Reviews in Endocrine & Metabolic Disorders 2002;3:325–338C© 2002 Kluwer Academic Publishers. Manufactured in The Netherlands.

Ghrelin: The Link Connecting Growth with Metabolismand Energy Homeostasis

Felipe F. Casanueva1 and Carlos Dieguez2

1Department of Medicine, Endocrine Section (FFC) and2Department of Physiology (CD), School of Medicine and ComplejoHospitalario Universitario de Santiago, University of Santiago deCompostela, Santiago de Compostela, Spain

Key Words. growth hormone, GH secretagogues, metabolism, foodintake, obesity, nutrition, GHRP-6

Introduction

Growth hormone (GH) is the sole pituitary hormone thatis tightly regulated by the metabolic milieu, and thisregulation appears to be superimposed over the classi-cal regulation by peptide hormones [1]. Furthermore, thehypothalamic regulation of the GH secreted by the so-matotroph is also peculiar, as it is exerted through thestimulatory action of GH releasing hormone (GHRH) andthe inhibitory one of somatostatin. Thus, GH is the onlyknown pituitary hormone regulated by two antagonistichypothalamic hormones, the others being controlled byjust one neurohormone. Peripherally, a liver-derived hor-mone, IGF-I, the synthesis and plasma levels of whichare stimulated in great part by GH, exerts classical neg-ative feedback on GH secretion, inhibiting it by actingboth at hypothalamic and pituitary levels. Other periph-eral hormones, such as thyroxine, gonadal steroids, glu-cocorticoids and leptin modulate the exact amount of GHand, in some way, connect the somatotroph status with theoverall hormonal balance of the body. Finally, metabolicsignals such as glucose, aminoacids, and free fatty acidsand their by-products, such as keto-acids, exert a rele-vant role in the control of GH secretion, food intake isthen a regulatory event of somatotroph function [2]. Theupshot of this picture is of one hormone whose actionsare implicated in a dual action on somatic growth andin the regulation of general metabolism, and which is, inturn, regulated by the energetic homeostasis of the indi-vidual. This dual field of action and regulation currentlyseems to be explained by a newly discovered hormone,ghrelin [3,4]. Ghrelin may well be the bridge connect-ing somatic growth and body composition with generalmetabolism.

Historical Background

In endocrinology, the normal path of discovery in a givenfield has been first to find the hormone, second to clonethe receptor, and third to develop clinically efficient ana-logues of this hormone. However, the saga of ghrelin hasbeen precisely the opposite: first the analogues (of a hith-erto unknown substance) were obtained; second the re-ceptor was cloned; and third the natural ligand for thisorphan receptor, i.e., ghrelin, was isolated. In brief, theendeavor started in 1975, at a time when the existence ofGHRH was still elusive, with some groups even thinkingthat such a compound would never be discovered, becauseit did not exist. At that time, Bowers’ group undertook atotally different approach to obtain a GH-releasing fac-tor. As enkephalins were short peptides able to liberateGH acting at a hypothalamic but not a pituitary level, thestrategy was to systematically introduce chemical modi-fications into the met-enkephalin structure and test for invitro, i.e., pituitary, GH secretion. The data obtained fromcomplex conformational energy calculations were used torelate structural features to the GH-releasing capabilitiesof the artificial compounds in order to introduce furthermodifications to increase potency and half-life. This iter-ative approach led, in 1980, to the development of the firsthighly potent GH-releasing hexapeptide, called GHRP-6[5,6]. GHRP-6 is the gold standard for all artificial GHsecretagogues, being a potent GH-releasing peptide bothin vitro and in vivo, and in all the species tested so far [7].GHRP-6 was the basis for the subsequent synthesis andtesting of different types of GH-releasing artificial com-pounds, of either peptidergic structure such as hexarelin[8], or non-peptidergic structure, such as MK-0677 [9], thedevelopment of which was a breakthrough in the modernendocrine-pharmacology. These different compounds are

Corresponding author: Dr. F.F. Casanueva.E-mail: endocrine@usc.es

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all known as GH-secretagogues (GHS). With the final iso-lation and characterization of GHRH in 1982, the interestin the whole field of GHS faded, rising again some yearslater when it was observed that GHS operated throughnon-GHRH and non-somatostatinergic mechanisms, andthat they acted on receptors different from those of GHRH[10]. The observation that the GHS have strong synergis-tic action when administered together with GHRH [11]was the definitive proof that such artificial compoundswere indeed not a surrogate of endogenous GHRH, andthe extensive amount of data generated in rodents and inhumans demonstrated that they are the activators of a newGH regulatory pathway endowed with high potential inthe clinical setting.

