drug-induced weight gain. Drugs 1980;19:268–278. 4 Arrang JM, Garbarg M, Lancelot JC, Lecomte JM, Pollard H, Robba M, et al: Highly potent and selective ligands for histamine H3receptors. Nature 1987;327:117–123. 5 Berne RM, Levy MN, Koeppen BM, Stanton BA: Physiology, ed 5. St Louis, Mosby, 2004. 6 Knigge U, Warberg J: The role of histamine in the neuroendocrine regulation of pituitary hormone secretion. Acta Endocrinol (Copenh) 1991;124:609–619. 7 Schwartz JC: Histamine receptors in the brain and their possible functions; in Ganellin CR, Parsons ME (eds): Pharmacology of Histamine Receptors, ed 1. Bristol, Wright, 1982, p 353. 8 Schwartz JC, Arrang JM, Garbarg M, Pollard H, Ruat M: Histaminergic transmission in the mammalian brain. Physiol Rev 1991; 71: 1–51. 9 Palacios JM, Wamsley JK, Kuhar MJ: The distribution of histamine H1-receptors in the rat brain: an autoradiographic study. Neuroscience 1981;6:15–37. 10 Ruat M, Traiffort E, Bouthenet ML, Schwartz JC, Hirschfeld J, Buschauer A, et al: Reversible and irreversible labeling and autoradiographic localization of the cerebral histamine H2 receptor using [125I]iodinated probes. Proc Natl Acad Sci USA 1990; 87: 1658–1662. 11 Smits R, Mulder A: Inhibitory effects of histamine on the release of serotonin and noradrenaline from rat brain slices. Neurochem Int 1991;18:215–220. 12 Clineschmidt BV, Lotti VJ: Histamine: intraventricular injection suppresses ingestive behavior of the cat. Arch Int Pharmacodyn Ther 1973;206:288–298. 13 Itowi N, Nagai K, Nakagawa H, Watanabe T, Wada H: Changes in the feeding behavior of rats elicited by histamine infusion. Physiol Behav 1988;44:221–226.

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.


213

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.

214

40 Yoshimatsu H, Itateyama E, Kondou S, Tajima D, Himeno K, Hidaka S, et al: Hypothalamic neuronal histamine as a target of leptin in feeding behavior. Diabetes 1999;48:2286– 2291. 41 Morimoto T, Yamamoto Y, Yamatodani A: Leptin facilitates histamine release from the hypothalamus in rats. Brain Res 2000; 868: 367–369. 42 Itateyama E, Chiba S, Sakata T, Yoshimatsu H: Hypothalamic neuronal histamine in genetically obese animals: its implication of leptin action in the brain. Exp Biol Med (Maywood ) 2003;228: 1132–1137. 43 Bugajski J, Janusz Z: Lipolytic responses induced by intracerebroventricular administration of histamine in the rat. Agents Actions 1981;11:147–150.


44 Tsuda K, Yoshimatsu H, Niijima A, Chiba S, Okeda T, Sakata T: Hypothalamic histamine neurons activate lipolysis in rat adipose tissue. Exp Biol Med (Maywood ) 2002; 227: 208–213. 45 Yoshimatsu H, Hidaka S, Niijima A, Sakata T: Histamine neurons down-regulate ob gene expression in rat white adipose tissue. Inflamm Res 2001;50(suppl 2):S72–S73. 46 Fulop AK, Foldes A, Buzas E, Hegyi K, Miklos IH, Romics L, et al: Hyperleptinemia, visceral adiposity, and decreased glucose tolerance in mice with a targeted disruption of the histidine decarboxylase gene. Endocrinology 2003;144:4306–4314. 47 Takahashi K, Suwa H, Ishikawa T, Kotani H: Targeted disruption of H3 receptors results in changes in brain histamine tone leading to an obese phenotype. J Clin Invest 2002; 110: 1791–1799.

Jørgensen /Knigge /Warberg /Kjær


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



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


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


Expression of Ghrelin in Pancreatic Islets

217

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


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

WT Ghrelin –/–

600

0.5 Plasma insulin (ng/ml)

Blood glucose (mg/dl)

500

*

400

**

300 200

**

100

**

0.4

*

**

0.3 0.2 0.1 0

0 0

a

WT Ghrelin –/–

0.6

15 30 45 60 75 90 105 Minutes after glucose injection (2.5 g/kg)

0

120

b

15

30 45 60 75 90 105 Minutes after glucose injection (2.5 g/kg)

120

Fig. 1. a, b Ghrelin deletion increases glucose-induced insulin secretion. Mice were fasted for 18 h and 2.5 g/kg glucose was injected intraperitoneally. * p ! 0.05, WT vs. KO; ** p ! 0.001 (produced with permission [65], © 2006 Elsevier).

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).



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

219

2.0

Glucose 8.3 mM

Glucose 8.3 mM

Glucose 8.3 mM

1.6

Fura-2 ratio (F340/F380)


2.0

1.2

0.8

1.6 Ghrelin 10 nM 1.2

0.8

0.4

0.4

2.0

2.0

Glucose 8.3 mM Glucose 8.3 mM

1.6

[D-Lys3]-GHRP-6 1 μM Fura-2 ratio (F340/F380)


Glucose 8.3 mM

Ghrelin 10 nM

1.2

0.8

Ghrelin 10 nM

1.6

1.2

0.8

0.4

0.4 0

10

a

20

30

40

50

0

20

b

Time (min)

40

60

80

Time (min)

120 80 40

*

0

a

0 30 60 90 120 150 Minutes after insulin injection

35 30 25 20 15 10

*

5 0 Basal

80 70 60 50 40 30 20 10 0

*

WT Ghrelin –/–

Glucose disposal rate (mg/kg/min)

Blood glucose (mg/dl)

160

WT Ghrelin –/–

Glucose infusion rate (mg/kg/min)

WT Ghrelin –/–

Glucose production rate (mg/kg/min)

2

140 120

*

WT Ghrelin –/–

100 80 60 40 20 0

Clamp

b

c

d

3 Fig. 2. Inhibitory effects of ghrelin on glucose-induced [Ca 2+]i

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

220


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).

Sun /Asnicar /Smith

WT

500

Ghrelin –/–

450

ob/ob

*

400

*

300 250 200 150

ob/ob

25 20 15 10

**

100 5

0

0 8

10 12 Age (weeks)

a

8w, fed 12w, fed 12w, fasted Weeks of age and feeding status

14

b

600

20

ob/ob

Plasma insulin (ng/ml)

400

* *

300

*

200 100 0

ob/ob Ghrelin–/– ob/ob

*

Ghrelin–/– ob/ob

500 Blood glucose (mg/dl)

**


50

*

16 12 8 4 0

0

c

Ghrelin –/–

30


350

WT

35

Plasma insulin (ng/ml)

Average blood glucose (mg/dl)

550

15

30

45

60

75

90

105

120

Minutes after glucose injection (0.625 g/kg)

0

d

15

30

45

60

75

90

105

120

Minutes after glucose injection (0.625 g/kg)

Fig. 4. Ghrelin deletion improves diabetic phenotype of ob/ob mice. The blood glucose (a) and plasma insulin (b) of WT, ghrelin–/–, ob/ob and ghrelin–/– ob/ob mice were measured under an ad libitum fed condition. Glucose and insulin responses of GTT (c, d) in ob/ob and ghrelin–/– ob/ob mice. GTT was performed on 18 h fasted

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-



221

Brain No change in feeding and weight Increased glucose production

Reduced insulin secretion

Ghrelin Liver

Pancreas Reduced glucose uptake

Muscle

Fig. 5. Schematic diagram of ghrelin’s role

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


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.



