Ghrelin, isolated from the human and rat stomach, is the endogenous ligand for the growth hormone (GH) secretagogue receptor, which is expressed in a variety of tissues, including the pancreatic islets. It has been shown that low plasma ghrelin levels correlates with elevated fasting insulin levels and type 2 diabetes. Here we show a physiological role of endogenous ghrelin in the regulation of insulin release and blood glucose in rodents. Acylated ghrelin, the active form of the peptide, was detected in the pancreatic islets. Counteraction of endogenous ghrelin by intraperitoneal injection of specific GH secretagogue receptor antagonists markedly lowered fasting glucose concentrations, attenuated plasma glucose elevation, and enhanced insulin responses during the glucose tolerance test (GTT). Conversely, intraperitoneal exogenous ghrelin GH-independently elevated fasting glucose concentrations, enhanced plasma glucose elevation, and attenuated insulin responses during GTT. Neither GH secretagogue receptor antagonist nor ghrelin affected the profiles of the insulin tolerance test. In isolated islets, GH secretagogue receptor blockade and antiserum against acylated ghrelin markedly enhanced glucose-induced increases in insulin release and intracellular Ca G hrelin, a novel acylated 28-amino acid peptide, was isolated from the human and rat stomach as the endogenous ligand (1) for the growth hormone (GH) secretagogue receptor (2). Circulating ghrelin is produced predominantly in the stomach (3). Ghrelin is a potent stimulator of GH release (1) and feeding (4). In addition, the plasma ghrelin level correlates inversely with obesity (5-7). A recent report of a cohort study has shown that low plasma ghrelin is associated with elevated fasting insulin levels, insulin resistance, and type 2 diabetes (8). These findings suggest that ghrelin could be involved in energy and glucose metabolism, in which insulin plays a crucial role. It has been well documented that systemic administration of ghrelin elevates blood glucose (9 -11). However, the physiological role of endogenous ghrelin in the regulation of blood glucose remains to be established. This question was addressed in the present study by examining the effects of ghrelin receptor blockade on blood glucose and insulin levels in rodents.It has been reported that ghrelin (12-16) and its mRNA (1,12,13,17), as well as GH secretagogue receptor mRNAs (1,12,13,17,18), are expressed in the pancreas and islet cells. The present study aimed to explore a role of endogenous ghrelin in islets in the regulation of insulin release. This was achieved by counteraction of the islet ghrelin with specific GH secretagogue receptor antagonists and antiserum against acylated ghrelin, the active form of the peptide (active ghrelin). Furthermore, although ghrelin reportedly influences insulin release in several different systems (12,19 -21), the signaling mechanisms of ghrelin in islet ␤-cells are unknown. We studied ghrelin's action mechanisms with special attention to the Ca 2ϩ signaling i...