GHS-receptors operate through the activation of thephospholipase-IP3 pathway, leading to a rise in intra-cellular Ca2+ [12]. These changes in Ca2+ were usedsome years later by the group of Roy Smith for thecloning of the GHS-receptor [13], a well characterizedseven-transmembrane-domain, G-protein-coupled recep-tor. The GHS-receptor is unique in being an orphan-receptor cloned with the help of compounds such as GHSwhich were never discovered, but invented. The last mile-stone in the field has been the isolation of the endogenousor natural ligand of the GHS-receptor, a peptide calledghrelin [3]. Ghrelin, releases GH both in vivo and in vitro;surprisingly, it was isolated from stomach, where its ex-pression is higher than in any other tissue. This marks thelatest phase of the saga, and unambiguously demonstratesthat ghrelin represents the natural hormone of a new phys-iological system implicated in GH regulation.

Ghrelin

In order to identify the endogenous ligand, the orphan-receptor strategy was employed, taking advantage of a cellline transfected with the GHS-receptor responding to itsactivation with a rapid Ca2+ rise. This bioassay was usedby the group of Kojima and Kangawa to screen differenttissue extracts, and the highest expression of the activatingfactor was found in stomach extracts, leading, in 1999, tothe isolation and characterization of the endogenous ligandfor the GHS-receptor, a 28-amino-acid peptide that wasnamed ghrelin, as ghre is the Proto-Indo-European root forgrow and relin indicates release [3]. Ghrelin specificallybinds to the GHS receptor and releases GH in vitro in adose-dependent manner. The observation that the stomachwas the main source of ghrelin was a striking surprise fora peptide acting in the release of GH. However, it is im-portant to stress that splanchnic areas provide most of thecirculating somatostatin, and that GHRH was isolated notfrom hypothalamus, but from pancreatic tumours. Thus,the three neurohormones implicated in the regulation of

GH, (ghrelin, GHRH and somatostatin) are heavily ex-pressed in gastrointestinal tissues.

Molecular Biology

The human ghrelin gene is located on chromosome 3, inparticular at the locus 3p25-26 [14], and is made up of4 exons and 3 introns. This genetic structure is identicalto the ghrelin gene in the rat, and very similar to that ofthe mouse (5 exons, 4 introns) [15–17]. It appears to be apeculiarity of the ghrelin gene that a short part of the firstintron is a non-coding region and the mature protein is en-coded in exons 1 and 2 (Fig. 1). The heteronuclear RNA(hnRNA) of the gene transcript is processed by alternativesplicing to yield two different mature mRNAs: one pro-duces the ghrelin precursor and a second, expressed in lowquantities, yields des-Gln 14-ghrelin, a molecule similarto ghrelin and possessing similar biological activities, butlacking one glutamine at position 14 (Gln 14) [18]. Thereason for this alternative splicing is that Gln 13 and Gln14 are separated by the first intron; at the 3′-end of thatintron there are two tandem CAG sequences and, whenthe first AG is used for the splicing signal [15], the secondCAG is translated into Gln 14 producing prepro-ghrelinmRNA. On the contrary, when the second AG is used forsplicing, prepro-des-Gln14-ghrelin mRNA is created toproduce a ghrelin molecule lacking Gln 14 [18]. This is anovel mechanism for producing multiple forms of a hor-mone. In fact, the usual mechanism for producing differentpeptide hormones from a single gene is through cleavageby processing proteases of the product of a single mRNA.Ghrelin provides the first example of production of twodifferent mature peptides from the alternative splicing ofthe peptide coding region.

The rat and human ghrelin precursors (prepro-ghrelin)are both composed of 117 amino-acids, and the ghrelinsequence of 28 amino-acids immediately follows the 23-residue signal peptide. Before being secreted, the ghre-lin molecule undergoes an enzymatic process in the cyto-plasm, a n-octanoyl addition at Ser 3. This esterificationby n-octanoic acid, which is essential for the biologicalactivity of both ghrelin and des-Gln14 ghrelin, yields thefinally secreted peptide of 3315 molecular weight [15].This process of acylation has no precedent in cell biologyeither. Acyl modifications have been described in recep-tors or in integral membrane proteins; however, ghrelin isthe first example of acylation in a secreted protein. Thestudy of the enzymatic regulation of this cytoplasmaticprocess will add great information to further understandthe cell biology of ghrelin at a large.

