Chicken ghrelin and growth hormone-releasing peptide-2 inhibit food intake of neonatal chicks

Ghrelin is an endogenous ligand for the growth hormone secretagogue (GHS) receptor. Ghrelin stimulates feeding in rats, however, intracerebroventricular (i.c.v.) injection of rat ghrelin inhibits feeding of neonatal chicks. In the present study, the effect of i.c.v. injection of different ghrelins including chicken and bullfrog ghrelin, and synthetic GH-releasing peptide (GHRP) on feeding of neonatal chicks was investigated. Chicken ghrelin strongly suppressed feeding. To compare the inhibitory effect, chicken and rat ghrelin were examined. The suppressive effect of feeding by chicken and rat ghrelin was almost identical. Bullfrog ghrelin contains a change in the acylated amino acid from Ser to Thr, strongly suppressed feeding. The i.c.v. injection of GHRP-2 (KP-102), a synthetic GHS, also inhibited feeding. These results indicate that the chicken GHS receptor is affected by several forms of GHS, and that food intake of neonatal chicks is inhibited by GHS receptor agonists.     

Effects of olanzapine on muscarinic M3 receptor binding density in the brain relates to weight gain, plasma insulin and metabolic hormone levels

The second generation antipsychotic drug (SGA) olanzapine has an efficacy to treat schizophrenia, but can cause obesity and type II diabetes mellitus. Cholinergic muscarinic M3 receptors (M3R) are expressed on pancreatic β-cells and in the brain where they influence insulin secretion and may regulate other metabolic hormones via vagal innervation of the gastrointestinal tract. Olanzapine's M3R antagonism is an important risk factor for its diabetogenic liability. However, the effects of olanzapine on central M3Rs are unknown. Rats were treated with 0.25, 0.5, 1.0 or 2.0 mg olanzapine/kg or vehicle (3×/day, 14-days). M3R binding densities in the hypothalamic arcuate (Arc) and ventromedial nuclei (VMH), and dorsal vagal complex (DVC) of the brainstem were investigated using [3H]4-DAMP plus pirenzepine and AF-DX116. M3R binding correlations to body weight, food intake, insulin, ghrelin and cholecystokinin (CCK) were analyzed. Olanzapine increased M3R binding density in the Arc, VMH and DVC, body weight, food intake, circulating plasma ghrelin and CCK levels, and decreased plasma insulin and glucose. M3R negatively correlated to insulin, and positively correlated to ghrelin, CCK, food intake and body weight. Increased M3R density is a compensatory up-regulation in response to olanzapine's M3R antagonism. Olanzapine acts on M3R in regions of the brain that control food intake and insulin secretion. Olanzapine's M3R blockade in the brain may inhibit the acetylcholine pathway for insulin secretion. These findings support a role for M3Rs in the modulation of insulin, ghrelin and CCK via the vagus nerve and provide a mechanism for olanzapine's diabetogenic and weight gain liability.     

Synthesis and pharmacological in vitro and in vivo evaluations of novel triazole derivatives as ligands of the ghrelin receptor. 1

A new series of growth hormone secretagogue (GHS) analogues based on the 1,2,4-triazole structure were synthesized and evaluated for their in vitro binding and their ability to stimulate intracellular calcium release to the cloned hGHS-1a ghrelin receptor expressed in LLC PK-1 cells. We have synthesized potent ligands of this receptor, some of them behaving as agonists, partial agonists, or antagonists. Some compounds among the most potent, i.e., agonist 29c (JMV2873), partial agonists including 21b (JMV2810), antagonists 19b (JMV2866) and 19c (JMV2844), were evaluated for their in vivo activity on food intake, after sc injection in rodents. Some compounds were found to stimulate food intake like hexarelin; some others were identified as potent hexarelin antagonists in this assay. Among the tested compounds, 21b was identified as an in vitro ghrelin receptor partial agonist, as well as a potent in vivo antagonist of hexarelin-stimulated food intake in rodents. Compound 21b was without effect on GH release from rat. However, in this series of compounds, it was not possible to find a clear correlation between in vitro and in vivo results.     

