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Regulatory Peptides 146

Hypothalamic gene expression following ghrelin therapyto gastrectomized rodents

Emil Egecioglu a, Björn Stenström b, Scarlett B. Pinnock c, Loraine Y.C. Tung c,Charlotta Dornonville de la Cour d, Andreas Lindqvist e, Rolf Håkanson d, Unni Syversen b,

Duan Chen b, Suzanne L. Dickson a,⁎

a Department of Physiology/Endocrinology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at Göteborg University,P.O. Box 434, SE-405 30 Göteborg, Sweden

b Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Laboratory Centre,Erling Skjalgssons Gate 1, NO-7006 Trondheim, Norway

c Department of Physiology, Development and Neuroscience, Anatomy School, Downing Street, Cambridge CB2 3DY, UKd Department of Pharmacology, Lund University, Solvegatan 10, S-223 62, Lund, Sweden

e Department of Experimental Medical Science, Lund University, Solvegatan 19, S-221 84, Lund, Sweden

Received 7 February 2007; received in revised form 30 August 2007; accepted 5 September 2007Available online 14 September 2007

Abstract

We investigated whether ghrelin depletion (by gastrectomy surgery) and/or treatment/replacement with the gastric hormone ghrelin alters theexpression of key hypothalamic genes involved in energy balance, in a manner consistent with ghrelin's pro-obesity effects. At 2 weeks after surgerymice were treated with ghrelin (12 nmol/mouse/day, sc) or vehicle for 8 weeks. Gastrectomy had little effect on the expression of these genes, with theexception of NPY mRNA in the arcuate nucleus that was increased. Ghrelin treatment (to gastrectomized and sham mice) increased the mRNAexpression of orexigenic peptides NPYandAgRPwhile decreasingmRNA expression of the anorexigenic peptide POMC. Twoweeks gavage treatmentwith the ghrelinmimetic,MK-0677, to rats increasedNPYand POMCmRNA in the arcuate nucleus andMCHmRNA in the lateral hypothalamus. Thus,while predicted pro-obesity ghrelin signalling pathways were activated by ghrelin and ghrelin mimetics, these were largely unaffected by gastrectomy.© 2007 Elsevier B.V. All rights reserved.

Keywords: Appetite; Growth hormone secretagogues; GHS-R1a; MK-0677; Melanin-concentrating hormone; Pro-opiomelanocortin; Neuropeptide Y; Agouti-relatedpeptide

1. Introduction

The hormone ghrelin was discovered in 1999 by Kojima etal., who isolated it from the rat stomach [1] where it is producedby the A-like cells of the oxyntic glands [2]. Indeed, thestomach is the major source of circulating ghrelin [2], althoughsmall amounts have been detected in other organs [3]. Inaddition, ghrelin is produced by hypothalamic neurones thatregulate the activity of the orexigenic neuropeptide Y (NPY)neurones of the arcuate nucleus [4].

Ghrelin is the first identified endogenous ligand for the growthhormone secretagogue receptor, GHS-R1a, cloned in 1996. This

⁎ Corresponding author. Tel.: +46 31 786 3568; fax: +46 31 786 35 12.E-mail address: suzanne.dickson@gu.se (S.L. Dickson).

0167-0115/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.regpep.2007.09.006

receptor is expressed in several tissues, notably at key CNS sitesthat regulate fat mass including the arcuate nucleus andventromedial nucleus of the hypothalamus and the mesolimbicsystem [5,6]. Ligands for this receptor, the growth hormonesecretagogues (GHSs), have been studied for over 20 years andhave provided a wealth of information about the mechanism ofaction of ghrelin. These agents include peptide and orally-activenon-peptide GHS-R1a agonists, such as MK-0677 [7]. Althoughboth ghrelin and GHS-R1a agonists induce growth hormonesecretion, recent interest largely focuses on their ability tostimulate food intake [8] and fat accumulation [9,10].