The main product of that original synthesis process ismature ghrelin, a peptide of 28 amino-acids n-acylatedwith an octanoic acid, which circulates in large quantities

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Fig. 1. Cartoon depicting the operation of the ghrelin gene in humans. Ghrelin is encoded in the two first exons and it is unique in the hnRNAprocessing that, by alternative splicing, two mature mRNAs are derived, one for ghrelin and another for des-Gln-14-ghrelin. The asterisk marks theboundary between the first intron and the second exon where the alternative splicing occurs. Before secretion, a n-octanoyl acylation occurs in Ser3through a novel and still undefined enzymatic mechanism.

and binds to the GHS receptor, leading to intracellularcalcium rise and GH secretion in the somatotrope cells.Human and rat ghrelin only differ in two amino-acids, aproof of the relevance of this peptide, as it has been wellpreserved throughout evolution. The active binding core ofghrelin is the 4-5 first amino-acids including the acylatedSer3, as short peptides containing this sequence efficientlybind to the GHS receptor [19], Nevertheless, and most im-portantly, those short peptides are devoid of GH secretorycapability [20], suggesting that, with the exception of oneor two amino-acids, the whole acylated molecule is essen-tial for eliciting GH secretion. As des-n-octanoyl ghrelindoes not bind to the GHS receptor and is devoid of biolog-ical activity [3], it is interesting to speculate as to how thefatty acid residue changes the physical properties of ghre-

lin to facilitate its coupling in the biomembrane-receptorstructure. In any case, this seems to be a mechanism thathas been preserved between species, as bullfrog ghrelinchanges the acylated amino-acid from Ser3 to Thr3, onceagain showing that the acylation being essential for bio-logical activity [21]. In the rat, biologically active ghre-lin is present in the quantities of 377 ± 55, 20 ± 0.6 and<0.05 fmol/mg of tissue in stomach, duodenum and hy-pothalamus, respectively [22].

The testis-specific expression of another species ofthe ghrelin gene transcript in mice, called ghrelin gene-derive transcript (GGDT) [16], has recently been reported.GGDT is not expressed in the stomach, only in testis,and the sequence acting as the unique exon is located atthe intron 3 of the mice ghrelin gene, indicating that it is

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generated by alternative usage of this intron as a testis-specific exon, for yielding a 12 amino-acid peptide [17].The regulatory mechanisms and physiological functionsof the GGDT product are, at present, unknown.

Most probably, ghrelin belongs to a new family ofmotilin-related peptides. In fact the motilin receptor wascloned and identified as a member of the GHS recep-tor family [23]. Human ghrelin and human motilin have36% identity and show complementary patterns of expres-sion along the gastrointestinal tract, as ghrelin is secretedby enterochromaffin cells of the stomach and motilin byenterochromaffin cells of the small intestine [24,25]. Itis most interesting to note that a peptide identified asm46, and later called motilin-related peptide (MTLRP),was isolated by classical molecular biology techniquesand the sequence communicated to the EMBL/GenBankdatabase only one day apart from the communication ofghrelin sequence [26]. It has now been demonstrated thatthe MTLRP and ghrelin sequence are identical. Ghrelinwas found through a classical purification method with aclear biological endpoint; on the contrary, MTLRP wasobtained in transfected COS cells without such an end-point. As COS cells probably lack the enzymatic machin-ery for acylation, MTLRP lacked the octanoyl residue andwas devoid of biological activity. Both ghrelin and motilinhave the same action on food intake and on gastrointesti-nal tract motility, but different ones on GH secretion. Itmay well be that motilin and ghrelin have evolved alonga similar process.

Distribution

Ghrelin is predominantly expressed and secreted by thestomach [3], with lower concentrations reported throgh-out the whole bowel, including the colon [24]. Four differ-ent types of cells have been reported in the stomach, i.e.,ECL, D, enterochromaffin and X/A-like cells. Ghrelin isproduced mainly in the fundus, in the X/A-like cells ofthe oxintic gland [27], cells that until now have had anundefined activity, and were devoid of known hormonalcontent. Close to 20% of ghrelin cells are associated withchromogranin A-immunoreactive cells, and it is notewor-thy that ghrelin positive cells have no contact with the lu-men of the oxintic gland; rather they are positioned closeto the capillaries, indicating that its main activity is thesecretion towards plasma and not to the intestinal tract[24,27]. Following the current rules of nomenclature theX/A-like cells should be renamed Ghr cells .

Other tissues expressing ghrelin are the pituitary[28], hypothalamus [3], heart, and kidney [29], althoughthe amount secreted and the physiological relevanceof its presence in these tissues is still undefined. Themost controversial point now is to understand whether

hypothalamic ghrelin is synthesized there or whether it ismerely transported from the blood. Ghrelin was identifiedby immunohistochemical methods in the arcuate nucleusof the hypothalamus [3], a region rich in GHRH neurons,but also implicated in the regulation of appetite. Ghrelinis present in several other hypothalamic areas, althoughthe presence of ghrelin mRNA has not been definitivelyproven. At present, it is still urgently needed to clarify thefollowing items: (a) whether hypothalamic ghrelin is lo-cally synthesized or merely transported from the blood, (b)whether, in addition to its role in energy balance regula-tion, it participates in the regulation of GHRH or somato-statin neurons, and (c) whether some ghrelin is secretedtoward the pituitary. The data available point toward reg-ulation of food intake as the main role for hypothalamicghrelin.

