Orexigenic effect of the melanocortin MC4 receptor antagonist HS014 is inhibited only partially by neuropeptide Y Y1 receptor selective antagonists

Neuropeptide Y (NPY) and melanocortin (MC) peptides have opposite effects on food intake: NPY-like peptides and MC receptor antagonists stimulate feeding and increase body weight, whereas melanocortins and NPY antagonists inhibit food intake. In this study we tested whether the orexigenic effect of the selective MC4 receptor antagonist HS014 (1 nmol) could be inhibited by three different NPY antagonists, (R)-N2-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]D-argininam ide (BIBP3226), (R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N2(diphenyl acetyl)-argininamidetrifluoroacetate (BIBO3304), and decapeptide [D-Tyr(27,36)D-Thr32]NPY(27-36), after icv administration in freely feeding male rats. All three NPY receptor antagonists inhibited the orexigenic effects of HS014 partially and with markedly different potency. [D-Tyr(27,36)D-Thr32]NPY(27-36) was active only in subconvulsive dose. The NPY Y1 selective antagonist BIBP3226 was more effective in inhibiting the effect of HS014 than BIBO3304 despite in vitro data indicating that BIBP3226 is about 10 times less potent than BIBO3304 at NPY Y1 receptor. An enantiomer of BIBO3304, BIBO3457, failed to inhibit HS014-induced feeding, indicating that the effects of BIBO3304 were stereoselective. These results suggest that stimulation of food intake caused by weakening of melanocortinergic tone at the MC4 receptor is partially but not exclusively related to NPY Y1 receptor activation.     

D-Trp(34)] neuropeptide Y is a potent and selective neuropeptide Y Y(5) receptor agonist with dramatic effects on food intake

The neuropeptide Y (NPY) Y(5) receptor has been proposed to mediate several physiological effects of NPY, including the potent orexigenic activity of the peptide. However, the lack of selective NPY Y(5) receptor ligands limits the characterization of the physiological roles of this receptor. Screening of several analogs of NPY revealed that [D-Trp(34)]NPY is a potent and selective NPY Y(5) receptor agonist. Unlike the prototype selective NPY Y(5) receptor agonist [D-Trp(32)]NPY, [D-Trp(34)]NPY markedly increases food intake in rats, an effect that is blocked by the selective NPY Y(5) receptor antagonist CGP 71683A. These data demonstrate that [D-Trp(34)]NPY is a useful tool for studies aimed at determining the physiological roles of the NPY Y(5) receptor.     

Effects of neuropeptide Y on food intake and brain biogenic amines in the rainbow trout (Oncorhynchus mykiss)

Neuropeptide Y (NPY) is one of the most potent stimulants of food intake in mammals, but very little is known about NPY actions in fish. The present study investigated the role of NPY in food intake in the rainbow trout (Oncorhynchus mykiss). Food intake was monitored at different times after intracerebroventricular administration of porcine NPY (4 or 8 microg). Both doses significantly increased food intake at 2 and 3 h, and this effect was dose-dependent. However, 50 h after administration of NPY, food intake was significantly lower than in control fish, and cumulative food intake had returned to levels similar to those seen in the control group. The NPY antagonist (D-Tyr27,36, D-Thr32)-NPY (10 microg) inhibited food intake 2 h after icv administration, but did not block the orexigenic effect of NPY when administered jointly with 4 microg NPY. To identify the NPY receptor subtypes involved in the effects of NPY on food intake, we studied the effects of the Y1 receptor agonist (Leu31, Pro34)-NPY (4 microg), the Y2 receptor agonist NPY(3-36) (4 microg), and the highly specific Y5 receptor agonist (cPP(1-7), NPY19-23, Ala31, Aib32, Gln34)-hPP (4 microg). Short-term (2 h) food intake was moderately stimulated by the Y1 agonist, more strongly stimulated by the Y2 agonist, and unaffected by the Y5 agonist. We found that administration of NPY (8 microg icv) had no effect on aminergic systems in several brain regions 2 and 50 h after NPY administration. These results indicate that NPY stimulates feeding in the rainbow trout, and suggest that this effect is cooperatively mediated by Y2- and Y1-like NPY receptors, not by Y5-like receptors.     