The stimulatory effects of ghrelin and GHS-R1a agonists onfood intake and fat accumulation are thought to be mediated byan interaction with the hypothalamic circuits that regulateenergy balance and also with mesolimbic pathways associated

Fig. 1. Integrated optical density, analyzed by in situ hybridization, for (A) NPY, (B) AgRP, (D) POMCmRNAs in the arcuate nucleus and (C) MCH mRNA in the lateralhypothalamus of either sham-operated (Sham) and gastrectomized (Gx) mice. The animals were given ghrelin (12 nmol/day) or vehicle by sc injections daily for 8 weeks.Representative bright field photomicrographs of sections incubated with 35S-labeled antisense oligonucleotide for each respective group and neuropeptide. The scale barrepresents A) 3 mm, B) 800 μm, C) 500 μm, D) 800 μm, respectively. Values are mean±SEM (n=5–6). Statistical comparison was made with a 2-way ANOVA. ⁎Pb0.05and ⁎⁎⁎Pb0.001 = significant difference between saline and ghrelin treatment. †Pb0.05 = significant difference between sham and gastrectomy surgery.

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Table 1Plasma ghrelin levels in Gx and sham-operated rats following treatment withMK-0677 or vehicle

Sham+vehicle Sham+MK-0677

Gx+vehicle Gx+MK-0677

Total ghrelin(pg/ml)

2078.4±214.0a 1537.1±118.0b 265.3±18.1c 245.0±14.2c

Results are expressed as mean±SEM. Statistics was analyzed with 2-wayANOVA. Means that do not share a common superscript are significantlydifferent; Pb0.05.

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with incentive behaviour [11,12]. In the hypothalamus, the NPYneurones represent a primary target for ghrelin [13]; theseneurones co-express AgRP and interact with key components ofthe energy balance pathways including pro-opiomelanocortin(POMC) cells in the arcuate nucleus and melanin-concentratinghormone (MCH) neurones in the lateral hypothalamus [14].Around 50% of the arcuate nucleus cells that are activated byGHS-R1a agonists also contain NPY mRNA [13]. Recordingsfrom identified NPY neurones following bath application ofghrelin confirm that NPY neurones are excited by ghrelin [4].Central ghrelin injection has also been shown to increase NPYand AgRP gene expression in the arcuate nucleus [15].

It has been proposed that ghrelin is a circulating hungerpeptide, partly because ghrelin levels increase between meals inexperimental animals [16] as well as in human subjects [17].Consistent with this view, ghrelin secretion increases in states ofnegative energy balance (e.g. fasting, anorexia nervosa) anddecreases in states of positive energy balance (e.g. after foodintake, obesity) [18].

The fact that 80–85% of circulating ghrelin disappears aftergastrectomy surgery (Gx) [2, 19] suggests that ghrelin is mainlyproduced by the stomach. However, it is not known whether thehypothalamic body fat-regulating circuits that respond to GHS-R1a agonists are affected inversely by the low plasma ghrelinconcentration that follows Gx. Previously we have shown thatghrelin treatment of Gx mice reverses the Gx-induced loss ofbody weight and body fat [19]. This finding may have clinicalimplications as it suggests that ghrelin therapy may be beneficialfor improving body composition in patients that have undergoneGx surgery. In the present study we sought to determine whethertreatment with ghrelin to intact animals or as a replacementtherapy after Gx alters the expression of key hypothalamic genesinvolved in body weight regulation, including mRNA for NPY,AgRP, POMC and MCH. According to the classic endocrineapproach, we hypothesised that the effects of Gx (that depletescirculating ghrelin by 80%) on the hypothalamic genesregulating energy balance would be reversed by ghrelinreplacement and/or treatment with a ghrelin mimetic.

2. Materials and methods

2.1. Animals

Female NMRI mice (30 g, B&K, Sollentuna, Sweden) andmale Sprague–Dawley rats (200–250 g, Möllegaard, Skensved,Denmark) were kept in a temperature-controlled environment

(∼20 °C), on a 12 h light/dark cycle, with free access tostandard food pellets (Lactamin, Vadstena, Sweden and B&KUniversal Ltd, Hull, England respectively) and tap water. Theexperiments were approved by the local Animal WelfareCommittee, Lund, Sweden and the Norwegian National AnimalResearch Authority (Forsøksdyrutvalget).

2.2. Eight weeks of ghrelin administration to Gx mice andsham-operated mice

Mice were subjected to Gx surgery (that includes sub-diaphragmatic vagotomy) or sham operation as describedpreviously [19]. Half of the Gx and half of the sham-operatedmice received daily subcutaneous injections of 12 nmol active(acylated) ghrelin for 8 weeks, starting at 2 weeks after surgery.This dose increases plasma ghrelin levels 1–2 fold higher thanuntreated intact mice at 16 h after injection. After 8 weekstreatment the mice were killed by decapitation (16–18 h afterthe last injection). Blood was collected for plasma ghrelinmeasurement and brains were frozen for in situ hybridization.Data on body weight, food intake, fat pad weight and plasmaconcentrations of ghrelin for these mice have been publishedelsewhere [19].