Interestingly, ghrelin has been found in placenta [30],an organ that contains all the main regulatory componentsof the somatotropic axis, i.e., GH, GHRH, somatostatin,IGF-I, and ghrelin. Although placental expression of ghre-lin changes significantly throughout pregnancy [30], thephysiological function, if any, of this new hormone is notknown at present.

Regulation of Secretion and CirculatingLevels of Ghrelin

Circulating ghrelin is produced mainly in the stomachand lower quantities in other segments of the intestine;other sources may compensate for the gastric produc-tion, as gastrectomy only reduces circulating ghrelin by65% [31]. Ghrelin circulates in healthy human blood ata plasma concentration of 117 ± 37 fmol/mL [3]; how-ever close to 80% of the total content is deamidatedghrelin, i.e., devoid of biological activity. In rats, thetotal plasma ghrelin concentration is 556 ± 43 fmol/mLand, for intact ghrelin, is 94 ± 14 fmol/mL [24], al-though values of 219 ± 71 and 4 ± 1 fmol/mL have alsobeen reported [22]. Interestingly, the integrated secre-tion of ghrelin over 24 hours correlates significantlywith the ghrelin values obtained in the basal state priorto breakfast [32]. These results have considerable rel-evance for clinical and experimental studies, as theymake it possible to use a single determination of thepeptide as a surrogate for the amount measured during24-h sampling. As current RIAs mostly measure totalghrelin, precaution is needed in the interpretation of data,as bioactive ghrelin does not have a fixed ratio withtotal ghrelin. Ghrelin radioimmunoassays are cumber-some, and considerable care should be taken in the ex-traction procedure of plasma, as ghrelin is easily deami-dated by plasma proteases [3]. Tissue content and plasmavalues will soon be redefined when more standardized

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procedures for blood sampling, plasma extraction andRIA become widely homogeneous. No binding proteinor transporting protein has been reported for ghrelinas yet.

Ghrelin mRNA expression is up-regulated in rat gas-tric fundus after fasting, hypoglycemia, or leptin ad-ministration, and plasma ghrelin concentration tends toincrease in parallel [33]. In humans, ghrelin increasestwofold preprandially, and decreases to 50% 120 min af-ter a meal [32,34] (Fig. 2). Plasma ghrelin is reducedin obese subjects compared with lean ones, and evenfurther reduced in Pima Indians, one of the populationswith highest prevalence of obesity and type 2 diabetesmellitus [35]. On the contrary, plasma ghrelin levels aremarkedly elevated in patients with malnutrition due toanorexia nervosa and return towards normal after refeed-ing [36]. As ghrelin is a GH releasing peptide, theseresults fit in well with the low GH values observed in

Fig. 2. Average plasma ghrelin, insulin and leptin concentrationsduring a 24-h period in 10 control volunteers. B = breakfast,L = lunch, D = dinner. From [32] with authorization.

obesity and high levels seen in malnutrition and fasting[2], and suggest that ghrelin expression and secretion isenhanced in situations of negative energy balance, whilethe contrary occurs in situations of positive energy bal-ance. However, it may be the case that the gender-baseddifferences in the pattern of GH secretion are not me-diated by ghrelin, as no gender-based differences in cir-culating ghrelin have been shown in humans [36]. Simi-larly, gender did not alter ghrelin mRNA expression in ratstomach [37].

Ghrelin Action

In order to function in the afferent loop controlling ei-ther energy homeostasis or GH secretion, circulating ghre-lin needs to reach the central nervous system (CNS).This has been demonstrated as the peripheral adminis-tration of ghrelin gains access to the CNS where it ac-tivates fos and Egr-1 proteins in neurons of the arcuatenucleus, paraventricular and dorsomedial nuclei, and areapostrema of the hypothalamus, while de-amidated ghrelinwas devoid of action [38]. This neuronal activation oc-curs through the specific activation of the GHS-receptorslocated upon GHRH and NPY neurons, as well as inadditional neurons, as was previously demonstrated forGHRP-6 [38].

As ghrelin was purified using as bioassay the recep-tor for the GHS MK-0677, it became obvious that ghre-lin must operate through it (Fig. 3). There is an ongo-ing controversy about whether the cloned secretagoguereceptor is “the” receptor or just “one of the” receptorsfor this family of compounds. Evident differences in thebinding activities of the peptidyl (GHRP-6, ghrelin) andnon-peptidyl (MK 0677) molecules have been reported.Similarly, adenosine has been reported to be an agonistof the GHS receptor, and ligand-binding studies and site-directed mutagenesis have shown that it binds to a bind-ing pocket in the receptor distinct from where GHRP-6and MK 0677 bind, although adenosine did not stimu-late GH secretion in vitro [39,40]. After the report of theSer3-acylation in the ghrelin molecule, the initial specu-lation was to relate this fatty acid addition with enhancedpermeability through the blood brain barrier due to anenhanced lipophilicity. This is not yet proven, but thereis no doubt that the acyl modification is essential forthe in vitro binding of ghrelin to the subtype-1a of theGHS [19].