Ghrelin, a novel growth hormone-releasing peptide isolated from human and rat stomach, is a 28 -amino acid peptide with a posttranslational acylation modification that is indispensable for stimulating growth hormone secretion by increasing intracellular Ca 2؉ concentration. It also functions in the regulation of feeding behavior, energy metabolism, and gastric acid secretion and motility. Using two different antibodies against the NH 2 -and COOH-terminal regions of ghrelin, we studied its localization in human and rat pancreas by immunohistochemistry. Ghrelin-immunoreactive cells were identified at the periphery of pancreatic islets in both species. Ghrelin co-localized exclusively with glucagon in rat islets, indicating that it is produced in ␣-cells. We identified ghrelin and des-acyl ghrelin in the rat pancreas using reverse-phase high-performance liquid chromatography combined with two radioimmunoassays. We also detected mRNA encoding ghrelin and its receptor in the rat pancreatic islets. Ghrelin increased the cytosolic free Ca 2؉ concentration in ␤-cells and stimulated insulin secretion when it was added to isolated rat pancreatic islets. These findings indicate that ghrelin may regulate islet function in an endocrine and/or paracrine manner. Diabetes 51:124 -129, 2002 G rowth-hormone secretagogues (GHSs) are small synthetic peptides and nonpeptide molecules that stimulate growth hormone (GH) release from the anterior pituitary through the GHS receptor (GHS-R) (1). GHS-R, a G protein-coupled receptor, promotes calcium release from the endoplasmic reticulum (2,3). Ghrelin, a 28 -amino acid peptide with an n-octanoylated Ser 3, was originally discovered in rat stomach as a cognate endogenous ligand for GHS-R by using an intracellular calcium influx assay on a stable cell line expressing rat GHS-R (4). The Ser 3 n-octanoylation is a unique modification necessary for ghrelin's activity. Ghrelin stimulates GH release when administered intravenously or intracerebroventricularly to rats and when applied directly to rat primary pituitary cells (4 -6). In addition, ghrelin increases food intake and body weight upon intracerebroventricular administration (7,8). Furthermore, intravenously or intracerebroventricularly administered ghrelin stimulates gastric acid secretion by activating the vagal system (9,10). These findings suggest that ghrelin is secreted in response to altered food intake or some other nutritional states and thereby plays a role in the regulation of feeding behavior, energy metabolism, and digestion. Ghrelin is produced primarily in the enteroendocrine cells of rats and humans (4,11,12). Many types of enteroendocrine cells, including insulin-and glucagonproducing cells of the pancreatic islets, develop from endodermal epithelium. Ghrelin, like insulin and glucagon, may be produced in the islets and involved in the regulation of energy metabolism.In the present study, we investigated the cellular source of ghrelin in rat and human pancreas by immunohistochemistry. Ghrelin molecules in rat pancreas we...

The hypothalamic paraventricular nucleus (PVN) functions as a center to integrate various neuronal activities for regulating feeding behavior. Nesfatin-1, a recently discovered anorectic molecule, is localized in the PVN. However, the anorectic neural pathway of nesfatin-1 remains unknown. Here we show that central injection of nesfatin-1 activates the PVN and brain stem nucleus tractus solitarius (NTS). In the PVN, nesfatin-1 targets both magnocellular and parvocellular oxytocin neurons and nesfatin-1 neurons themselves and stimulates oxytocin release. Immunoelectron micrographs reveal nesfatin-1 specifically in the secretory vesicles of PVN neurons, and immunoneutralization against endogenous nesfatin-1 suppresses oxytocin release in the PVN, suggesting paracrine/autocrine actions of nesfatin-1. Nesfatin-1-induced anorexia is abolished by an oxytocin receptor antagonist. Moreover, oxytocin terminals are closely associated with and oxytocin activates pro-opiomelanocortin neurons in the NTS. Oxytocin induces melanocortin-dependent anorexia in leptin-resistant Zucker-fatty rats. The present results reveal the nesfatin-1-operative oxytocinergic signaling in the PVN that triggers leptin-independent melanocortin-mediated anorexia.

Recent studies suggest that oxytocin (Oxt) is implicated in energy metabolism. We aimed to explore acute and sub-chronic effects of peripheral Oxt treatment via different routes on food intake and energy balance. Intraperitoneal (ip) injection of Oxt concentration-dependently decreased food intake in mice. Ip Oxt injection induced c-Fos expression in the hypothalamus and brain stem including arcuate nucleus (ARC), paraventricular nucleus (PVN) and nucleus tractus solitarius (NTS). Subcutaneous (sc) injection of Oxt suppressed food intake in normal and high fat diet-induced obese (DIO) mice. Daily sc injection of Oxt for 17 days in DIO mice reduced food intake for 6 days and body weight for the entire treatment period and additional 9 days after terminating Oxt. Oxt infusion by sc implanted osmotic minipumps for 13 days in DIO mice reduced food intake, body weight, and visceral fat mass and adipocyte size. Oxt infusion also decreased respiratory quotient specifically in light phase, ameliorated fatty liver and glucose intolerance, without affecting normal blood pressure in DIO mice. These results demonstrate that peripheral Oxt treatment reduces food intake and visceral fat mass, and ameliorates obesity, fatty liver and glucose intolerance. Peripheral Oxt treatment provides a new therapeutic avenue for treating obesity and hyperphagia.