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


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.

References 1 Smith RG, Pong S-S, Hickey GJ, Jacks TM, Cheng K, Leonard RJ, Cohen CJ, Arena JP, Chang CH, Drisko JE, Wyvratt MJ Jr, Fisher MH, Nargund RP, Patchett AA: Modulation of pulsatile GH release through a novel receptor in hypothalamus and pituitary gland. Recent Prog Horm Res 1996;51:261–286. 2 Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum CI, Hamelin M, Hreniuk DL, Palyha OC, Anderson J, Paress PS, Diaz C, Chou M, Liu KK, McKee KK, Pong S-S, Chaung L-YP, Elbrecht A, Dashkevicz M, Heavens R, Rigby M, Sirinathsinghji DJS, Dean DC, Melillo DG, Patchett AA, Nargund RP, Griffin PR, DeMartino JA, Gupta SK, Schaeffer JM, Smith RG, Van der Ploeg LHT: A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 1996;273:974–977. 3 Smith RG, Van der Ploeg LH, Howard AD, Feighner SD, Cheng K, Hickey GJ, Wyvratt MJ Jr, Fisher MH, Nargund RP, Patchett AA: Peptidomimetic regulation of growth hormone secretion. Endocr Rev 1997; 18: 621– 645. 4 Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K: Ghrelin is a growthhormone-releasing acylated peptide from stomach. Nature 1999;402:656–660.


5 Peino R, Baldelli R, Rodriguez-Garcia J, Rodriguez-Segade S, Kojima M, Kangawa K, Arvat E, Ghigo E, Dieguez C, Casanueva FF: Ghrelin-induced growth hormone secretion in humans. Eur J Endocrinol 2000;143:R11– R14. 6 Hataya Y, Akamizu T, Takaya K, Kanamoto N, Ariyasu H, Saijo M, Moriyama K, Shimatsu A, Kojima M, Kangawa K, Nakao K: A low dose of ghrelin stimulates GH release synergistically with GH-releasing hormone in humans. J Clin Endocrinol Metab 2001; 86: 4552. 7 Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, Matsukura S: A role for ghrelin in the central regulation of feeding. Nature 2001;409:194–198. 8 Wren AM, Small CJ, Abbott CR, Dhillo WS, Seal LJ, Cohen MA, Batterham RL, Taheri S, Stanley SA, Ghatei MA, Bloom SR: Ghrelin causes hyperphagia and obesity in rats. Diabetes 2001;50:2540–2547. 9 Wren AM, Seal LJ, Cohen MA, Brynes AE, Frost GS, Murphy KG, Dhillo WS, Ghatei MA, Bloom SR: Ghrelin enhances appetite and increases food intake in humans. J Clin Endocrinol Metab 2001;86:5992. 10 Smith RG: Development of growth hormone secretagogues. Endocr Rev 2005; 26: 346– 360.

11 Smith RG, Jiang H, Sun Y: Developments in ghrelin biology and potential clinical relevance. Trends Endocrinol Metab 2005; 16: 436–442. 12 Broglio F, Prodam F, Me E, Riganti F, Lucatello B, Granata R, Benso A, Muccioli G, Ghigo E: Ghrelin: Endocrine, metabolic and cardiovascular actions. J Endocrinol Invest 2005;28:23–25. 13 Sun Y, Wang P, Zheng H, Smith RG: Ghrelin stimulation of growth hormone release and appetite is mediated through the growth hormone secretagogue receptor. Proc Natl Acad Sci USA 2004;101:4679–4684. 14 Sun Y, Ahmed S, Smith RG: Deletion of ghrelin impairs neither growth nor appetite. Mol Cell Biol 2003; 23:7973–7981. 15 Zhang JV, Ren PG, Avsian-Kretchmer O, Luo CW, Rauch R, Klein C, Hsueh AJ: Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin’s effects on food intake. Science 2005;310:996–999. 16 Broglio F, Gottero C, Prodam F, Gauna C, Muccioli G, Papotti M, Abribat T, Van Der Lely AJ, Ghigo E: Non-acylated ghrelin counteracts the metabolic but not the neuroendocrine response to acylated ghrelin in humans. J Clin Endocrinol Metab 2004; 89: 3062–3065.


225

17 Gauna C, Delhanty PJ, Hofland LJ, Janssen JA, Broglio F, Ross RJ, Ghigo E, van der Lely AJ: Ghrelin stimulates, while des-octanoyl ghrelin inhibits, glucose output by primary hepatocytes. J Clin Endocrinol Metab 2005; 90:1055–1060. 18 Ariyasu H, Takaya K, Iwakura H, Hosoda H, Akamizu T, Arai Y, Kangawa K, Nakao K: Transgenic mice overexpressing des-acyl ghrelin show small phenotype. Endocrinology 2005;146:355–364. 19 Toshinai K, Yamaguchi H, Sun Y, Smith RG, Yamanaka A, Sakurai T, Date Y, Mondal MS, Shimbara T, Kawagoe T, Murakami N, Miyazato M, Kangawa K, Nakazato M: Des-acyl ghrelin induces food intake by a mechanism independent of the growth hormone secretagogue receptor. Endocrinology 2006; 147: 2306–2314. 20 Levin BE, Routh VH, Kang L, Sanders NM, Dunn-Meynell AA: Neuronal glucosensing: What do we know after 50 years? Diabetes 2004;53:2521–2528. 21 Burdakov D, Luckman SM, Verkhratsky A: Glucose-sensing neurons of the hypothalamus. Philos Trans R Soc Lond B Biol Sci 2005;360:2227–2235. 22 Penicaud L, Leloup C, Lorsignol A, Alquier T, Guillod E: Brain glucose sensing mechanism and glucose homeostasis. Curr Opin Clin Nutr Metab Care 2002;5:539–543. 23 Thorens B: A gene knockout approach in mice to identify glucose sensors controlling glucose homeostasis. Pflügers Arch 2003; 445:482–490. 24 Marty N, Dallaporta M, Foretz M, Emery M, Tarussio D, Bady I, Binnert C, Beermann F, Thorens B: Regulation of glucagon secretion by glucose transporter type 2 (glut2) and astrocyte-dependent glucose sensors. J Clin Invest 2005;115:3545–3553. 25 Kim MS, Yoon CY, Park KH, Shin CS, Park KS, Kim SY, Cho BY, Lee HK: Changes in ghrelin and ghrelin receptor expression according to feeding status. Neuroreport 2003; 14:1317–1320. 26 Chen X, Ge YL, Jiang ZY, Liu CQ, Depoortere I, Peeters TL: Effects of ghrelin on hypothalamic glucose-responding neurons in rats. Brain Res 2005;1055:131–136. 27 Cowley MA, Smith RG, Diano S, Tschop M, Pronchuk N, Grove KL, Strasburger CJ, Bidlingmaier M, Esterman M, Heiman ML, Garcia-Segura LM, Nillni EA, Mendez P, Low MJ, Sotonyi P, Friedman JM, Liu H, Pinto S, Colmers WF, Cone RD, Horvath TL: The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis. Neuron 2003;37:649–661.