2.3. Two weeks of MK-0677 administration to Gx rats andsham-operated rats

In a follow-up study, we sought to determine whether theghrelin receptor agonist, MK-0677, influences the expression ofkey hypothalamic genes involved in body weight regulation inGx and sham-operated rats. Starting 4 weeks after surgery, Gxrats and sham-operated rats received daily oral gavage withMK-0677 (gift from R. Deghenghi, Europeptides, Paris,France) at a dose of 4 mg/kg per day for 2 weeks, by whichtime fat-accumulating effects of ghrelin became evident in aprevious study [19]. Two additional groups of rats were givenan equal volume (0.5 ml) of tap water vehicle by gavage. After2 weeks of treatment rats were killed by decapitation. Bloodwas collected and brains were removed as before.

2.4. Measurement of plasma ghrelin

Rat ghrelin was measured in plasma using radioimmunoas-say kits (Phoenix Pharmaceuticals, Belmont, California, USA)with rat ghrelin-28 as standard. This assay detects both activeacetylated and des-acetyl rat ghrelin-28.

2.5. In situ hybridization

In situ hybridization was performed on 10 μm mouse or ratbrain sections as described previously [20]. Antisense oligonu-cleotide probes designed for NPY, AGRP, POMC and MCHmRNA were used. NIH image software was used to obtain anintegrated optical density measurement for each section andstandardised by comparison with radioactive standards (C14microscales, range 30–862 mCi/g; Amersham). The opticaldensities were measured in six consecutive sections. The

Fig. 2. Integrated optical density, analyzed by in situ hybridization, for (A) NPY, (B) AgRP, (D) POMC mRNAs in the arcuate nucleus and (C) MCH mRNA in thelateral hypothalamus of sham-operated (Sham) or gastrectomized (Gx) rats. The rats were given MK-0677 (4 mg/kg) or vehicle daily by oral treatment for 12 days.Representative bright field photomicrographs of sections incubated with 35S-labeled antisense oligonucleotide for each respective group and neuropeptide. The scalebar represents A) 1.6 mm, B) 1.6 mm, C) 1.5 mm, D) 1.5 mm, respectively. Values are mean±SEM (n=7 for all groups). Comparisons between groups were made witha 2-way ANOVA followed by HSD Turkey post hoc test. ⁎Pb0.05, ⁎⁎Pb0.01, ⁎⁎⁎Pb0.001.

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duration of exposure to the X-ray film varied according to themRNA transcript under investigation (5 days–2 months).

2.6. Statistical analysis

Comparisons between groups were made by 2-way ANOVAusing treatment (i.e. ghrelin or MK-0677 versus vehicle) andsurgery (Gx or sham operation) as fixed factors. To investigatewhether there was a significant difference between the factorsan interaction term was used. If significant, the interaction termwas kept in the model and the outcome analyzed by HSDTurkey post hoc test. A significant interaction describes that thetreatment response differed significantly between the surgicalgroups. Values were transformed to logarithms when appropri-ate. P-valuesb0.05 were considered significant.

3. Results

3.1. Effects of Gx in mice, with or without ghrelin replacement, onthe expression of hypothalamic genes involved in energy balance

There was an overall effect of both ghrelin treatment(Pb0.001) and Gx surgery (Pb0.05) on the expression ofNPY mRNA in the arcuate nucleus. At 10 weeks followingsurgery the expression of NPY mRNA was increased in Gxmice compared to sham mice. Furthermore, ghrelin treatmentfor 8 weeks increased NPY mRNA expression compared tovehicle treatment (Fig. 1A). Ghrelin treatment increased AgRPmRNA expression in the arcuate nucleus compared to vehicletreatment (Pb0.05) whereas Gx per se was without effect onAgRP mRNA expression (Fig. 1B). MCH mRNA expression inthe lateral hypothalamus was not affected by ghrelin treatmentor Gx (Fig. 1C). POMC mRNA expression in the arcuatenucleus was reduced by ghrelin treatment compared to vehicletreatment (Pb0.01). Gx was without effect (Fig. 1D).