Regulation of food intake in the goldfish by interaction between ghrelin and orexin

Intracerebroventricular (ICV) administration of ghrelin, orexin and neuropeptide Y (NPY) stimulates food intake in goldfish. Orexin and NPY interact with each other in the regulation of feeding, while ghrelin-induced feeding has also shown to be mediated by NPY in the goldfish model. To investigate the interaction between ghrelin and orexin, we examined the effects of a selective orexin receptor-1 antagonist, SB334867, and a growth hormone secretagogue-receptor antagonist, [D-Lys(3)]-GHRP-6, on ghrelin- and orexin-A-induced feeding. Ghrelin-induced food intake was completely inhibited for 1h following ICV preinjection of SB334867, while [D-Lys(3)]-GHRP-6 attenuated orexin-A stimulated feeding. Furthermore, ICV administration of ghrelin or orexin-A at a dose sufficient to stimulate food intake increased the expression of each other's mRNA in the diencephalon. These results indicate that, in goldfish, ghrelin and orexin-A have interacting orexigenic effects in the central nervous system. This is the first report that orexin-A-induced feeding is mediated by the ghrelin signaling in any animal model.     

Central prostaglandin D(2) stimulates food intake via the neuropeptide Y system in mice

We found that prostaglandin (PG) D(2), the most abundant PG in the central nervous system, stimulates food intake after intracerebroventricular administration in mice. The orexigenic effect of PGD(2) was mimicked by a selective agonist for the DP(1) receptor among two receptor subtypes for PGD(2), and abolished by its antagonist. Central administration of an antagonist or antisense oligodeoxynucleotide for the DP(1) receptor remarkably decreased food intake, body weight and fat mass. Hypothalamic mRNA levels of lipocalin-type PGD synthase were up-regulated after fasting. The orexigenic activity of PGD(2) was also abolished by an antagonist for neuropeptide Y (NPY) Y(1) receptor. Taken together, PGD(2) may stimulate food intake through central DP(1) receptor coupled to the NPY system.     

Effects of ghrelin administration on decreased growth hormone status in obese animals

Obesity is characterized by markedly decreased ghrelin and growth hormone (GH) secretion. Ghrelin is a GH-stimulating, stomach-derived peptide that also has orexigenic action. Ghrelin supplement may restore decreased GH secretion in obesity, but it may worsen obesity by its orexigenic action. To reveal effects of ghrelin administration on obese animals, we first examined acute GH and orexigenic responses to ghrelin in three different obese and/or diabetic mouse models: db/db mice, mice on a high-fat diet (HFD mice), and Akita mice for comparison. GH responses to ghrelin were significantly suppressed in db/db, HFD, and Akita mice. Food intake of db/db and Akita mice were basally higher, and further stimulation of food intake by ghrelin was suppressed. Pituitary GH secretagogue receptor mRNA levels in db/db and HFD mice were significantly decreased, which may partly contribute to decreased GH response to ghrelin in these mice. In Akita mice for comparison, decreased hypothalamic GH-releasing hormone (GHRH) mRNA levels may be responsible for decreased GH response, since maximum GH response to ghrelin needs GHRH. When ghrelin was injected into HFD mice with GHRH coadministrated, GH responses to ghrelin were significantly emphasized. HFD mice injected with low-dose ghrelin and GHRH for 10 days did not show weight gain. These results indicate that low-dose ghrelin and GHRH treatment may restore decreased GH secretion in obesity without worsening obesity.     