226

28 Chen HY, Trumbauer ME, Chen AS, Weingarth DT, Adams JR, Frazier EG, Shen Z, Marsh DJ, Feighner SD, Guan XM, Ye Z, Nargund RP, Smith RG, Van Der Ploeg LH, Howard AD, MacNeil DJ, Qian S: Orexigenic action of peripheral ghrelin is mediated by neuropeptide Y and agouti-related protein. Endocrinology 2004; 145:2607–2612. 29 Bailey ART, Von Englehardt N, Smith RG, Leng G, Dickson SL: Growth hormone secretagogue activation of the arcuate nucleus and brainstem occurs via a non-noradrenergic pathway. J Neuroendocrinol 2000; 12: 191– 198. 30 Dickson SL, Viltart O, Bailey AR, Leng G: Attenuation of the growth hormone secretagogue induction of fos protein in the rat arcuate nucleus by central somatostatin action. Neuroendocrinology 1997;66:188–194. 31 Zheng H, Bailey ART, Jiang M-H, Honda K, Chen HY, Trumbauer ME, Van der Ploeg LHT, Schaeffer JM, Leng G, Smith RG: Somatostatin receptor subtype-2 knockout mice are refractory to growth hormone negative feedback on arcuate neurons. Mol Endocrinol 1997;11:1709–1717. 32 Solomon A, De Fanti BA, Martinez JA: The nucleus tractus solitari participates in peripheral ghrelin glucostatic hunger signalling mediated by insulin. Neuropeptides 2006;40:169–175. 33 Poykko SM, Kellokoski E, Horkko S, Kauma H, Kesaniemi YA, Ukkola O: Low plasma ghrelin is associated with insulin resistance, hypertension, and the prevalence of type 2 diabetes. Diabetes 2003;52:2546–2553. 34 Broglio F, Gottero C, Benso A, Prodam F, Volante M, Destefanis S, Gauna C, Muccioli G, Papotti M, van der Lely AJ, Ghigo E: Ghrelin and the endocrine pancreas. Endocrine 2003;22:19–24. 35 Date Y, Nakazato M, Hashiguchi S, Dezaki K, Mondal MS, Hosoda H, Kojima M, Kangawa K, Arima T, Matsuo H, Yada T, Matsukura S: Ghrelin is present in pancreatic ␣ cells of humans and rats and stimulates insulin secretion. Diabetes 2002;51:124–129. 36 Volante M, Allia E, Gugliotta P, Funaro A, Broglio F, Deghenghi R, Muccioli G, Ghigo E, Papotti M: Expression of ghrelin and of the GH secretagogue receptor by pancreatic islet cells and related endocrine tumors. J Clin Endocrinol Metab 2002;87:1300–1308. 37 Wierup N, Yang S, McEvilly RJ, Mulder H, Sundler F: Ghrelin is expressed in a novel endocrine cell type in developing rat islets and inhibits insulin secretion from INS-1 (832/13) cells. J Histochem Cytochem 2004; 52:301–310. 38 Prado CL, Pugh-Bernard AE, Elghazi L, Sosa-Pineda B, Sussel L: Ghrelin cells replace insulin-producing ␤ cells in two mouse models of pancreas development. Proc Natl Acad Sci USA 2004;101:2924–2929.


39 Guan XM, Yu H, Palyha OC, McKee KK, Feighner SD, Sirinathsinghji DJ, Smith RG, Van der Ploeg LH, Howard AD: Distribution of mRNA encoding the growth hormone secretagogue receptor in brain and peripheral tissues. Brain Res Mol Brain Res 1997;48:23– 29. 40 Gnanapavan S, Kola B, Bustin SA, Morris DG, McGee P, Fairclough P, Bhattacharya S, Carpenter R, Grossman AB, Korbonits M: The tissue distribution of the mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans. J Clin Endocrinol Metab 2002; 87: 2988. 41 Kageyama H, Funahashi H, Hirayama M, Takenoya F, Kita T, Kato S, Sakurai J, Lee EY, Inoue S, Date Y, Nakazato M, Kangawa K, Shioda S: Morphological analysis of ghrelin and its receptor distribution in the rat pancreas. Regul Pept 2005; 126:67–71. 42 Cummings DE, Purnell JQ, Frayo RS, Schmidova K, Wisse BE, Weigle DS: A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans. Diabetes 2001;50: 1714–1719. 43 Holdstock C, Ludvigsson J, Karlsson FA: Abnormal ghrelin secretion in new onset childhood type 1 diabetes. Diabetologia 2004; 47: 150–151. 44 Soriano-Guillen L, Barrios V, Lechuga-Sancho A, Chowen JA, Argente J: Response of circulating ghrelin levels to insulin therapy in children with newly diagnosed type 1 diabetes mellitus. Pediatr Res 2004; 55: 830– 835. 45 Katsuki A, Urakawa H, Gabazza EC, Murashima S, Nakatani K, Togashi K, Yano Y, Adachi Y, Sumida Y: Circulating levels of active ghrelin is associated with abdominal adiposity, hyperinsulinemia and insulin resistance in patients with type 2 diabetes mellitus. Eur J Endocrinol 2004; 151: 573– 577. 46 Murdolo G, Lucidi P, Di Loreto C, Parlanti N, De Cicco A, Fatone C, Fanelli CG, Bolli GB, Santeusanio F, De Feo P: Insulin is required for prandial ghrelin suppression in humans. Diabetes 2003;52:2923–2927. 47 Kahn BB, Flier JS: Obesity and insulin resistance. J Clin Invest 2000;106:473–481. 48 Booth DA: Some characteristics of feeding during streptoxotocin-induced diabetes in the rat. J Comp Physiol Psychol 1972;80:238– 249. 49 Ishii S, Kamegai J, Tamura H, Shimizu T, Sugihara H, Oikawa S: Role of ghrelin in streptozotocin-induced diabetic hyperphagia. Endocrinology 2002; 143:4934–4937. 50 Dong J, Peeters TL, De Smet B, Moechars D, Delporte C, Vanden Berghe P, Coulie B, Tang M, Depoortere I: Role of endogenous ghrelin in the hyperphagia of mice with streptozotocin-induced diabetes. Endocrinology 2006; 147:2634–2642.