In a detailed analysis using labeled hexarelin as tracer,it has been demonstrated that GHS have specific receptorsin a wide range of endocrine and non-endocrine humantissues and that most probably different receptor subtypesexist for GH secretagogues, with different tissue distribu-tion [41] (Fig. 4).

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Fig. 3. Model of the GHS receptor. Amino acid residues in the deduced human protein sequence are illustrated by a schematic representation ofreceptor TM topology (N-terminus, extracellular; C-terminus intracellular). It is interesting the homology between the human sequence and thePufferfish 78B7 sequence, being the conserved aminoacids depicted on white circles. Remarkable conservation appears considering that the twospecies are separated by 400 million years. From [13] with authorization.

Intracellular Signalling

The transducing pathways through which ghrelin exert itsregulatory actions over different cell types are far fromunderstood. It is currently known that ghrelin binding tothe G-protein-coupled GHS-receptor, activates the phos-pholipase C signaling pathway leading to the generationof inositol phosphates plus protein kinase C activation.The inositols release Ca2+ from internal stores and thesubsequent entry of Ca2+ from the extracellular milieuthrough voltage-operated T- and L-type channels initiatesthe cellular response to the stimulus [42]. Similarly to thatreported for GHS, ghrelin is able to induce a homolo-gous desensitization, or down regulation, of its receptor.Of particular relevance are recent data showing that ghre-lin activates the IRS-1-GRB 2-MAP kinase pathways inhepatocytes, leading to cell proliferation. Unlike insulin,ghrelin inhibited Akt kinase activity and up-regulated glu-coneogenesis [43]. Studies aiming to identify the diverse

intracellular pathways in different cell types activated byghrelin are urgently needed.

Ghrelin Role in the Regulation ofSomatotrope Cell Function and GH Secretion

As artificial GHS were developed based in their abilityto release GH in vitro and in vivo, and these GHSsserved to clone the GHS-receptor, which in turn was thebioassay to identify the new peptide, the observation thatghrelin induced GH secretion in vitro in a dose-dependentmanner was the definitive proof of the identification ofthe natural ligand of the system [3]. Not only was ghrelinreasonably effective in vitro, it was also specific, as theother pituitary hormones were not affected at all [3].Shortly afterwards, it was reported that ghrelin was ableto release GH in freely moving rats, with a very rapidresponse of 5–10 minutes, returning to basal at 30 minutes

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Fig. 4. Distribution of GHS receptors in different human body tissues.From [41] with authorization.

[44] (Fig. 5). As ghrelin is able to release GH in vivo whenadministered directly via an intracerebroventricular route[45] and is able to enter the CNS from the periphery [38],it is possible, although not proven, that stomach-derivedghrelin may participate physiologically in GH regulation.Shorter ghrelin molecules bind to the GHS receptor, butnearly the entire ghrelin molecule is necessary for theeffective release of GH.

Ghrelin is also a potent GH releaser in humans (Fig. 5)[46–49] and, similar to previous reports with GHS, noside-effects are reported after the administration of largedoses of this new compound. The potency of ghrelin, mea-sured as its GH releasing capability, is higher than forGHRH and comparable to GHS [20], although, when an-alyzed on a molar basis, the relative potency of ghrelin vs.GHS has still not been settled. It now seems most prob-able that GHS and ghrelin are acting on similar receptorand pathways, as saturating doses of both compounds ad-ministered together elicit the same GH response as any ofthem separately [20]. Identical to that reported for GHS[50,51], GHRH antiserum or GHRH antagonists preventor diminish the GH-releasing possibilities of ghrelin [25],

A

B

C

Fig. 5. Effects of ghrelin on GH secretion in different models. (A) Invitro dose-response relationship of GH secretion by dispersed anteriorpituitary cells exposed to either ghrelin or GHRH. Redrawn from [3].(B) GH secretion in freely moving male rats after stimulation withdifferent doses of ghrelin or GHRH. Redrawn from [44]. (C) GHsecretion in normal subjects after the administration of ghrelin, theGHS hexarelin, and GHRH (all at 1 µg/Kg intravenously). Redrawnfrom [53].

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demonstrating that, in order for ghrelin to be operative onGH secretion, the normal functioning of the GHRH re-ceptor is necessary [51]. Ghrelin-mediated GH secretionis partially insensitive to the inhibitory action of either so-matostatin or of metabolic compounds, such as glucose orfree fatty acids [52].