Complement C5a stimulates food intake via a prostaglandin D2- and neuropeptide Y-dependent mechanism in mice

We have recently found that prostaglandin (PG) D₂ stimulates food intake via DP₁ receptor. Here we show that complement C5a stimulates food intake by activating the orexigenic PGD₂ system. C5a (30–100 pmol/mouse), after intracerebroventricular administration, stimulated food intake in non-food-deprived mice. The orexigenic activity of C5a was blocked by co-administration of a DP₁ receptor antagonist, BWA868C. Central administration of C5a elevated the hypothalamic mRNA expression of COX-2 but not COX-1, and the food intake stimulation of C5a was inhibited by pretreatment with a COX-2 inhibitor, celecoxib, suggesting that C5a activates COX-2 upstream of the PGD₂–DP₁ system. The orexigenic activity of C5a was also inhibited by an antagonist for neuropeptide Y (NPY) Y₁ receptor, which was activated downstream of the PGD₂–DP₁ system. These results suggest that C5a stimulates food intake via a PGD₂- and NPY-dependent mechanism. C5a is the first example of orexigenic peptides acting through the PGD₂–NPY system in the central nervous system.     

Effects of homologous ghrelins on the growth hormone/insulin-like growth factor-I axis in the tilapia, Oreochromis mossambicus

Ghrelin is a gut-brain peptide synthesized mainly in the oxyntic mucosal cells of the stomach, and has potent growth hormone (GH)-releasing and orexigenic activities. Recently, two forms of ghrelin, ghrelin-C8 and -C10, were identified in the Mozambique tilapia (Oreochromis mossambicus). The present study describes in vitro and in vivo effects of these endogenous ghrelins on the GH/insulin-like growth factor-I (IGF-I) axis. Ghrelin-C8 (100 nM) stimulated GH release from primary cultures of pituitary cells after 4 and 8 h of incubation, whereas no effect was seen on prolactin (PRL) release. Stimulatory effects of ghrelin-C8 and -C10 (100 nM) on GH release during 6 h of incubation were blocked by pre-incubation with GHS receptor antagonist, [D-Lys(3)]-GHRP-6 (10 microM). Intraperitoneal injection of ghrelin-C8 (1 ng/g body weight) and -C10 (0.1 and 1 ng/g body weight) significantly increased plasma GH levels after 5 h. Significant increases were observed also in hepatic expression of IGF-I and GH receptor (GHR) mRNA following injections of both forms of ghrelin (0.1 and 1 ng/g body weight), although there was no effect on plasma levels of IGF-I. In the next experiment, both forms of ghrelin (1 ng/g body weight) significantly increased plasma IGF-I levels 10 h after the injection. No significant effect of either ghrelin was observed on plasma PRL levels. Both forms of GHS receptor (GHSR-1a and -1b) were found in the pituitary, clearly indicating that tilapia ghrelins stimulate primarily GH release through the GHS receptor. Stimulation of hepatic expression of IGF-I and GHR suggests metabolic roles of ghrelin in tilapia.     

Ghrelin -- a new endogenous growth hormone secretagogue

Ghrelin is a new endogenous peptide, discovered in 1999 by Kojima et al., as the result of a search for an endogenous ligand for an orphan receptor of known structure and function. Ghrelin is composed of 28 amino acids and is produced mostly by cells of the stomach, hypothalamus, and hypophysis, but it has also been detected in other tissues. Its discovery is related to the development of a new hypothesis regarding the regulation of growth hormone secretion. It is an antagonist of somatostatin. Ghrelin activates the release of growth hormone from the somatotrophic cells of the hypophysis. It participates in the regulation of energy homeostasis, increases food intake, decreases energy output and exerts a lipogenetic effect. Its metabolic effects do not depend on the GH/IGF-I system, but are mediated by the NPY/Y1 and AGRP receptor system. Ghrelin influences the secretion and motility of the gastrointestinal tract, especially the stomach. The presence of ghrelin and its receptors has also been demonstrated in many other tissues. Its function in these tissues has not yet been studied, thus providing many possibilities for further research.     