Sun /Asnicar /Smith

51 Wortley KE, Del Rincon JP, Murray JD, Garcia K, Iida K, Thorner MO, Sleeman MW: Absence of ghrelin protects against earlyonset obesity. J Clin Invest 2005; 115: 3573– 3578. 52 Wortley KE, Anderson KD, Garcia K, Murray JD, Malinova L, Liu R, Moncrieffe M, Thabet K, Cox HJ, Yancopoulos GD, Wiegand SJ, Sleeman MW: Genetic deletion of ghrelin does not decrease food intake but influences metabolic fuel preference. Proc Natl Acad Sci USA 2004;101:8227–8232. 53 Broglio F, Gottero C, Prodam F, Destefanis S, Gauna C, Me E, Riganti F, Vivenza D, Rapa A, Martina V, Arvat E, Bona G, van der Lely AJ, Ghigo E: Ghrelin secretion is inhibited by glucose load and insulin-induced hypoglycaemia but unaffected by glucagon and arginine in humans. Clin Endocrinol (Oxf) 2004;61:503–509. 54 Lee HM, Wang G, Englander EW, Kojima M, Greeley GH Jr: Ghrelin, a new gastrointestinal endocrine peptide that stimulates insulin secretion: enteric distribution, ontogeny, influence of endocrine, and dietary manipulations. Endocrinology 2002; 143:185–190. 55 Egido EM, Rodriguez-Gallardo J, Silvestre RA, Marco J: Inhibitory effect of ghrelin on insulin and pancreatic somatostatin secretion. Eur J Endocrinol 2002;146:241–244. 56 Reimer MK, Pacini G, Ahren B: Dose-dependent inhibition by ghrelin of insulin secretion in the mouse. Endocrinology 2003; 144:916–921. 57 Broglio F, Arvat E, Benso A, Gottero C, Muccioli G, Papotti M, van der Lely AJ, Deghenghi R, Ghigo E: Ghrelin, a natural GH secretagogue produced by the stomach, induces hyperglycemia and reduces insulin secretion in humans. J Clin Endocrinol Metab 2001; 86:5083–5086. 58 Broglio F, Gottero C, Benso A, Prodam F, Destefanis S, Gauna C, Maccario M, Deghenghi R, van der Lely AJ, Ghigo E: Effects of ghrelin on the insulin and glycemic responses to glucose, arginine, or free fatty acids load in humans. J Clin Endocrinol Metab 2003;88:4268–4272. 59 Bagnasco M, Kalra PS, Kalra SP: Ghrelin and leptin pulse discharge in fed and fasted rats. Endocrinology 2002; 143:726–729. 60 Bagnasco M, Dube MG, Katz A, Kalra PS, Kalra SP: Leptin expression in hypothalamic PVN reverses dietary obesity and hyperinsulinemia but stimulates ghrelin. Obes Res 2003;11:1463–1470. 61 Seufert J: Leptin effects on pancreatic ␤-cell gene expression and function. Diabetes 2004;53(suppl 1):S152–S158. 62 Murata M, Okimura Y, Iida K, Matsumoto M, Sowa H, Kaji H, Kojima M, Kangawa K, Chihara K: Ghrelin modulates the downstream molecules of insulin signaling in hepatoma cells. J Biol Chem 2002; 277:5667– 5674.


63 Anderwald C, Muller G, Koca G, Furnsinn C, Waldhausl W, Roden M: Short-term leptin-dependent inhibition of hepatic gluconeogenesis is mediated by insulin receptor substrate-2. Mol Endocrinol 2002; 16: 1612– 1628. 64 Ceddia RB, Koistinen HA, Zierath JR, Sweeney G: Analysis of paradoxical observations on the association between leptin and insulin resistance. FASEB J 2002;16:1163–1176. 65 Sun Y, Asnicar M, Saha PK, Chan L, Smith RG: Ablation of ghrelin improves the diabetic but not obese phenotype of ob/ob mice. Cell Metab 2006;3:379–386. 66 Dezaki K, Hosoda H, Kakei M, Hashiguchi S, Watanabe M, Kangawa K, Yada T: Endogenous ghrelin in pancreatic islets restricts insulin release by attenuating Ca 2+ signaling in ␤ cells: implication in the glycemic control in rodents. Diabetes 2004; 53:3142–3151. 67 Gauna C, Meyler FM, Janssen JA, Delhanty PJ, Abribat T, van Koetsveld P, Hofland LJ, Broglio F, Ghigo E, van der Lely AJ: Administration of acylated ghrelin reduces insulin sensitivity, whereas the combination of acylated plus unacylated ghrelin strongly improves insulin sensitivity. J Clin Endocrinol Metab 2004;89:5035–5042. 68 Ariyasu H, Takaya K, Hosoda H, Iwakura H, Ebihara K, Mori K, Ogawa Y, Hosoda K, Akamizu T, Kojima M, Kangawa K, Nakao K: Delayed short-term secretory regulation of ghrelin in obese animals: evidenced by a specific RIA for the active form of ghrelin. Endocrinology 2002; 143:3341–3350. 69 Asakawa A, Inui A, Kaga T, Katsuura G, Fujimiya M, Fujino MA, Kasuga M: Antagonism of ghrelin receptor reduces food intake and body weight gain in mice. Gut 2003; 52: 947–952. 70 Poitout V, Robertson RP: An integrated view of ␤-cell dysfunction in type 2 diabetes. Annu Rev Med 1996;47:69–83. 71 Zhang CY, Baffy G, Perret P, Krauss S, Peroni O, Grujic D, Hagen T, Vidal-Puig AJ, Boss O, Kim YB, Zheng XX, Wheeler MB, Shulman GI, Chan CB, Lowell BB: Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, ␤cell dysfunction, and type 2 diabetes. Cell 2001;105:745–755. 72 Erlanson-Albertsson C: The role of uncoupling proteins in the regulation of metabolism. Acta Physiol Scand 2003;178:405–412. 73 Blander G, Guarente L: The Sir2 family of protein deacetylases. Annu Rev Biochem 2004;73:417–435. 74 Bordone L, Motta MC, Picard F, Robinson A, Jhala US, Apfeld J, McDonagh T, Lemieux M, McBurney M, Szilvasi A, Easlon EJ, Lin SJ, Guarente L: Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic ␤ cells. PLoS Biol 2006; 4:e31.

75 Moynihan KA, Grimm AA, Plueger MM, Bernal-Mizrachi E, Ford E, Cras-Meneur C, Permutt MA, Imai S: Increased dosage of mammalian Sir2 in pancreatic ␤ cells enhances glucose-stimulated insulin secretion in mice. Cell Metab 2005;2:105–117. 76 Doi A, Shono T, Nishi M, Furuta H, Sasaki H, Nanjo K: IA-2␤, but not IA-2, is induced by ghrelin and inhibits glucose-stimulated insulin secretion. Proc Natl Acad Sci USA 2006;103:885–890. 77 Fagan SP, Azizzadeh A, Moldovan S, Ray MK, Adrian TE, Ding X, Coy DH, Brunicardi FC: Insulin secretion is inhibited by subtype five somatostatin receptor in the mouse. Surgery 1998;124:254–259. 78 Tirone TA, Norman MA, Moldovan S, DeMayo FJ, Wang XP, Brunicardi FC: Pancreatic somatostatin inhibits insulin secretion via SSTR-5 in the isolated perfused mouse pancreas model. Pancreas 2003; 26:e67–e73. 79 Nagaya N, Kojima M, Uematsu M, Yamagishi M, Hosoda H, Oya H, Hayashi Y, Kangawa K: Hemodynamic and hormonal effects of human ghrelin in healthy volunteers. Am J Physiol 2001;280:R1483–R1487. 80 Nagaya N, Miyatake K, Uematsu M, Oya H, Shimizu W, Hosoda H, Kojima M, Nakanishi N, Mori H, Kangawa K: Hemodynamic, renal, and hormonal effects of ghrelin infusion in patients with chronic heart failure. J Clin Endocrinol Metab 2001;86:5854–5859. 81 Nonogaki K, Ohashi-Nozue K, Oka Y: A negative feedback system between brain serotonin systems and plasma active ghrelin levels in mice. Biochem Biophys Res Commun 2006;341:703–707. 82 Adeghate E, Ponery AS, Pallot D, Parvez SH, Singh J: Distribution of serotonin and its effect on insulin and glucagon secretion in normal and diabetic pancreatic tissues in rat. Neuroendocrinol Lett 1999; 20:315–322. 83 Iwakura H, Hosoda K, Son C, Fujikura J, Tomita T, Noguchi M, Ariyasu H, Takaya K, Masuzaki H, Ogawa Y, Hayashi T, Inoue G, Akamizu T, Hosoda H, Kojima M, Itoh H, Toyokuni S, Kangawa K, Nakao K: Analysis of rat insulin II promoter-ghrelin transgenic mice and rat glucagon promoter-ghrelin transgenic mice. J Biol Chem 2005; 280: 15247–15256. 84 Granata R, Settanni F, Catapano F, Trovato L, Destefanis S, Gallo D, Isgaard J, Muccioli G, Ghigo E: [p2–166] acylated and unacylated ghrelin promote proliferation and inhibit serum starvation- and cytokine-induced apoptosis of pancreatic ␤ cells through camp/pka, erk1/2 and pi3k/akt: ENDO 2006. Boston/Mass, The Endocrine Society, 2006. 85 Toshinai K, Date Y, Murakami N, Shimada M, Mondal MS, Shimbara T, Guan JL, Wang QP, Funahashi H, Sakurai T, Shioda S, Matsukura S, Kangawa K, Nakazato M: Ghrelininduced food intake is mediated via the orexin pathway. Endocrinology 2003; 144: 1506– 1512.