3.2. Plasma ghrelin levels in Gx rats, with or without MK-0677 treatment

Gx decreased circulating ghrelin by approx 85% in rat,confirming that the Gx surgery was successful (Pb0.01, Table 1).In sham rats the plasma ghrelin concentration was reduced by26% by 2 weeks MK-0677 treatment compared to vehiclecontrols (Pb0.01, Table 1).

3.3. Effects of Gx in rats, with or without MK-0677 treatment,on the expression of hypothalamic genes involved in energybalance

At 6 weeks after Gx in rats the expression of NPY mRNA inthe arcuate nucleus was increased 2.6 fold (Pb0.01). Further-more, NPY mRNA levels were almost 2 fold higher in Gx ratstreated with MK-0677 than in sham-operated rats receivingMK-0677 (Pb0.001). Two weeks of MK-0677 treatmentincreased NPY mRNA expression in sham and Gx rats by 5.1and 5.4 fold respectively (Pb0.05 and Pb0.01 respectively,Fig. 2A).

Compared to vehicle treatment, 2 weeks of MK-0677treatment raised AgRP mRNA expression by 2.6 fold in Gxrats (Pb0.001). There was a significant interaction betweentreatment and surgery such that AgRP expression levels were2.5 fold higher in Gx rats treated with MK-0677 than in sham-operated rats receiving the same treatment (Pb0.001, Fig. 2B).

MK-0677 raised MCH mRNA expression by 7 fold in sham-operated rats (Pb0.001) and by 2.9 fold in Gx rats compared totheir respective vehicle control group (Pb0.001). The MCHmRNA response to MK-0677 treatment was stronger in sham-operated rats than in Gx rats (Pb0.05, Fig. 2C). Treatment withMK-0677 raised POMC mRNA expression in the arcuatenucleus of sham-operated rats by 3.3 fold (Pb0.01). Gx per sedid not affect POMC mRNA expression but prevented the raiseinduced by MK-0677 (Pb0.001, Fig. 2D).

4. Discussion

Recently, we reported that treatment with ghrelin reverses theloss of bodyweight and body fat that follows Gx inmice [19].Wenow report that long-term ghrelin treatment (including ghrelin as areplacement therapy to Gxmice) influences the expression of keyhypothalamic genes involved in energy balance in a mannerconsistent with ghrelin's orexigenic and pro-obesity effects. Thus,in sham-operated and Gx mice, daily ghrelin administration for8 weeks increased the expression of the orexigenic peptides NPYand AgRP and decreased the expression of the anorexigenicpeptide POMC in the arcuate nucleus. Moreover we found thatremoval of the stomach, that reduces circulating ghrelin by around85% [2,19], had little effect on the expression levels of thehypothalamic genes measured.

There is a general belief that ghrelin's acute orexigeniceffects contribute to the fat accumulation observed during long-term ghrelin exposure. However, it is rather striking that, duringchronic ghrelin therapy to mice (by daily sc injections for8 weeks), the fat-accumulating effects are evident without anydetectable change in daily food intake [19]. This could indicatethat the hypothalamic circuits mediating ghrelin's acute effectson food intake are not identical, or at least show only partialoverlap, to those mediating ghrelin's effects on adiposity.Alternatively, it may be that during chronic ghrelin exposure,ghrelin's orexigenic effects are masked or suppressed due toundetermined compensatory mechanisms that may be second-ary to the fat accumulation and associated endocrine changes. Insupport of this hypothesis, we show that during chronic ghrelintreatment ghrelin's effects to increase gene expression of theorexigenic peptides NPY and AGRP occur (this study) withoutany detectable change in food intake [19].

We also observed a reduced expression of mRNA for theanorexigenic peptide POMC in the chronic ghrelin treatment/replacement groups. However, such an effect is most likelyindirect since there are few ghrelin receptors on POMCneurones [21]. Recordings from identified POMC neuronesshow that ghrelin increases the GABAergic inhibitory inputonto POMC neurones [4]. The net effect of POMC deletion inmice is obesity [22] and loss of function mutations in the POMCgene in humans also cause obesity [23]. It seems likely that

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these pro-obesity changes in hypothalamic gene expressionobserved during chronic ghrelin treatment/replacement contrib-ute to the fat-accumulating effects of ghrelin, that occur in theabsence of altered food intake.