As GHRP-6 plus GHRH is a very potent GH releaser[11], attempts to verify whether this synergistic action alsooccurred for ghrelin were undertaken. In fact, ghrelin andthe GHS hexarelin, showed a strong potentiation of theirGH secretory capability when injected together in humans[53], and more strikingly when ghrelin was employed avery low doses [49]. This peculiar activity occurs due to asimultaneous ghrelin activation of pituitary and hypothala-mic structures [54]. Homologous and heterologous desen-sitization occur after the administration of different GHSat high dosages [55,56], and could also occur when usingghrelin. As ghrelin is tonically released into the plasmafrom the stomach, it is of foremost importance to clarifywhether this situation partially desensitizes central struc-tures regulating GH secretion.

As opposed to the in vitro data, in vivo ghrelin induceda significant secretion of prolactin and ACTH/cortisol[47,53], without altering the secretion of LH, FSH andTSH (Fig. 6), a fact previously observed for artificial GHS.These data demonstrate that, at the pituitary level, onlynontumoral somatotrope cells have functional GHS recep-tors while, at the hypothalamic level and via other unde-fined pathways, ghrelin also activates ACTH and PRL se-cretion. Contrary to the rodent data [57], ghrelin-inducedACTH secretion seems to be mediated by hypothalamicvasopressin release [58]. As the above responses have beenobserved when testing with large doses of either GHSor ghrelin, it remains to be observed what happens af-ter more physiological administration. Furthermore, it ismost probable that similar to what was reported for GHS[59–61], long-term administration of ghrelin may enhanceGH secretion without significantly disturbing PRL andACTH/cortisol levels. From a physiological point of view,and in trying to understand the participation of this newhormone in somatotropic axis regulation, it is worth men-tioning that adult patients with GH deficiency have ghrelinlevels similar to control subjects, both before and after GHreplacement therapy [62].

In addition to its regulatory role on GH secretion, it hasrecently been reported that ghrelin induces an activationof pit-1 expression in anterior pituitary cells, an actionwhich appears to be developmentally regulated as it isobserved only in infant but not in adult rats [63]. Ghrelinbinds to the GHS receptor being fully able to activate genetranscription without requiring new protein synthesis, theeffect being exerted at a promoter region that contains twoCRE elements. Furthermore, the ghrelin action on Pit-1

Fig. 6. The administration of the GHS hexarelin, and ghrelin inducesthe release of ACTH/cortisol/aldosterone and of PRL in normalsubjects. Redrawn from [53].

transcription was dependent on MAPK, PKC, and PKAactivation [63].

Ghrelin and the Regulationof Energy Homeostasis

The fact that GHS were orexigenic agents when admin-istered centrally or peripherally has previously been pub-lished [64–66], although the topic was not further devel-oped by researchers in the field. This was given impetusafter the publication of clinical studies oriented towardsunderstanding the action of ghrelin on GH secretion,in which ghrelin administration powerfully induced the

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sensation of hunger in 75% of subjects tested [46], an ob-servation which has subsequently been confirmed [53,67].It is worth noting that Ghigo’s group in Turin has observedon repeated occasions over the last few years, that bothhexarelin and ghrelin enhanced hunger sensation, whileour group working in parallel in Santiago de Compostelahas never observed it. This difference, along with the factthat, in those groups observing a positive response it wasseen in only 75% of subjects, points toward genetic or en-vironmental differences in the actions of ghrelin and GHSon food intake regulation. Nevertheless, the whole topicreceived a powerful boost after the report that, in rodents,ghrelin stimulates food intake while reducing fat depot

Fig. 7. Ghrelin-stimulated adiposity in mice. Mice treated once a daywith ghrelin did not change food intake (a) but gained more than 10%of body weight (b) due to the increase in fat mass as measured by DXA;white figures controls, black figures ghrelin-treated. Redrawn from[68].

utilization [68] (Fig. 7). This finding has been confirmedby other groups [69–71], indicating the involvement of thenew hormone in the regulation of energy balance. The ac-tivity of ghrelin and GHS over food intake most probablyoccurs through mechanisms other that those implicatedin GH regulation [65,68,69,72]. Ghrelin stimulates foodintake in rodents when administered by any route, eithercentrally or peripherally, a relevant observation consider-ing that other orexigenic peptides are devoid of action viathe periphery. Ghrelin is the most powerful stimulator ofappetite of all known peptides, and this action is eliminatedwhen NPY and agouti-related protein (AGRP) are antago-nized [70]. It is more interesting to observe the similaritiesand complementary actions between leptin and ghrelin.Leptin has been reported to be able to reduce food intakeand, at the same time, to selectively reduce fat mass with-out altering lean body mass [73], while ghrelin has theexact opposite actions, i.e., it increases food intake, selec-tively enhances fat mass causing animals to selectively usecarbohydrate as primary source of energy, as is observedby the increment in the respiratory quotient [68]. Further-more, ghrelin reverses the anorexigenic actions of leptin,probably through the activation of the NPY/Y1 receptorpathway [72]. Interestingly, ghrelin is the unique gastroin-testinal pepide which stimulates food intake, as all otherpeptides of such origin are anorexigenic.