227

86 Cai XJ, Widdowson PS, Harrold J, Wilson S, Buckingham RE, Arch JR, Tadayyon M, Clapham JC, Wilding J, Williams G: Hypothalamic orexin expression: modulation by blood glucose and feeding. Diabetes 1999;48: 2132–2137. 87 Nowak KW, Mackowiak P, Switonska MM, Fabis M, Malendowicz LK: Acute orexin effects on insulin secretion in the rat: In vivo and in vitro studies. Life Sci 2000; 66: 449– 454. 88 Nakabayashi M, Suzuki T, Takahashi K, Totsune K, Muramatsu Y, Kaneko C, Date F, Takeyama J, Darnel AD, Moriya T, Sasano H: Orexin-A expression in human peripheral tissues. Mol Cell Endocrinol 2003; 205: 43– 50.

228

89 Ouedraogo R, Naslund E, Kirchgessner AL: Glucose regulates the release of orexin-A from the endocrine pancreas. Diabetes 2003; 52:111–117. 90 Kirchgessner AL: Orexins in the brain-gut axis. Endocr Rev 2002;23:1–15. 91 Gauna C, Delhanty PJ, van Aken MO, Janssen JA, Themmen AP, Hofland LJ, Culler M, Broglio F, Ghigo E, van der Lely AJ: Unacylated ghrelin is active on the INS-1E rat insulinoma cell line independently of the growth hormone secretagogue receptor type 1a and the corticotropin-releasing factor-2 receptor. Mol Cell Endocrinol 2006;251:103–111.


92 Depoortere I, Thijs T, Peeters T: The contractile effect of the ghrelin receptor antagonist, D -Lys3-GHRP-6, in rat fundic strips is mediated through 5-HT receptors. Eur J Pharmacol 2006;537:160–165. 93 Ohkura M, Tanaka N, Kobayashi H, Wada A, Nakai J, Yamamoto R: Insulin induces internalization of the 5-HT2a receptor expressed in HEK293 cells. Eur J Pharmacol 2005; 518: 18–21. 94 Irako T, Akamizu T, Hosoda H, Iwakura H, Ariyasu H, Tojo K, Tajima N, Kangawa K: Ghrelin prevents development of diabetes at adult age in streptozotocin-treated newborn rats. Diabetologia 2006;49:1264–1273. 95 Ahima RS: Ghrelin - A new player in glucose homeostasis? Cell Metab 2006;3:301–302.

Sun /Asnicar /Smith


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



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


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


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


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

235

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


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.

References 1 Casanueva FF, Dieguez C: Neuroendocrine regulation and actions of leptin. Front Neuroendocrinol 1999; 20:317–363. 2 Fink G: Neuroendocrine regulation of pituitary function: general principles; in Conn PM, Freeman ME (eds): Neuroendocrinology in Physiology and Medicine. Totowa/NJ, Humana Press, 2000, pp 107–134. 3 Tena-Sempere M, Huhtaniemi I: Gonadotropins and gonadotropin receptors; in Fauser BCJM (ed): Reproductive Medicine – Molecular, Cellular and Genetic Fundamentals. New York, Parthenon Publishing, 2003, pp 225–244. 4 Friedman JM, Halaas JL: Leptin and regulation of body weight in mammals. Nature 1998;395:763–770. 5 Rosenbaum M, Leibel RL: Leptin; a molecule integrating somatic energy stores, energy expenditure and fertility. Trends Endocrinol Metab 1998;9:117–124.

238

6 Ahima RS, Saper CB, Flier JS, Elmquist JK: Leptin regulation of neuroendocrine systems. Front Neuroendocrinol 2000; 21: 263– 307. 7 Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM: Positional cloning of the mouse obese gene and its human homologue. Nature 1994;372:425–432. 8 Wauters M, Considine RV, van Gaal LF: Human leptin: From an adipocyte hormone to an endocrine mediator. Eur J Endocrinol 2000;143:293–311. 9 Sahu A: A hypothalamic role in energy balance with special emphasis on leptin. Endocrinology 2004;145:2613–2620. 10 Zigman JM, Elmquist JK: From anorexia to obesity – the Yin and Yang of body weight control. Endocrinology 2003; 144: 3749– 3756.


11 Chehab FF, Mounzih K, Lim ME: Early onset of reproductive function in normal female mice treated with leptin. Science 1997; 275, 88–90. 12 Mounzih K, Lu R, Chehab FF: Leptin treatment rescues the sterility of genetically obese ob/ob males. Endocrinology 1997;138:1190– 1193. 13 Barash IA, Cheung CC, Weigle DS, Ren H, Kabigting EB, Kuijper JL, Clifton DK, Steiner RA: Leptin is a metabolic signal to the reproductive system. Endocrinology 1996; 137:3144–3147. 14 Cheung CC, Thornton JE, Nurani SD, Clifton DK, Steiner RA: A reassessment of leptin’s role in triggering the onset of puberty in the rat and mouse. Neuroendocrinology 2001;74:12–21.