Alpha-melanocyte-stimulating hormone mediates melanin-concentrating hormone-induced anorexigenic action in goldfish

In goldfish, intracerebroventricular (ICV) administration of melanin-concentrating hormone (MCH) inhibits feeding behavior, and fasting decreases hypothalamic MCH-like immunoreactivity. However, while MCH acts as an anorexigenic factor in goldfish, in rodents MCH has an orexigenic effect. Therefore, we examined the involvement of two anorexigenic neuropeptides, alpha-melanocyte-stimulating hormone (alpha-MSH) and corticotropin-releasing hormone (CRH), in the anorexigenic action of MCH in goldfish, using an alpha-MSH receptor antagonist, HS024, and a CRH receptor antagonist, alpha-helical CRH((9-41)). ICV injection of HS024, but not alpha-helical CRH((9-41)), suppressed MCH-induced anorexigenic action for a 60-min observation period. We then examined, using a real-time PCR method, whether MCH affects the levels of mRNAs encoding various orexigenic neuropeptides, including neuropeptide Y (NPY), orexin, ghrelin and Agouti-related peptide (AgRP), in the goldfish diencephalon. ICV administration of MCH at a dose sufficient to inhibit food consumption decreased the expression of mRNAs for NPY and ghrelin, but not for orexin and AgRP. These results indicate that the anorexigenic action of MCH in the goldfish brain is mediated by the alpha-MSH signaling pathway and is accompanied by inhibition of NPY and ghrelin synthesis.     

Growth hormone secretion: molecular and cellular mechanisms and in vivo approaches

Growth hormone (GH) release is under the direct control of hypothalamic releasing hormones, some being also produced peripherally. The role of these hypothalamic factors has been understood by in vitro studies together with such in vivo approaches as stalk sectioning. Secretion of GH is stimulated by GH-releasing hormone (GHRH) and ghrelin (acting via the GH secretagogue [GHS] receptor [GHSR]), and inhibited by somatostatin (SRIF). Other peptides/proteins influence GH secretion, at least in some species. The cellular mechanism by which the releasing hormones affect GH secretion from the somatotrope requires specific signal transduction systems (cAMP and/or calcium influx and/or mobilization of intracellular calcium) and/ or tyrosine kinase(s) and/or nitric oxide (NO)/cGMP. At the subcellular level, GH release (at least in response to GHS) is accomplished by the following. The GH-containing secretory granules are moved close to the cell surface. There is then transient fusion of the secretory granules with the fusion pores in the multiple secretory pits in the somatotrope cell surface.     

Deterministic construct of amplifying actions of ghrelin on pulsatile growth hormone secretion

Ghrelin is a native ligand for the growth hormone secretagogue (GHS) receptor that stimulates pulsatile GH secretion markedly. At present, no formal construct exists to unify ensemble effects of ghrelin, GH-releasing hormone (GHRH), somatostatin (SRIF), and GH feedback. To model such interactions, we have assumed that ghrelin can stimulate pituitary GH secretion directly, antagonize inhibition of pituitary GH release by SRIF, oppose suppression of GHRH neurons in the arcuate nucleus (ArC) by SRIF, and induce GHRH secretion from ArC. The dynamics of such connectivity yield self-renewable GH pulse patterns mirroring those in the adult male and female rat and explicate the following key experimental observations. 1) Constant GHS infusion stimulates pulsatile GH secretion. 2) GHS and GHRH display synergy in vivo. 3) A systemic pulse of GHS stimulates GH secretion in the female rat at any time and in the male more during a spontaneous peak than during a trough. 4) Transgenetic silencing of the neuronal GHS receptor blunts GH pulses in the female. 5) Intracerebroventricular administration of GHS induces GH secretion. The minimal construct of GHS-GHRH-SRIF-GH interactions should aid in integrating physiological data, testing regulatory hypotheses, and forecasting innovative experiments.     

Nonpeptidyl somatostatin agonists demonstrate that sst2 and sst5 inhibit stimulated growth hormone secretion from rat anterior pituitary cells.