2,4-diaminopyrimidine derivatives as potent growth hormone secretagogue receptor antagonists

Ghrelin, a gut-derived orexigenic hormone, is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R). Centrally administered ghrelin has been shown to cause hunger and increase food intake in rodents. Inhibition of ghrelin actions with ghrelin antibody, peptidyl GHS-R antagonists, and antisense oligonucleosides resulted in weight loss and food intake decrease in rodents. Here we report the effects of GHS-R antagonists, some of which were potent, selective, and orally bioavailable. A structure-activity relationship study led to the discovery of 8a, which was effective in decreasing food intake and body weight in several acute rat studies.     

Recent developments in ghrelin receptor (GHS-R1a) agonists and antagonists

The 28 amino acid polypeptide, ghrelin, is a novel neuroendocrine hormone synthesised primarily in the stomach. It stimulates growth hormone (GH) secretion, increases food intake and appetite and promotes body weight gain by decreasing fat mass metabolism, particularly in rodent models. The pharmacological properties of this peptide are mediated by the type 1a growth hormone secretagogue receptor (GHS-R1a), a G-protein-coupled receptor localised predominantly in the pituitary gland and hypothalamus. The discovery of new agents that either mimic or modulate the actions of ghrelin via the GHS-R1a has attracted considerable interest in recent years. Many peptidyl and peptidomimetic GHS-R1a agonists such as growth hormone-releasing peptides (GHRPs) and growth hormone secretagogues (GHSs) that stimulate GH secretion and increase plasma levels of insulin-like growth factor-1 (IGF-1) have been identified. First generation GHSs have undergone extensive clinical evaluation in humans, but have shown disappointing results in the treatment of diseases such as frailty, the age-related decline in body composition. Several of these compounds were found to increase adiposity in rodent models. Second generation dipeptidyl and third generation small molecule GHSs have recently been profiled in various animal models. A third generation of GHS with partial agonist activity at the GHS-R1a showed anabolic activity in rodent models, increasing fat-free (muscle) mass rather than fat mass. A few GHS-R1a receptor antagonists have been disclosed, although these compounds have exhibited poor selectivities or weak affinities for the receptor. Since endogenous ghrelin appears to play an important role in the long-term regulation of energy balance, GHS-R1a antagonists may be useful in the prevention of weight gain, following weight loss, or in the treatment of obesity, a pathological condition characterised by excess adiposity. This review describes the pharmacology of select ghrelin receptor modulators and highlights new ghrelin receptor agonists and antagonists that have appeared in the literature from 1999 – 2002.     

Serum ghrelin concentrations in patients receiving olanzapine or risperidone

Rationale Although enhanced appetite and weight gain are potential side effects of treatment with antipsychotic agents, particularly olanzapine and clozapine, the mechanism is poorly understood.Objectives To test the hypothesis that ghrelin, a gastrointestinal hormone that enhances appetite, is involved in increased food intake and weight gain during treatment with antipsychotics.Methods Serum ghrelin concentrations were investigated in schizophrenic patients receiving olanzapine or risperidone, and in healthy volunteers.Results Serum ghrelin concentrations did not increase, but rather decreased, in patients treated with olanzapine or risperidone in comparison with healthy volunteers. No significant difference was found in serum ghrelin concentration between patients treated with olanzapine and risperidone.Conclusions Our results indicate that ghrelin is not a direct cause of increased food intake and weight gain during treatment with olanzapine or risperidone, whereas ghrelin is associated with metabolic change in patients receiving these agents.     

Dual age-dependent effect of ghrelin administration on serum level of insulin-like growth factor-1 and gastric growth in young rats