To verify and extend our initial observations using ghrelin asa treatment/replacement therapy in mice, we examined theeffects of the ghrelin receptor agonist MK-0677 on theexpression of hypothalamic genes involved in body weightregulation in rats. MK-0677 increased the expression ofhypothalamic NPY and AgRP mRNA in a similar manner toghrelin. In contrast to sham rats, where MK-0677 induced alarge increase in both POMC and MCH expression, MK-0677treatment of Gx rats did not influence expression of these genes.The simplest explanation for this difference is that MK-0677′seffects on POMC and MCH expression require the presence of afunctionally intact stomach, an intact vagus (as Gx surgeryincludes sub-diaphragmatic vagotomy) or the presence ofanother endocrine signal, presumably from the stomach. Thepattern of hypothalamic expression for NPY, AgRP and MCHfollowing MK-0677 treatment in rat and ghrelin treatment inmice is rather similar. In view of the effects of ghrelin on POMCexpression in mice as well as the fat-accumulating effects ofboth ghrelin and MK-0677 it was surprising to find thattreatment with MK-0677 increased the expression of POMC inthe arcuate nucleus. Although we are not aware of any reports ofthe effects of ghrelin on POMC mRNA expression, this findingis at odds with the view that ghrelin acts presynaptically toincrease the inhibitory input onto identified POMC neurones[4]. The difference could be a consequence of elevated desacylghrelin levels which was seen in mice treated with ghrelin butnot in rats treated with MK-0677. Induction of MCH mRNAexpression by MK-0677 is consistent with ghrelin's orexigenicand fat-accumulating effects. However, the fact that we did notsee a significant effect of ghrelin treatment on MCH mRNA inmice, suggests that MCH neurones may not represent a ghrelintarget. Consistent with this, we showed that ghrelin is able tostimulate food intake in mice lacking the MCH receptor [24].Certainly, our data suggest a complex and partly contradictoryinfluence by MK-0677 on both fat-accumulating and fat-suppressing systems in the hypothalamus. It is noteworthy alsothat, as the ghrelin study was carried out in female mice, whilethe MK-0677 used male rats, the hypothalamic genes studiedmay be influenced by gender and by differing influences ofgonadal steroids. Consistent with this, female GHS-R1aknockout mice fed a normal diet become leaner than the malemice [25].

The weight loss that follows Gx and gastric bypass surgery isin line with the emerging view that the stomach signals to thehypothalamic circuits that regulate energy balance. We weretherefore surprised that loss of the stomach did not affect thehypothalamic genes studied to any large extent. As the Gxsurgery includes sub-diaphragmatic vagotomy, we envisagedthat this in itself could also impact upon the hypothalamic genesstudied. Accordingly, the Gx animals are not only depleted ofgastric hormones but also lack an intact vagus, which providesan important route through which many of these hormones suchas ghrelin signal [26]. Even though circulating ghrelin levels

were strongly depleted by Gx in both mice and rats, only NPYmRNA was slightly increased following Gx, whereas AgRP,MCH and POMC mRNA were unaffected. The simplestexplanation is that by 10 weeks post-surgery, Gx animalshave reached a new body weight homeostasis. Gastrectomyleads to an altered endocrine status, reflecting the absence of anumber of gastric hormones and also secondary changes thatreflect, for example, altered enteric signalling or reduced fatmass. Thus, the hypothalamic response to ghrelin depletion maybe opposed by suppressed levels of adipose-derived satietysignals such as leptin [27] or by as yet unidentified gastricfactors. The paradoxical increase of mRNA expression of theorexigenic peptide NPY in the ghrelin-depleted Gx animalscould even reflect a compensatory increase in hunger drives thatare secondary to the weight loss and/or to the loss of thecontainer function of the stomach.

In light of reports concerning the loss of effect ofperipherally administered ghrelin on food intake followingvagotomy or bilateral midbrain transaction [26,28], it isinteresting to note that subcutaneous ghrelin treatment increasedboth NPY and AgRP mRNA expression and decreased POMCmRNA expression in both sham and Gx mice. The effect ofMK-0677 on NPY and AgRP mRNA expression was evenpotentiated by Gx surgery whilst the effect of MK-0677 onMCH and POMC expression was ablated by Gx in rats.