Somatostatin (SST) regulates growth hormone (GH) secretion from pituitary somatotrophs by interacting with members of the SST family of G-protein-coupled receptors (sst1-5). We have used potent, nonpeptidyl SST agonists with sst2 and sst5 selectivity to determine whether these receptor subtypes are involved in regulating growth hormone releasing hormone (GHRH) stimulated secretion. GHRH stimulated GH release from pituitary cells in a dose-dependent manner, and this secretion was inhibited by Tyr(11)-SST-14, a nonselective SST analog. A sst2 selective agonist, L-779,976, potently inhibited GHRH-stimulated GH release. In addition, L-817, 818, a potent sst5 receptor selective agonist, also inhibited GH secretion, but was approximately 10-fold less potent (P < 0.01, ANOVA) in inhibiting GH release than either Tyr(11)-SST-14 or L-779, 976. These results show that both sst2 and sst5 receptor subtypes regulate GHRH-stimulated GH release from rat pituitary cells.     

The effect of agouti-related protein on growth hormone secretion in adult male rats

Agouti-related protein (AGRP) and neuropeptide Y (NPY) are synthesized in the same neurons in the hypothalamic arcuate nucleus. We have previously shown that NPY/AGRP neurons contain growth hormone (GH) receptor mRNA, and are activated following systemic GH administration. We also reported that NPY inhibits GH secretion when administered centrally. In this study, we have examined the effect of AGRP on GH secretion. Central administration of AGRP (83-132) as a single injection of 1 or 10 microg/rat, or chronic treatment of 1 microg/rat, every 12 h for 7 days, did not alter the GH secretory pattern of adult male rats. AGRP (83-132) at doses of 1-100 nM (4 h) did not alter baseline- and GHRH-induced GH secretion from the rat pituitary cell cultures. These results suggest that AGRP does not play a significant role in the feedback regulation of the GH secretion.     

Regulation of Neuropeptide Y Release by Neuropeptide Y Receptor Ligands and Calcium Channel Antagonists in Hypothalamic Slices

Neuropeptide Y (NPY) is an important regulator of energy balance in mammals through its orexigenic, antithermogenic, and insulin secretagogue actions. We investigated the regulation of endogenous NPY release from rat hypothalamic slices by NPY receptor ligands and calcium channel antagonists. High-potassium stimulation (60 mM) of the slices produced a calcium-dependent threefold increase in NPY release above basal release. The Y2 receptor agonists NPY(13-36) and N-acetyl[Leu28,Leu31]NPY(24-36), the Y4 agonist rat pancreatic polypeptide (rPP), and the Y4/Y5 agonist human pancreatic polypeptide (hPP) significantly reduced both basal and stimulated NPY release. NPY(13-36)-induced reduction of NPY release could be partially prevented in the presence of the weak Y2 antagonist T4-[NPY(33-36)]4, whereas the hPP- and rPP-induced inhibition of release was not affected by the Y5 antagonist CGP71683A or the Y1 antagonist BIBP3226. The selective Y1, Y2, and Y5 antagonists had no effect on either basal or potassium-stimulated release when administered alone. The calcium channel inhibitors omega-conotoxin GVIA (N-type), omega-agatoxin TK (P/Q-type), and omega-conotoxin MVIIC (Q-type) all significantly inhibited potassium-stimulated NPY release, without any effect on basal release, whereas nifedipine had no effect on either basal or stimulated release. Addition of both omega-conotoxin GVIA and omega-agatoxin TK together completely inhibited the potassium-stimulated release. In conclusion, we have demonstrated that NPY release from hypothalamic slices is calcium-dependent, involving N-, P-, and Q-type calcium channels. NPY release is also inhibited by Y2 agonists and rPP/hPP, suggesting that Y2 and Y4 receptors may act as autoreceptors on NPY-containing nerve terminals.     