Ghrelin is a circulating growth hormone-releasing peptide primarily isolated from human and rat stomach. The aim of present study was to investigate the effect of ghrelin administration on gastric growth in suckling, and young peripubertal 7-week-old rats. Rats were treated for 7 days with saline or ghrelin (4, 8 or 16 nmol/kg/dose) intraperitoneally twice a day. Suckling rats were treated from the fist postnatal day. Results: Treatment with ghrelin did not affect animal weight in suckling rats; whereas in 7-week-old animals, administration of ghrelin caused a significant increase in body weight. In suckling rats, ghrelin decreased the gastric weight, DNA synthesis and DNA content. In young 7-week-old peripubertal rats, treatment with ghrelin increased food intake and animal body weight. This effect was accompanied with a significant increase in gastric mucosa weight, DNA synthesis and DNA content. Treatment with ghrelin increased serum level of growth hormone in all rats tested, but this result was much higher in 7-week-old peripubertal rats than in suckling rats. Serum level of insulin-like growth factor-1 was not affected by ghrelin administration in suckling rats. In contrast, ghrelin caused a significant increase in serum level of insulin-like growth factor-1 in 7-week-old peripubertal rats. We conclude that administration of ghrelin exhibits biphasic effect on gastric growth in young rats: in suckling rats, ghrelin reduces gastric growth, whereas in young 7-week-old animals, treatment with ghrelin stimulates gastric growth. The growth-promoting effect of ghrelin in the stomach seems to depend on the stimulation of food intake and the release of insulin-like growth factor-1.     

Pharmacological characterization of the ghrelin receptor antagonist, GSK1614343 in rat RC-4B/C cells natively expressing GHS type 1a receptors

A novel growth hormone secretagogues type 1a (GHS1a) receptors antagonist (2R)-N'-[3,5- bis(trifluoromethyl)phenyl]-2-[(8aR)-hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl]-2-(3- pyridinyl)ethanohydrazide (GSK1614343) was functionally characterised in rat pituitary adenoma cell line, RC-4B/C endogenously expressing GHS1a receptors. The antagonism profile of GSK1614343 was compared with that of 6-[(4-fluorophenyl)oxy]-2-methyl-3-{[(3S)-1-(1-methylethyl)-3- piperidinyl]methyl}-4(3H)-quinazolinone (YIL-781) another ghrelin receptor antagonist recently published. The activity of both compounds was also evaluated at rat recombinant GHS1a receptors. The characterization of the two antagonists was performed by intracellular calcium mobilization measurements by using fluorometric imaging plate reader (FLIPR) technology and inositol phosphate (IP) turnover measurements by [3H]-IP accumulation assay. RC-4B/C and U2-OS cells transiently transduced with rat GHS1a receptors virus were used. In RC-4B/C cells, GSK1614343 and YIL-781, depressed the ghrelin maximal response in FLIPR assay as result of hemi-equilibria phenomenon. When using the [3H]-IP accumulation assay both compounds behaved as competitive antagonist with pKB values of 8.03 for GSK1614343 and 7.54 for YIL-781. In rat recombinant receptor, GSK1614343 and YIL-781 inhibited the calcium response induced by ghrelin with pIC50 values of 7.90 and 8.27, respectively. GSK1614343 and YIL-781 did not show intrinsic activity in both endogenously expressed and recombinant rat GHS1a receptors. The new ghrelin receptor antagonist GSK1614343 is a potent competitive antagonist in rat pituitary RC-4B/C cells endogenously expressing GHS1a receptors when equilibrium conditions between ligand and receptor are reached in the test assay. GSK1614343 represents a useful tool to investigate the physiological relevance of the ghrelin system in rat models.     

The role of ghrelin in the control of energy balance

Ghrelin is the only potent orexigenic peptide in circulation. It stimulates food intake and leads to positive energy balance, adipogenesis, and body weight gain. However, the physiological significance of ghrelin in the regulation of energy homeostasis is controversial, since loss of ghrelin function in rodents does not necessarily lead to anorexia and weight loss. In this chapter, we discuss the metabolic function of ghrelin and are highlighting recent findings including the discovery and function of ghrelin-acylating enzyme ghrelin O-acyltransferase (GOAT). Based on available published data, we conclude that ghrelin is a principally important endogenous regulator of energy balance, which however may affect both food intake and systemic metabolism via independent mechanisms. Importantly, ghrelin, when acylated by GOAT, might represent a key molecular link between the sensing of consumed calories and the neuroendocrine control of energy homeostasis. Thus, agents antagonizing the action of ghrelin may have therapeutic potential in the therapy of obesity.     