Tena-Sempere

15 García Mayor RV, Andrade MA, Rios M, Lage M, Dieguez C, Casanueva FF: Serum leptin levels in normal children: relationship to age, gender, body mass index, pituitarygonadal hormones and pubertal stage. J Clin Endocrinol Metab 1997;82:2849–2855. 16 Carro E, Pinilla L, Seoane LM, Considine RV, Aguilar E, Casanueva FF, Dieguez C: Influence of endogenous leptin tone on the estrous cycle and luteinizing hormone pulsatility in female rats. Neuroendocrinology 1997;66:375–377. 17 Gonzalez LC, Pinilla L, Tena-Sempere M, Aguilar E: Leptin116–130 stimulates prolactin and LH secretion in fasted adult male rats. Neuroendocrinology 1999;70:213–220. 18 Chan JL, Mantzoros CS: Leptin and the hypothalamic-pituitary regulation of the gonadotropin-gonadal axis. Pituitary 2001; 4: 87–92. 19 Schurgin S, Canavan B, Koutkia P, DePaoli AM, Grinspoon S: Endocrine and metabolic effects of physiologic r-metHuLeptin administration during acute caloric deprivation in normal-weight women. J Clin Endocrinol Metab 2004;89:5402–5409. 20 Welt CK, Chan JL, Bullen J, Murphy R, Smith P, DePaoli AM, Karalis A, Mantzoros CS: Recombinant human leptin in women with hypothalamic amenorrhea. N Engl J Med 2004;351:987–997. 21 Watanobe H: Leptin directly acts within the hypothalamus to stimulate gonadotropinreleasing hormone secretion in vivo in rats. J Physiol 2002;545:255–268. 22 Nagatani S, Guthikonda P, Thompson RC, Tsukamura H, Maeda KI, Foster DL: Evidence for GnRH regulation by leptin: leptin administration prevents reduced pulsatile LH secretion during fasting. Neuroendocrinology 1998; 67:370–376. 23 Magni P, Vettor R, Pagano C, Calcagno A, Beretta E, Messi E, Zanisi M, Martini L, Motta M: Expression of a leptin receptor in immortalized gonadotropin-releasing hormone-secreting neurons. Endocrinology 1999;140:1581–1585. 24 Smith JT, Acohido BV, Clifton DK, Steiner RA: KiSS-1 neurons are direct targets for leptin in the ob/ob mouse. J Neuroendocrinol 2006;18:298–303. 25 Tena-Sempere M: GPR54 and kisspeptin in reproduction. Hum Reprod Update 2006; 12:631–639. 26 Castellano JM, Navarro VM, FernandezFernandez R, Nogueiras R, Tovar S, Roa J, Vazquez MJ, Vigo E, Casanueva FF, Aguilar E, Pinilla L, Dieguez C, Tena-Sempere M: Changes in hypothalamic KiSS-1 system and restoration of pubertal activation of the reproductive axis by kisspeptin in undernutrition. Endocrinology 2005; 146:3917–3925. 27 Yu WH, Kimura M, Walczewska A, Karanth S, McCann SM: Role of leptin in hypothalamic-pituitary function. Proc Natl Acad Sci USA 1997;94:1023–1028.

Control of Reproduction by Ghrelin and Leptin

28 Tena-Sempere M, Pinilla L, González LC, Navarro J, Dieguez C, Casanueva FF, Aguilar E: In vitro pituitary and testicular effects of leptin-related synthetic peptide, leptin116–130 amide, involve actions both similar to and distinct from those of the native leptin molecule in the adult rat. Eur J Endocrinol 2000;142:406–410. 29 Tena-Sempere M, Pinilla L, González LC, Dieguez C, Casanueva FF, Aguilar E: Leptin inhibits testosterone secretion from adult rat testis in vitro. J Endocrinol 1999; 161: 211– 218. 30 Zamorano PL, Mahesh VB, de Sevilla LM, Chorich LP, Bhat GK, Brann DW: Expression and localization of the leptin receptor in endocrine and neuroendocrine tissues of the rat. Neuroendocrinology 1997;65: 223–228. 31 Spicer LJ, Francisco CC: The adipose obese gene product, leptin: evidence of a direct inhibitory role in ovarian function. Endocrinology 1991; 138:3374–3379. 32 Zachow RJ, Magoffin DA: Direct intraovarian effects of leptin: impairment of the synergistic action of insulin-like growth factorI on follicle-stimulating hormone-dependent estradiol-17␤ production by rat ovarian granulosa cells. Endocrinology 1997; 138: 847–850. 33 Agarwal SK, Vogel K, Weitsman SR, Magoffin DA: Leptin antagonizes the insulin-like growth factor-I augmentation of steroidogenesis in granulosa and theca cells of the human ovary. J Clin Endocrinol Metab 1999; 84:1072–1076. 34 Ghizzoni L, Barreca A, Mastorakos G, Furlini M, Vottero A, Ferrari B, Chrousos GP, Bernasconi S: Leptin inhibits steroid biosynthesis by human granulosa-lutein cells. Horm Metab Res 2001;33:323–328. 35 Kendall NR, Gutierrez CG, Scaramuzzi RJ, Baird DT, Webb R, Campbell BK: Direct in vivo effects of leptin on ovarian steroidogenesis in sheep. Reproduction 2004; 128: 757– 765. 36 Kitawaki J, Kusuki I, Koshiba H, Tsukamoto K, Honjo H: Leptin directly stimulates aromatase activity in human luteinized granulosa cells. Mol Hum Reprod 1999; 5: 708– 713. 37 Swain JE, Dunn RL, McConnell D, Gonzalez-Martinez J, Smith GD: Direct effects of leptin on mouse reproductive function: regulation of follicular, oocyte, and embryo development. Biol Reprod 2004; 71:1446–1452. 38 Karlsson C, Lindell K, Svensson E, Bergh C, Lind P, Billig H, Carlsson LM, Carlsson B: Expression of functional leptin receptors in the human ovary. J Clin Endocrinol Metab 1997;82: 4144–4148. 39 Duggal PS, Weitsman SR, Magoffin DA, Norman RJ: Expression of the long (Ob-RB) and the short (Ob-RA) forms of leptin receptor throughout the oestrous cycle in the mature rat ovary. Reproduction 2002; 123: 899– 905.

40 Archanco M, Muruzabal FJ, Llopiz D, Garayoa M, Gomez-Ambrosi J, Fruhbeck G, Burell MA: Leptin expression in the rat ovary depends on estrous cycle. J Histochem Cytochem 2003;51:1269–1277. 41 Loffler S, Aust G, Kohler U, Spanel-Borowski K: Evidence of leptin expression in normal and polycystic human ovaries. Mol Hum Reprod 2001;7:1143–1149. 42 Ryan NK, Woodhouse CM, van der Hoek KH, Gilchrist RB, Armstrong DT, Norman RJ: Expression of leptin and its receptor in the murine ovary: possible role in the regulation of oocyte maturation. Biol Reprod 2002; 66:1548–1554. 43 Ryan NK, van der Hoek KH, Robertson SA, Norman RJ: Leptin and leptin receptor expression in the rat ovary. Endocrinology 2003;144:5006–5013. 44 Kikuchi N, Andoh K, Abe Y, Yamada K, Mizunuma H, Ibuki Y: Inhibitory action of leptin on early follicular growth differs in immature and adult female mice. Biol Reprod 2001;65: 66–71. 45 Duggal PS, van der Hoek KH, Milner CR, Ryan NK, Armstrong DT, Magoffin DA, Norman RJ: The in vivo and in vitro effects of exogenous leptin on ovulation in the rat. Endocrinology 2000; 141:1971–1976. 46 Craig J, Zhu H, Dyce PW, Petrik J, Li J: Leptin enhances oocyte nuclear and cytoplasmatic maturation via the mitogen-activated protein kinase pathway. Endocrinology 2004; 145:5355–5363. 47 Olatinwo MO, Bhat GK, Stah CD, Mann DR: Impact of gonadotropin administration on folliculogenesis in prepubertal ob/ob mice. Mol Cell Endocrinol 2005;245:121–127. 48 Barkan D, Hurgin V, Dekel N, Amsterdam A, Rubinstein M: Leptin induces ovulation in GnRH-deficient mice. FASEB J 2005;19:133– 135. 49 Brannian JD, Hansen KA: Leptin and ovarian folliculogenesis: Implications for ovulation induction and ART outcomes. Semin Reprod Med 2002;20:103–112. 50 Tena-Sempere M, Pinilla L, Zhang F-P, González LC, Huhtaniemi I, Casanueva FF, Dieguez C, Aguilar E: Developmental and hormonal regulation of leptin receptor (ObR) messenger ribonucleic acid expression in rat testis. Biol Reprod 2001; 64:634–643. 51 Caprio M, Fabbrini E, Ricci G, Basciani S, Gnessi L, Arizzi M, Carta AR, De Martino MU, Isidori AM, Frajese GV, Fabbri A: Ontogenesis of leptin receptor in rat Leydig cells. Biol Reprod 2003; 68:1199–1207. 52 El-Hefnawy T, Ioffe S, Dym M: Expression of the leptin receptor during germ cell development in the mouse testis. Endocrinology 2000;141:2624–2630. 53 Caprio M, Isidori AM, Carta AR, Moretti C, Dufau ML, Fabbri A: Expression of functional leptin receptors in rodent Leydig cells. Endocrinology 1999; 140:4939–4947.