Bis(31/31')[[Cys(31), Nva(34)]NPY(27-36)-NH(2)]: a neuropeptide Y (NPY) Y(5) receptor selective agonist with a latent stimulatory effect on food intake in rats

The actions of neuropeptide Y (NPY) are mediated by at least six G-protein coupled receptors denoted as Y(1), Y(2), Y(3), Y(4), Y(5), and y(6). Investigations using receptor selective ligands and receptor knock-out mice suggest that NPY effects on feeding are mediated by both Y(1) and Y(5) receptors. We have previously shown that Cys-dimers of NPY C-terminal peptides exhibit Y(1) selectivity relative to Y(2) receptors. Re-investigation of their selectivity with respect to the newly cloned receptors, has identified bis(31/31') [[Cys(31), Nva(34)]NPY(27-36)-NH(2)] (BWX-46) as a Y(5) receptor selective agonist. BWX-46 selectively bound Y(5) receptors, and inhibited cAMP synthesis by Y(5) cells with potencies comparable to that of NPY. Moreover, BWX-46 (10 microM) exhibited no significant effect on the cAMP synthesis by Y(1), Y(2), and Y(4) cells. Thus, BWX-46 constitutes the lowest molecular weight Y(5) selective agonist reported to date. Intrahypothalamic (i.h.t)-injection of 30 and 40 microg of BWX-46 stimulated the food intake by rats in a gradual manner, reaching maximal level 8 h after injection. This response was similar to that exhibited by other Y(5) selective agonists, but differed from that of NPY, which exhibited a rapid orexigenic stimulus within 1 h. It is suggested that the differences in the orexigenic stimuli of NPY and Y(5) agonists may be due to their differences in the signal transduction mechanisms.     

Growth hormone (GH)-independent stimulation of adiposity by GH secretagogues

Growth hormone secretagogues (GHSs) stimulate growth hormone (GH) secretion, which is lipolytic. Here we compared the effects of twice daily s.c. treatment of GH and the GHS, ipamorelin, on body fat in GH-deficient (lit/lit) and in GH-intact (+/lit and +/+) mice. In +/lit and lit/lit mice ipamorelin induced a small (15%) increase in body weight by 2 weeks, that was not further augmented by 9 weeks. GH treatment markedly enhanced body weight in both groups. Ipamorelin also increased fat pad weights relative to body weight in both lit/lit and +/lit mice. Two weeks GHS treatment (ipamorelin or GHRP-6) also increased relative body fat, quantified by in vivo dual energy X-ray absorpiometry (DEXA) in GH-intact mice. GH decreased relative fat mass in lit/lit mice and had no effect in GH-intact mice. Treatment with GHS, but not GH, increased serum leptin and food intake in GH-intact mice. Thus, GHSs increase body fat by GH-independent mechanisms that may include increased feeding.     

Endocrine and orexigenic actions of growth hormone secretagogues in rainbow trout (Oncorhynchus mykiss)

The effects of growth hormone secretagogues (GHSs) on the teleost somatotropic axis are poorly understood, particularly with respect to insulin-like growth factor-I (IGF-I) and the IGF-binding proteins (IGFBPs). To assess the endocrine and orexigenic responses of rainbow trout (Oncorhynchus mykiss) to GHS treatment, animals were injected with human GHRH(1-29)-amide, KP-102 or rat ghrelin at 0, 1 or 10 pmol/g body mass. Feed intake was tested at 2 and 5 h post-injection and plasma levels of growth hormone (GH), IGF-I and the IGFBPs were determined at 3, 6 and 12 h post-injection. Feed intake was significantly elevated by all of the GHSs tested at both post-injection time points. All GHSs elevated plasma GH levels in a time-dependent manner. Plasma IGF-I levels were elevated by all GHSs at 3 h post-injection, whereas those animals treated with KP-102 and ghrelin exhibited depressions at 6 h. Four IGFBPs were identified in the plasma by western blotting. Levels of the 20 kDa IGFBP decreased over the sampling time. Levels of the 32 kDa IGFBP were significantly depressed by all GHSs tested. Levels of the 42 kDa IGFBP were significantly elevated by all GHSs tested. Plasma levels of the 50 kDa IGFBP was decreased in some treatment groups at 3 h, but elevated by 6 h in the ghrelin-treated groups and elevated in all treatment groups by 12 h post-injection. The endocrine and orexigenic responses demonstrate that GHSs influence the teleost neuroendocrine system beyond short-term actions (<3 h post-injection) on GH release and the responses of the IGFBPs to GHS treatment support this notion and clarify their identification as functional homologues to mammalian IGFBPs.