Changes of ghrelin and leptin levels in plasma by cigarette smoke in rats

Cigarettes smoke (CS) limits food intake and body weight increase. Ghrelin and leptin are hormones regulating appetite and energy balance. While ghrelin increases food intake and causes a positive energy balance, leptin decreases food intake and enhances a negative energy balance. To investigate the possible role of ghrelin and leptin regarding the negative energy balance caused by CS, 10-week old male Wistar rats (n = 10) were exposed to CS from 30 cigarettes twice a day for 5 days a week for four weeks. In the smoking group, food intake and body weight gain were less than those in the non-smoking group (n = 10) during the entire CS exposure. In the smoking group, the plasma levels of acyl ghrelin were significantly higher (75.9 ± 5.1 fmol/ml versus 46.5 ± 3.3 fmol/ml, p < 0.01), while those of leptin were significantly lower than those in the non-smoking group (434.9 ± 41.1 ng/ml versus 744.0 ± 45.4 ng/ml, p < 0.01) after the final CS exposure. However, the plasma des-acyl ghrelin levels were not affected by CS exposure. These results suggested that acyl ghrelin and leptin levels in plasma may change to compensate for the negative energy balance by CS.     

Review article: The gastrointestinal tract: neuroendocrine regulation of satiety and food intake

Background The gastrointestinal tract elicits numerous signals regulating food intake and satiety, and recently many studies have been performed to elucidate the mechanisms regulating these signals. Aim To describe the effects of the gastrointestinal tract on satiety, satiation and food intake. Methods A PubMed search was performed to identify and select the relevant literature using search terms including ‘gastric satiety, intestine + satiety, satiation, cholecystokinin, ghrelin, peptide YY, glucagon‐like peptide‐1 and ileal brake’. Results Satiation, satiety and food intake result, among other factors, from signals originating in the stomach caused by distension and signals from the small intestine. These intestinal signals result from nutrient sensing in the gut and activate neural and humoral pathways. Activation of the distal part of the gut, the so called ileal brake, leads to reduction in hunger and food intake, and models of chronic ileal brake activation lead to massive weight loss. Conclusion Gastrointestinal signals are crucial for the regulation of food intake, satiety and satiation. The ileal brake deserves special attention, as both ileal intubation studies and surgical studies demonstrate that activation of the ileal brake reduces food intake. In the surgical models, weight loss occurs without adaptation to the anorectic effects of ileal brake activation.     

Potential role of the growth hormone secretagogues in clinical practice

Growth hormone secretagogues (GHS) are synthetic molecules that promote secretion of growth hormone by activation of the GHS receptor (GHSR). Ghrelin is the endogenous ligand of this receptor, which was isolated from gastric mucosa. This peptide is widely expressed in different tissues and has a broad spectrum of effects. This review summarises: i) structure and pharmacokinetics of the more important GHS; ii) structure, distribution and subcellular pathways of ghrelin; iii) structure, distribution and signal transduction of the GHS receptors; and iv) effects of GHS in different tissues and organs and the potential therapeutic role of ghrelin and GHS. There are increasing evidences that GHS and ghrelin have a wide range of actions both in physiological and pathological conditions. Potential therapeutic applications of these GHS are constantly increasing and clinical trials are needed to establish usefulness of these compounds.     

Cardiac effects of ghrelin and its endogenous derivatives des-octanoyl ghrelin and des-Gln14-ghrelin

The mechanisms underlying the cardiac activities of synthetic growth hormone secretagogues (GHS) are still unclear. The natural ligand of the GHS receptors, i.e. ghrelin, classically binds the GHS receptor and exerts endocrine actions in acylated forms only; its cardiovascular actions still need to be investigated further. In order to clarify these aspects, we studied the effects of either the synthetic peptidyl GHS hexarelin (1 microM), or the natural ghrelin (50 nM) and the endogenous ghrelin derivatives des-Gln14-ghrelin (1-100 nM) and des-octanoyl ghrelin (50 nM), on the tension developed by guinea pig papillary muscle and on L-type Ca2+ current (ICa) of isolated ventricular cells. The binding of these molecules to ventricular cell membrane homogenates was also studied. We observed that all peptides reduced the tension developed at low frequencies (60-120 beats/min) in a dose-dependent manner. No alteration in cardiac contractility was induced by des-Gln14-ghrelin or des-octanoylated ghrelin when the endocardial endothelium had been removed or after cyclooxygenase blockade. Pretreatment with tyramine (2 microM) had no effect on the inotropic response induced by des-Gln(14)-ghrelin. No significant effect on I(Ca) of isolated ventricular cells was observed in the presence of des-Gln14-ghrelin (100 nM). The order of potency on the tension of papillary muscle was: des-octanoyl ghrelin > ghrelin = des-Gln14-ghrelin > hexarelin. This gradient of potency was consistent with the binding experiments performed on ventricular membranes where either acylated or unacylated ghrelin forms, and hexarelin, recognized a common high-affinity binding site. In conclusion, ghrelin, des-Gln14-ghrelin and des-octanoyl ghrelin, show similar negative inotropic effect on papillary muscle; as des-octanoyl ghrelin is peculiarly devoid of any GH-releasing activity, the cardiotropic action of these molecules is independent of GH release. The binding studies and the experiments performed both on the isolated cells and on papillary muscle after endothelium removal or cyclooxygenase blockade indicate that the cardiotropic action of natural and synthetic ghrelin analogues reflects the interaction with a novel GHS receptor (peculiarly common for ghrelin and des-octanoyl ghrelin), leading to release of cyclooxygenase metabolites from endothelial cells, as indicated by direct measurement of prostacyclin metabolite 6-keto-PGF(1alpha).     