These findings suggest that stomach-derived ghrelinmay cross the blood brain barrier to regulate central ap-petite mechanisms [25], although this fact has not beenproven. However, relevant changes in circulating ghre-lin appear to fit the hypothesis: for example, in rodents,ghrelin mRNA in stomach and ghrelin levels in plasmawere increased by fasting and reduced by feeding, actionsunrelated to gastric volume changes [33,68,74]. Passiveimmunoneutralization with ghrelin antibodies inhibitedstarvation-induced as well as natural food intake in ro-dents, clearly indicating a tonic ghrelin action at hypotha-lamic receptors [74], data which fit well with the provenability of ghrelin to cross the blood-brain-barrier and im-mediately activate arcuate nucleus structures, better infasted than in fed animals [38].

Ghrelin levels are decreased in obese subjects [35]while elevated in malnutrition states such as cachexia [75]and anorexia nervosa in the latter group, weight recoverynormalizes plasma ghrelin values [36]. Considerable inter-est raised the possibility that ghrelin could be implicatedin the etiology of obesity as mutations in the ghrelin geneare associated with obesity in humans [76]. Basal ghre-lin levels are more alike in identical twins than betweenpairs, indicating a genetic basis for these levels based ei-ther on identical production or clearance, although basalghrelin levels did not predict the ultimate weight gain afterartificial overfeeding [77].

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Circulating ghrelin undergoes relevant changes in re-lation to food intake, being elevated before and decreasedafter feeding [32,34]. In this manner, plasma ghrelin lev-els increased twofold before each meal and fell to troughlevels 60 minutes after eating in a reciprocal pattern withinsulin and with intermeal changes that are in phase withleptin (Fig. 2), results that overall suggest that preprandialghrelin increases have a role in meal initiation in humans[32]. The orexigenic effects of ghrelin may, in part, bemediated by the vagal nerve, as its administration at rel-atively low doses enhanced the efferent activity of thisnerve [74]. It has been reported that, in rodents, ghrelininjected centrally or peripherally stimulated gastric con-traction, secretion, and emptying [74,78], and inhibitedpancreatic protein secretion [79], probably through thevagal cholinergic pathway.

Ghrelin Action on other Hormonal Systems

If the main endocrinological activity of ghrelin is the regu-lation of GH secretion, the induced changes in ACTH andPRL have always been viewed as part of the “noise” of thesystem, and no physiological role has been attributed tothem. One should remember that even GHRH elicits smallchanges in the secretion of ACTH and PRL in humans.However, recent reports in rodents indicate that ghrelincould be implicated in the neuroendocrine and behavioralresponse to stress [80] and in reducing LH secretion [81].Ghrelin may have relevant actions on non neuroendocrineareas and, in particular, in the endocrine pancreas, as itsacute administration induces hyperglycemia due to a re-duction in insulin secretion, an effect that is not mediatedby GH [67], and which could be explained by the pertur-batory action exerted by ghrelin in the downstream sig-nalling of insulin at a post-receptor level [43]. Although adiabetogenic action of GHS was reported in rodents, theseeffects were probably blurred by the parallel secretion ofGH [82].

Preliminary Reports of Ghrelin Actionswhich Merit Further Explorations

Ghrelin mRNA and ghrelin receptor mRNA are expressedin medulary thyroid carcinoma [83] and in all types of pi-tuitary adenomas, with different expressions depending onthe type [28,84]. The implication of this observation is thatlocal synthesis of ghrelin may have an autocrine-paracrinerole on pituitary hormone release. Similarly, the presenceof ghrelin receptors other than GHS-receptors in humanbreast carcinomas and neoplastic lung tissues opens newareas of research and therapy in these areas [85,86]. Ofparticular relevance is the production of ghrelin by en-docrine tumors of the stomach and intestine, as well as

related neuroendocrine cell hyperplasias, which representa clinical model to further clarify the impact of ghrelinhypersecretion on endocrine and non endocrine functions[87]. The widespread ghrelin expression in human T-cells,B-cells, and neutrophils may indicate unknown biologicalfunctions involving non-GH-mediated modulation of theimmune system [88]. Testicular ghrelin gene expressionwas demonstrated through early postnatal development aswell as in adult rat testis, with the expression restricted toLeydig cells [89]. Functionally, ghrelin inhibits testicularsecretion of testosterone regulating the expression of sev-eral steroid regulatory proteins, an observation that sug-gests new actions for this regulatory peptide [89].

The promising results of GHS action on heart functionand protection [90] seem to be further supported by theactions of ghrelin on the cardiovascular system. In hu-mans, ghrelin has been shown to have beneficial hemody-namic effects, by reducing cardiac afterload and increas-ing cardiac output without increasing heart rate [91]. Thewide distribution of ghrelin receptors in cardiovasculartissues and in particular its high density in atheroscleroticcoronary arteries may implicate a role for ghrelin as aneurotransmitter in such pathological processes [92].