239

54 Giovambattista A, Suescun MO, Nessralla CC, Franca LR, Spinedi E, Calandra RS: Modulatory effects of leptin on Leydig cell function of normal and hyperleptinemic rats. Neuroendocrinology 2003;78:270–279. 55 Tena-Sempere M, Manna PR, Zhang F-P, Pinilla L, González LC, Dieguez C, Huhtaniemi I, Aguilar E: Molecular mechanisms of leptin action in adult rat testis: potential targets for leptin-induced inhibition of steroidogenesis and pattern of leptin receptor messenger ribonucleic acid expression. J Endocrinol 2001;170:413–423. 56 Isidori AM, Caprio M, Strollo F, Moretti C, Frajese G, Isidori A, Fabbri A: Leptin and androgens in male obesity: evidence for leptin contribution to reduced androgen levels. J Clin Endocrinol Metab 1999;84:3673–3680. 57 Caprio M, Fabbrini E, Isidori AM, Aversa A, Fabbri A: Leptin in reproduction. Trends Endocrinol Metab 2001;12:65–72. 58 Luukkaa V, Pesonen U, Huhtaniemi I, Lehtonen A, Tilvis R, Tuomilehto J, Koulu M, Huupponen R: Inverse correlation between serum testosterone and leptin in men. J Clin Endocrinol Metab 1998; 83: 3243– 3246. 59 Kojima M, Hosada H, Date Y, Nakazato M, Matsuo H, Kangawa K: Ghrelin is a growth hormone-releasing acylated peptide from stomach. Nature 1999;402:656–660. 60 Casanueva FF, Dieguez C: Growth hormone secretagogues: physiological role and clinical utilities. Trends Endocrinol Metab 1999; 10:30–38. 61 Van der Lely AJ, Tschop M, Heiman ML, Ghigo E: Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin. Endocr Rev 2004;25:426–457. 62 Korbonits M, Goldstone AP, Gueorguiev M, Grossman AB: Ghrelin – a hormone with multiple functions. Front Neuroendocrinol 2004;25:27–68. 63 Gualillo O, Lago F, Gomez-Reino J, Casanueva FF, Dieguez C: Ghrelin, a widespread hormone: insights into molecular and cellular regulation of its expression and mechanism of action. FEBS Lett 2003; 552: 105– 109. 64 Toshinai K, Yamaguchi H, Sun Y, Smith RG, Yamanaka A, Sakurai T, Date Y, Mondal MS, Shimbara T, Kawagoe T, Murakami N, Miyazato M, Kangawa K, Nakazato M: Des-acyl ghrelin induces food intake by a mechanism independent of the growth hormone secretagogue receptor. Endocrinology 2006; 147: 2306–2314. 65 Gauna C, Delhanty PJ, van Aken MO, Janssen JA, Themmen AP, Hofland LJ, Culler M, Broglio F, Ghigo E, van der Lely AJ: Unacylated ghrelin is active on the INS-1E rat insulinoma cell line independently of the growth hormone secretagogue receptor type 1a and the corticotropin-releasing factor 2 receptor. Mol Cell Endocrinol 2006;251:103–111.

240

66 Martini AC, Fernandez-Fernandez R, Tovar S, Navarro VM, Vigo E, Vazquez MJ, Davies JS, Thompson NM, Aguilar E, Pinilla L, Wells T, Dieguez C, Tena-Sempere M: Comparative analysis of the effects of ghrelin and unacylated ghrelin upon luteinizing hormone secretion in male rats. Endocrinology 2006;147:2374–2382. 67 Cummings DE: Ghrelin and the short- and long-term regulation of appetite and body weight. Physiol Behav 2006;89:71–74. 68 Furuta M, Funabashi T, Kimura F: Intracerebroventricular administration of ghrelin rapidly suppresses pulsatile luteinizing hormone secretion in ovariectomized rats. Biochem Biophys Res Commun 2001; 288: 780– 785. 69 Vulliemoz NR, Xiao E, Xia-Zhang L, Germond M, Rivier J, Ferin M: Decrease in luteinizing hormone pulse frequency during a five-hour peripheral ghrelin infusion in the ovariectomized rhesus monkey. J Clin Endocrinol Metab 2004;89:5718–5723. 70 Fernandez-Fernandez R, Tena-Sempere M, Aguilar E, Pinilla L: Ghrelin effects on gonadotropin secretion in male and female rats. Neurosci Lett 2004;362:103–107. 71 Iqbal J, Kurose Y, Canny B, Clarke IJ: Effects of central infusion of ghrelin on food intake and plasma levels of growth hormone, luteinizing hormone, prolactin, and cortisol secretion in sheep. Endocrinology 2006;147: 510–519. 72 Lanfranco F, Bonelli L, Broglio F, Me E, Baldi M, di Bisceglie C, Tagliabue, Manieri C, Ghigo E: Ghrelin inhibits LH pulsatility in humans. 88th Endocrine Society Meeting (ENDO2006), Boston 2006, P3-807. 73 Fernandez-Fernandez R, Tena-Sempere M, Navarro VM, Barreiro ML, Castellano JM, Aguilar E, Pinilla L: Effects of ghrelin upon gonadotropin-releasing hormone and gonadotropin secretion in adult female rats: in vivo and in vitro studies. Neuroendocrinology 2005;82: 245–255. 74 Fernandez-Fernandez R, Navarro VM, Barreiro ML, Vigo EM, Tovar S, Sirotkin AV, Casanueva FF, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M: Effects of chronic hyperghrelinemia on puberty onset and pregnancy outcome in the rat. Endocrinology 2005;146:3018–3025. 75 Caminos JE, Nogueiras R, Blanco M, Seoane LM, Bravo S, Alvarez CV, Garcia-Caballero T, Casanueva FF, Dieguez C: Cellular distribution and regulation of ghrelin messenger ribonucleic acid in the rat pituitary gland. Endocrinology 2003; 144:5089–5097. 76 Unniappan S, Peter RE: In vitro and in vivo effects of ghrelin on luteinizing hormone and growth hormone release in goldfish. Am J Physiol 2004;286:R1093–R1101.