Early tolerance to the hypophagic effect of the cannabinoid receptor antagonist SR141716 does not impede blockade of an orexigenic stimulus

The cannabinoid CB1 receptor antagonist SR141716 (Rimonabant) is known to reduce food intake by central and peripheral mechanisms. Recently, SR141716 has been reported to block the orexigenic effect of ghrelin, a potent orexigenic peptide produced by the stomach. This study investigated whether in rats, made tolerant to the hypophagic effect of SR141716, the drug was still capable to block the orexigenic activity of another non-natural (hypothalamic) peptide, i.e., the growth hormone releasing peptide (GHRP) hexarelin, a ghrelin mimetic. In the acute experiments, each dose of SR141716 (1, 5 and 10 mg/kg i.p.) reduced food intake with respect to vehicle-treated rats, whereas hexarelin (160 microg/kg s.c.) markedly stimulated feeding. All doses of SR141716 were capable to reduce the orexigenic effect of the GHRP. A 15-day administration of SR141716 (10 mg/kg i.p.) reduced both food intake and body weight. Tolerance to the hypophagic effect of SR141716 developed within 5 days, but in contrast, body weight remained markedly below that of vehicle-treated group throughout the entire treatment period. Interestingly, despite development of tolerance to its hypophagic effect, SR141716 was capable to suppress the orexigenic effect of repeated hexarelin challenge tests performed throughout the chronic experiments. In conclusion, the results of the present study confirm and broaden the existence of a functional relationship between ghrelin and endocannabinoids in the control of food intake, and bespeak the ability of a CB1 receptor antagonist to suppress orexia caused by stimuli alien to direct stimulation of the cannabinoid system.     

Growth hormone secretagogue receptor antagonists

The patent claims peptidic/nonpeptidic inhibitors of the ghrelin receptor, the growth hormone secretagogue receptor (GHSR) 1A. Among these compounds, it was disclosed that the addition in some compounds of a GlyMetAla tripeptide at the N-terminus of the ghrelin peptide agonists converts them into ghrelin receptor antagonists. One of these peptides, among the few that have been studied in vivo, was shown to be able to reduce food intake and body weight gain in rats.     

糖肝煎对2型糖尿病大鼠摄食及摄食抑制因子1、饥饿素的影响

目的 探究糖肝煎对2型糖尿病（T2DM）大鼠摄食及血浆摄食抑制因子1(Nesfatin-1)、饥饿素（Ghrelin）的影响。方法将50只SD大鼠采用随机数字表法分成正常组、T2DM组、利格列汀组（50 mg/kg利格列汀）、低剂量组（2.8 g/kg糖肝煎）、高剂量组（5.6 g/kg糖肝煎）,10只/组;通过尾静脉注射链脲佐菌素（STZ）及高脂饲料进行T2DM大鼠模型构建;利格列汀组、低剂量组及高剂量组连续4周给予相应药物灌胃,正常组及T2DM组给予等量生理盐水,1次/天,于实验结束后观察大鼠一般情况,并称取大鼠体质量,测定摄食量、摄水量、尿量;全自动生化分析仪测定大鼠血清空腹血糖（FBG）、低密度脂蛋白胆固醇（LDLC）、高密度脂蛋白胆固醇（HDL-C）、三酰甘油（TG）及总胆固醇（TC）水平;计算大鼠口服葡萄糖耐量试验（OGTT）曲线下面积（AUC）;酶联免疫吸附试验（ELISA）检测血清胰岛素（INS）水平及血浆核连蛋白2(NUCB2)、Ghrelin、Nesfatin-1水平;实时荧光定量逆转录聚合酶链式反应（qRT-PCR）检测血浆Nesfatin-1、Ghrelin m...     