Ghrelin Analogues and Clinical Implications

Usually, analogues are developed after the discovery andtesting of a putative new hormone or factor. In the peculiararea of GHS, the analogues were invented before the keyhormone of the system was known. In fact, over the past15 years, potent drugs able to activate the ghrelin-receptor,i.e., the GHS have been developed, namely GHRP-6,hexarelin, MK-0677, ipamorelin, CP-424,391, etc. [7].These compounds have large bioavailability, stimulate GHsecretion in all species tested so far, and are active byany route, including intravenously, intramuscularly, in-tranasally, subcutaneously, orally and transdermally. In thenext few months, testing of the biological actions of ghre-lin will be completed, and, if they are similar to those of theexisting GHS, these later compounds would remain betterchoices for clinical settings, since ghrelin, a molecule of 28amino-acids, presents no advantage over shorter, plasmaprotease-resistant ones. In brief, GHS used as ghrelin ana-logues would be preferred to the direct administration ofGH, as they should induce a more physiological profile ofplasma GH, and one potential target may be children withidiopathic or non-organic GH deficiency [93]. MK-0677has successfully been used by oral route to elderly sub-jects to increase 24-h GH secretion [59,94] or to preventthe catabolic state of diet-induced weight loss in obesesubjects [59]. Potentially the ghrelin analogues could beused in all pathological states in which the administrationof moderate GH doses has been shown to be effective,

Ghrelin 335

such as infertility and ovulation induction, mild catabolicstates, wound and fracture healing, and osteoporosis, andwould become an alternative to exogenous GH in catabolicstates such as strokes, severe burns and AIDS [7].

GHS have potential uses in the testing of the soma-totrope axis and, taking advantage of their extraordinarypotency when used in synergy, it has been shown that thecombined administration of GHRP-6 plus GHRH couldbe an effective stimulus for diagnosing GH deficiency inadults [95,96]. In the unlikely situation that ghrelin shouldshow clinical activities that are different from the avail-able GHS, new cheaper, specific ghrelin-analogues withenhanced bioavailability would be developed. It is inter-esting to consider that more stable ether or thioether bondsare capable of replacing the octanoyl ester bond in ghre-lin eliminating the enhanced fragility to proteases in themolecule. These should prove to be advantageous for thegeneration of pharmaceuticals with longer half-lives andhigher activites.

Summary and Integrative View

A positive energy balance is necessary to maximize the an-abolic actions of GH. As ghrelin anticipates the initiationof meals and releases GH, one could share the teleologicalview that, in fact, ghrelin integrates anabolic changes inthe body. In catabolic situations, enhanced ghrelin levelsmay induce a combination of enhanced food intake, in-creased gastric emptying and food assimilation coupledwith GH levels that would promote a prompt nutrient in-corporation to muscles and to fat reserves. The selectiveactions of ghrelin enhancing food intake and fat mass arethe mirror opposite actions of leptin which reduces foodintake and selectively eliminates fat mass. Thus, both pep-tides may act as physiological regulators of energy balancein the long run and, interestingly, both come from periph-eral organs such as stomach or white adipose tissue. Withthe incorporation of ghrelin to the group of physiologicalregulators of GH secretion that until now, have includedGHRH, somatostatin, and IGF-I, we are dealing with ahormone, GH, that appears to be one of the most tightlyregulated in the body, a fact which merits explanation.

Several facts are widely assumed, but unproven. Forexample, it is not known whether stomach-derived circu-lating ghrelin indeed acts at a hypothalamic or a pituitarylevel to regulate GH secretion. Using the same line of rea-soning, although most of the hypothalamic ghrelin seemsto be dedicated to the regulation of food intake and energyhomeostasis, its role in regulating hypothalamic GHRHand somatostatinergic neurons or a direct effect on the pi-tuitary need to be fully evaluated. In any case, until now,ghrelin has provided considerable insights showing howclassical purification methods may have an advantage over

modern molecular biology techniques or that new syn-thetic mechanisms and new configurations of biologicalmolecules can still be discovered.

The study of ghrelin will provide increased knowl-edge of the intricate regulation of GH secretion and itcan be foreseen that its most important contributions willbe to provide new physiological insights into the regula-tion of pituitary somatotrophs. At this point, it appears thatghrelin could be the heretofore unknown link connectinggrowth with metabolism and energy homeostasis.

Acknowledgments

The technical collaboration of Ms Mary Lage is grate-fully acknowledged. The results presented were sup-ported by research grants from Fondo de InvestigacionSanitaria, Spanish Ministry of Health, from SecretariaXeral de Investigacion e Desenvolvemento, Conselleriade Educacion Xunta de Galicia and from DGICYT.

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