77 Tanaka M, Hayashida Y, Nakao N, Nakai N, Nakashima K: Testis-specific and developmentally induced expression of a ghrelin gene-derived transcript that encodes a novel polypeptide in the mouse. Biochim Biophys Acta 2001;1522:62–65. 78 Tena-Sempere M, Barreiro ML, Gonzalez LC, Gaytan F, Zhang FP, Caminos JE, Casanueva FF, Dieguez C, Aguilar E: Novel expression and functional role of ghrelin in rat testis. Endocrinology 2002;143:717–725. 79 Gnanapavan S, Kola B, Bustin SA, Morris DG, McGee P, Fairclough P, Bhattacharya S, Carpenter R, Grossman AB, Korbonits M: The tissue distribution of the mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans. J Clin Endocrinol Metab 2002; 87: 2988–2991. 80 Barreiro ML, Gaytan F, Caminos JE, Pinilla L, Casanueva FF, Aguilar E, Dieguez C, Tena-Sempere M: Cellular location and hormonal regulation of ghrelin expression in rat testis. Biol Reprod 2002; 67:1768–1776. 81 Gaytan F, Barreiro ML, Caminos JE, Chopin LK, Herington AC, Morales C, Pinilla L, Paniagua R, Nistal M, Casanueva FF, Aguilar E, Dieguez C, Tena-Sempere M: Expression of ghrelin and its functional receptor, the type 1a growth hormone secretagogue receptor, in normal human testis and testicular tumors. J Clin Endocrinol Metab 2004; 89: 400–409. 82 Barreiro ML, Suominen JS, Gaytan F, Pinilla L, Chopin LK, Casanueva FF, Diéguez C, Aguilar E, Toppari J, Tena-Sempere M: Developmental, stage-specific and hormonally regulated expression of growth hormone secretagogue receptor messenger RNA in rat testis. Biol Reprod 2003; 68:1631–1640. 83 Caminos JE, Tena-Sempere M, Gaytan F, Sanchez-Criado JE, Barreiro ML, Nogueiras R, Casanueva FF, Aguilar E, Dieguez C: Expression of ghrelin in the cyclic and pregnant rat ovary. Endocrinology 2003; 144: 1594– 1602. 84 Gaytan F, Barreiro ML, Chopin LK, Herington AC, Morales C, Pinilla L, Casanueva FF, Aguilar E, Dieguez C, Tena-Sempere M: Immunolocalization of ghrelin and its functional receptor, the type 1a growth hormone secretagogue receptor, in the cyclic human ovary. J Clin Endocrinol Metab 2003; 88: 879–887. 85 Gaytan F, Morales C, Barreiro ML, Jeffery P, Chopin LK, Herington AC, Casanueva FF, Aguilar E, Dieguez C, Tena-Sempere M: Expression of growth hormone secretagogue receptor type 1a, the functional ghrelin receptor, in human ovarian surface epithelium, mullerian duct derivatives, and ovarian tumors. J Clin Endocrinol Metab 2005; 90: 1798–1804. 86 Ishikawa T, Fujioka H, Ishimura T, Takenaka A, Fujisawa M: Ghrelin expression in human testis and serum testosterone level. J Androl 2007;28:320–324.

Tena-Sempere

87 Barreiro ML, Gaytan F, Castellano JM, Suominen JS, Roa J, Gaytan M, Aguilar E, Dieguez C, Toppari J, Tena-Sempere M: Ghrelin inhibits the proliferative activity of immature Leydig cells in vivo and regulates stem cell factor messenger ribonucleic acid expression in rat testis. Endocrinology 2004; 145:4825–4834. 88 Sirotkin AV, Grossmann R, María-Peon MT, Roa J, Tena-Sempere M, Klein S: Novel expression and functional role of ghrelin in chicken ovary. Mol Cell Endocrinol 2006; 257–258:5–25. 89 Gualillo O, Caminos JE, Blanco M, GarciaCaballero T, Kojima M, Kangawa K, Dieguez C, Casanueva FF: Ghrelin, a novel placentalderived hormone. Endocrinology 2001; 142: 788–794. 90 Cortelazzi D, Cappiello V, Morpurgo PS, Ronzoni S, Nobile de Santis MS, de Santis MS, Cetil I, Beck-Peccoz P, Spada A: Circulating levels of ghrelin in human fetuses. Eur J Endocrinol 2003;149:111–116.

Control of Reproduction by Ghrelin and Leptin

91 Tanaka K, Minoura H, Isobe T, Yonaha H, Kawato H, Wang DF, Yoshida T, Kojima M, Kangawa K, Toyoda N: Ghrelin is involved in the decidualization of human endometrial stromal cells. J Clin Endocrinol Metab 2003; 88:2335–2340. 92 Kawamura K, Sato N, Fukuda J, Kodama H, Kumegai J, Tanikawa H, Nakamura A, Honda Y, Sato T, Tanaka T: Ghrelin inhibits the development of mouse preimplantation embryos in vitro. Endocrinology 2003; 144: 2623–2633. 93 Smith RG, Jiang H, Sun Y: Developments in ghrelin biology and potential clinical relevance. Trends Endocrinol Metab 2005: 16: 436–442. 94 Tena-Sempere M, Barreiro ML: Leptin in male reproduction: the testis paradigm. Mol Cell Endocrinol 2002; 188:9–13.

95 Barreiro ML, Tena-Sempere M: Ghrelin and reproduction: a novel signal linking energy status and fertility? Mol Cell Endocrinol 2005;226:1–9. 96 Tena-Sempere M: Exploring the role of ghrelin as novel regulator of gonadal function. Growth Horm IGF Res 2005;15:83–88. 97 Garcia JM, Li H, Mann D, Epner D, Hayes TG, Marcelli M, Cunningham GR: Hypogonadism in male patients with cancer. Cancer 2006;106:2583–2591. 98 Sun Y, Ahmed S, Smith RG: Deletion of ghrelin impairs neither growth nor appetite. Mol Cell Biol 2003; 23:7973–7981. 99 Sun Y, Wang P, Zheng H, Smith RG: Ghrelin stimulation of growth hormone release and appetite is mediated through the growth hormone secretagogue receptor. Proc Natl Acad Sci USA 2004;101:4679–4684.


241

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



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

Neuro-endocrinology of Growth and Energy Balance

Energy Balance and Gastrointestinal Cancer

Sensory and Metabolic Control of Energy Balance

Cancer and Energy Balance, Epidemiology and Overview (Energy Balance and Cancer)

Neuroendocrinology of Leptin

Fish Neuroendocrinology

An Introduction to Neuroendocrinology

Neuroendocrinology, Volume 182: Pathological Situations and Diseases

The Limits to Growth Revisited (SpringerBriefs in Energy Energy Analysis)

Peptides in Energy Balance and Obesity (Frontiers in Nutritional Science)

Neuroendocrinology: An Integrated Approach

Balance

Balance

Balance

Low Energy Ion Assisted Film Growth

Low Energy Ion Assisted Film Growth

Balance

Balance

Balance

Balance

Growth Theory and Growth Policy

Balance of Nature

Balance of Trade

Out of Balance

Balance of Nature

Balance Of Power

The Balance of Silence

Balance of power

Balance of Power

Balance of Na

Balance of Nature

Neuro-endocrinology of Growth and Energy Balance

Energy Balance and Gastrointestinal Cancer

Sensory and Metabolic Control of Energy Balance

Cancer and Energy Balance, Epidemiology and Overview (Energy Balance and Cancer)

Neuroendocrinology of Leptin

Fish Neuroendocrinology

An Introduction to Neuroendocrinology

Neuroendocrinology, Volume 182: Pathological Situations and Diseases

The Limits to Growth Revisited (SpringerBriefs in Energy Energy Analysis)

Peptides in Energy Balance and Obesity (Frontiers in Nutritional Science)

Neuroendocrinology: An Integrated Approach

Balance

Balance

Balance

Low Energy Ion Assisted Film Growth

Low Energy Ion Assisted Film Growth

Balance

Balance

Balance

Balance

Growth Theory and Growth Policy

Balance of Nature

Balance of Trade

Out of Balance

Balance of Nature

Balance Of Power

The Balance of Silence

Balance of power

Balance of Power

Balance of Na

Balance of Nature

Recommend Documents