In summary we show that daily long-term treatment withghrelin or a GHS-R1a agonist stimulates pro-obesity pathwaysin the hypothalamus (including activation of the arcuate NPY/AgRP neurones) consistent with ghrelin's effects to increasebody weight and fat accumulation in rodents. Furthermore,ghrelin also inhibited POMC gene expression providing a newindirect reason for the stimulation of fat accumulation byghrelin. Surprisingly, Gx surgery, that depletes circulatingghrelin by ∼85%, had little impact on the hypothalamic genes,measured 10 weeks post-operatively. This may reflect theestablishment of a new endocrine milieu after Gx surgery,involving depletion of other gastric factors, adipose-derivedfactors or indeed a new homeostasis at the level of the adipostat.

Acknowledgments

Anna Themner-Persson performed Gx surgery in mice.Staffan Nilsson gave statistical advice. Supported by EU 6thFramework (LSHM-CT-2003-503041), Eva och Oscar Ahrénsstiftelse, Vetenskapsrådet (04x-1007 and 04x-14610), Novo-nordisk Fonden (2798) and The Sahlgrenska Academy Centrefor Cardiovascular and Metabolic Research. Björn Stenströmhas a PhD fellowship from the Central Norway Regional HealthAuthority.

References

[1] Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K.Ghrelin is a growth-hormone-releasing acylated peptide from stomach.Nature 1999;402:656–60.

[2] Dornonville de la Cour CD, Björkqvist M, Sandvik AK, Bakke I, ZhaoCM, Chen D, Håkanson R. A-like cells in the rat stomach contain ghrelinand do not operate under gastrin control. Regul Pept 2001;99:141–50.

182 E. Egecioglu et al. / Regulatory Peptides 146 (2008) 176–182

[3] Horvath TL, Diano S, Sotonyi P, Heiman M, Tschöp M. Minireview:ghrelin and the regulation of energy balance—a hypothalamic perspective.Endocrinology 2001;142:4163–9.

[4] Cowley MA, Smith RG, Diano S, Tschöp M, Pronchuk N, Grove KL,Strasburger CJ, Bidlingmaier M, Esterman M, Heiman ML, Garcia-SeguraLM, Nillni EA, Mendez P, Low MJ, Sotonyi P, Friedman JM, Liu H, PintoS, Colmers WF, et al. The distribution and mechanism of action of ghrelinin the CNS demonstrates a novel hypothalamic circuit regulating energyhomeostasis. Neuron 2003;37:649–61.

[5] Guan XM, Yu H, Palyha OC, McKee KK, Feighner SD, SirinathsinghjiDJ, Smith RG, Van der Ploeg LH, Howard AD. Distribution of mRNAencoding the growth hormone secretagogue receptor in brain andperipheral tissues. Brain Res Mol Brain Res 1997;48:23–9.

[6] Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, RosenblumCI, 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, et al. A receptor in pituitary and hypothalamusthat functions in growth hormone release. Science 1996;273:974–7.

[7] Patchett AA, Nargund RP, Tata JR, Chen MH, Barakat KJ, Johnston DB,Cheng K, Chan WW, Butler B, Hickey G, et al. Design and biologicalactivities of L-163,191 (MK-0677): a potent, orally active growth hormonesecretagogue. Proc Natl Acad Sci U S A 1995;92:7001–5.

[8] Wren AM, Small CJ, Abbott CR, Dhillo WS, Seal LJ, Cohen MA,Batterham RL, Taheri S, Stanley SA, Ghatei MA, Bloom SR. Ghrelincauses hyperphagia and obesity in rats. Diabetes 2001;50:2540–7.

[9] Lall S, Tung LYC, Ohlsson C, Jansson JO, Dickson SL. Growth hormone(GH)-independent stimulation of adiposity by GH secretagogues. BiochemBiophys Res Commun 2001;280:132–8.

[10] Tschöp M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents.Nature 2000;407:908–13.

[11] Jerlhag E, Egecioglu E, Dickson SL, Andersson M, Svensson L, Engel JA.Ghrelin stimulates locomotor activity and accumbal dopamine-overflowvia central cholinergic systems in mice: implications for its involvement inbrain reward. Addict Biol 2006;11:45–54.