Growth hormone secretagogue receptor antagonists

The patent claims peptidic/nonpeptidic inhibitors of the ghrelin receptor, the growth hormone secretagogue receptor (GHSR) 1A. Among these compounds, it was disclosed that the addition in some compounds of a GlyMetAla tripeptide at the N-terminus of the ghrelin peptide agonists converts them into ghrelin receptor antagonists. One of these peptides, among the few that have been studied in vivo, was shown to be able to reduce food intake and body weight gain in rats.     

Central manipulation of dopamine receptors attenuates the orexigenic action of ghrelin

Objective

Recent evidence suggests that ghrelin, a peptidic hormone stimulating food intake, interacts with the dopamine signaling. This interaction has been demonstrated to modulate several effects of ghrelin, such as locomotor activity, memory, and food intake. Ghrelin increases dopamine levels in the shell of the nucleus accumbens stimulating food intake, while ablation of the ghrelin receptor attenuates the hypophagia caused by the activation of dopamine receptor 2. However, it is not known whether the orexigenic action of ghrelin is due to changes in central dopamine receptors.

Materials and methods

We used Sprague–Dawley rats injected with different dopamine receptor agonists, antagonists, and ghrelin.

Results

We demonstrate that the specific central blockade of dopamine receptor 1, 2, and 3 (D1, D2, and D3, respectively) reduces the orexigenic action of ghrelin. Similarly, specific central stimulation, either singly of dopamine receptor 1 or dopamine receptors 2 and 3 simultaneously, causes a significant decrease in ghrelin-induced food intake. Co-stimulation of all three receptors (D1, D2, and D3) also led to a marked attenuation in ghrelin-induced food intake. Importantly, the reduction in ghrelin-induced feeding was not caused by malaise or any type of behavioral alteration.

Conclusion

Taken together, these data indicate that dopamine receptors play an important role in acute stimulation of feeding behavior induced by central injection of ghrelin.     

Ghrelin and ghrelin receptor inhibitors: agents in the treatment of obesity

Background: Current medical treatment of obesity is highly ineffective. Soon after its discovery as the endogenous ligand for the growth hormone secretagogue-receptor (GHS-R), ghrelin was shown to stimulate food intake (including in humans) and promote body weight gain and adipogenesis. Objectives: This review discusses the role of the ghrelin/GHS-R pathway in energy homeostasis regulation and its role as a novel molecular target for the treatment of obesity. Methods: Medline was searched for relevant articles published in English. Results/conclusion: A large series of animal studies shows that inhibition of the ghrelin/GHS-R pathway reduces food intake, body weight and adiposity, through reduction of appetite and augmentation of energy expenditure and fat catabolism. This suggests that inhibition of this novel pathway may be used to treat/prevent obesity and its complications.     

Cardiovascular effects of ghrelin and growth hormone secretagogues

Apart from promoting GH secretion and to regulate appetite and metabolism, it has become increasingly clear that GH secretagogues (GHS) and ghrelin exert a number of effects on the cardiovascular system. The main cardiovascular actions of GHS are possible inotropic effects, vasodilation, reported cardioprotective effects against ischemia, and in vitro effects on cardiomyocytes involving cell proliferation and anti-apoptotic actions. An interesting and intriguing feature of the cardiovascular effects of GHS is that they may be exerted directly on the heart and vasculature rather than being mediated by growth hormone. Evidence to suggest this is the finding of GHS binding sites on cardiomyocytes and the fact that some of the effects of GHS can be expressed also in the absence of GH. Taken together, these results offer an interesting perspective on the future where further studies aiming at evaluating a role of GHS and ghrelin in the treatment of cardiovascular disease are warranted.