[12] Jerlhag E, Egecioglu E, Dickson SL, Douhan A, Svensson L, Engel JA.Ghrelin administration into tegmental areas stimulates locomotor activityand increases extracellular concentration of dopamine in the nucleusaccumbens. Addict Biol 2007;12:6–16.

[13] Dickson SL, Luckman SM. Induction of c-fos messenger ribonucleic acid inneuropeptide Yand growth hormone (GH)-releasing factor neurons in the ratarcuate nucleus following systemic injection of the GH secretagogue, GH-releasing peptide-6. Endocrinology 1997;138:771–7.

[14] Schwartz MW, Woods SC, Porte D, Seeley RJ, Baskin DG. Centralnervous system control of food intake. Nature 2000;404:661–71.

[15] Kamegai J, Tamura H, Shimizu T, Ishii S, Sugihara H, Wakabayashi I.Chronic central infusion of ghrelin increases hypothalamic neuropeptide Yand agouti-related protein mRNA levels and body weight in rats. Diabetes2001;50:2438–43.

[16] Tolle V, Bassant MH, Zizzari P, Poindessous-Jazat F, Tomasetto C,Epelbaum J, Bluet-Pajot MT. Ultradian rhythmicity of ghrelin secretion inrelation with GH, feeding behavior, and sleep–wake patterns in rats.Endocrinology 2002;143:1353–61.

[17] Cummings DE, Purnell JQ, Frayo RS, Schmidova K, Wisse BE, WeigleDS. A preprandial rise in plasma ghrelin levels suggests a role in mealinitiation in humans. Diabetes 2001;50:1714–9.

[18] Shiiya T, Nakazato M, Mizuta M, Date Y, Mondal MS, Tanaka M, NozoeS, Hosoda H, Kangawa K, Matsukura S. Plasma ghrelin levels in lean andobese humans and the effect of glucose on ghrelin secretion. J ClinEndocrinol Metab 2002;87:240–4.

[19] Dornonville de la Cour C, Lindqvist A, Egecioglu E, Tung YCL, Surve V,Ohlsson C, Jansson JO, Erlanson-Albertsson C, Dickson SL, Håkanson R.Ghrelin treatment reverses the reduction in weight gain and body fat ingastrectomised mice. Gut 2005;54:907–13.

[20] Challis BG, Pinnock SB, Coll AP, Carter RN, Dickson SL, O'Rahilly S.Acute effects of PYY3-36 on food intake and hypothalamic neuropeptideexpression in themouse. BiochemBiophys Res Commun 2003;311:915–9.

[21] Willesen MG, Kristensen P, Rømer J. Co-localization of growth hormonesecretagogue receptor and NPY mRNA in the arcuate nucleus of the rat.Neuroendocrinology 1999;70:306–16.

[22] Yaswen L, Diehl N, Brennan MB, Hochgeschwender U. Obesity in themouse model of pro-opiomelanocortin deficiency responds to peripheralmelanocortin. Nat Med 1999;5:1066–70.

[23] Krude H, Biebermann H, Luck W, Horn R, Brabant G, Gruters A. Severeearly-onset obesity, adrenal insufficiency and red hair pigmentation causedby POMC mutations in humans. Nat Genet 1998;19:155–7.

[24] Bjursell M, Egecioglu E, Gerdin AK, Svensson L, Oscarsson J, Morgan D,Snaith M, Tornell J, Bohlooly-Y M. Importance of melanin-concentratinghormone receptor for the acute effects of ghrelin. Biochem Biophys ResCommun 2005;326:759–65.

[25] Zigman JM, Nakano Y, Coppari R, Balthasar N, Marcus JN, Lee CE, JonesJE, Deysher AE, Waxman AR, White RD, Williams TD, Lachey JL,Seeley RJ, Lowell BB, Elmquist JK. Mice lacking ghrelin receptors resistthe development of diet-induced obesity. J Clin Invest 2005;115:3564–72.

[26] Date Y, Murakami N, Toshinai K, Matsukura S, Niijima A, Matsuo H,Kangawa K, Nakazato M. The role of the gastric afferent vagal nerve inghrelin-induced feeding and growth hormone secretion in rats. Gastroen-terology 2002;123:1120–8.

[27] Zhang YY, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM.Positional cloning of the mouse obese gene and its human homolog.Nature 1994;372:425–32.

[28] 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 thenoradrenergic pathway from the hindbrain to the hypothalamus. Cell Metab2006;4:323–31.