The gastric hormone ghrelin and its receptor, growth hormone secretagogue receptor (GHSR), are expressed in pancreas. Here, we report that ghrelin is released from pancreatic islets to regulate glucose-induced insulin release. Plasma concentrations of ghrelin, as well as insulin, were higher in pancreatic veins than in arteries. GHSR antagonist and immunoneutralization of endogenous ghrelin enhanced glucose-induced insulin release from perfused pancreas, whereas exogenous ghrelin suppressed it. GHSR antagonist increased plasma insulin levels in gastrectomized and normal rats to a similar extent. Ghrelin knockout mice displayed enhanced glucose-induced insulin release from isolated islets, whereas islet density, size, insulin content, and insulin mRNA levels were unaltered. Glucose tolerance tests (GTTs) in ghrelin knockout mice showed increased insulin and decreased glucose responses. Treatment with high-fat diet produced glucose intolerance in GTTs in wild-type mice. In ghrelin knockout mice, the high-fat diet-induced glucose intolerance was largely prevented, whereas insulin responses to GTTs were markedly enhanced. These findings demonstrate that ghrelin originating from pancreatic islets is a physiological regulator of glucose-induced insulin release. Antagonism of the ghrelin function can enhance insulin release to meet increased demand for insulin in high-fat diet-induced obesity and thereby normalize glycemic control, which may provide a potential therapeutic application to counteract the progression of type 2 diabetes. Diabetes 55: 3486 -3493, 2006

Nesfatin-1 is a novel satiety molecule in the hypothalamus and is also present in peripheral tissues. Here we sought to identify the active segment of nesfatin-1 and to determine the mechanisms of its action after peripheral administration in mice. Intraperitoneal injection of nesfatin-1 suppressed food intake in a dose-dependent manner. Nesfatin-1 has three distinct segments; we tested the effect of each segment on food intake. Injection of the midsegment decreased food intake under leptin-resistant conditions such as db/db mice and mice fed a high-fat diet. After injection of the midsegment, expression of c-Fos was significantly activated in the brainstem nucleus tractus solitarius (NTS) but not in the hypothalamic arcuate nucleus; the nicotinic cholinergic pathway to the NTS contributed to midsegment-induced anorexia. Midsegment injection significantly increased expression of proopiomelanocortin and cocaine- and amphetamine-regulated transcript genes in the NTS but not in the arcuate nucleus. Investigation of mutant midsegments demonstrated that a region with amino acid sequence similarity to the active site of agouti-related peptide was indispensable for anorexigenic induction. Our findings indicate that the midsegment of nesfatin-1 causes anorexia, possibly by activating POMC and CART neurons in the NTS via a leptin-independent mechanism after peripheral stimulation.

Ghrelin is a newly discovered peptide that is released from the stomach and from neurons in the hypothalamic arcuate nucleus (ARC) and potently stimulates growth hormone release and food intake. Neuropeptide-Y (NPY) neurons in the ARC play an important role in the stimulation of food intake. The present study aimed to determine whether ghrelin directly activates NPY neurons and, if so, to explore its signaling mechanisms. Whether the neurons that respond to ghrelin could be regulated by orexin and leptin was also examined. G hrelin, an endogenous ligand for the growth hormone secretagogue (GHS) receptor (GHSR), is synthesized abundantly in the stomach and to a much lesser extent in the hypothalamic arcuate nucleus (ARC) (1). Peripheral or intracerebroventricular (ICV) injection of ghrelin releases growth hormone, stimulates food intake, and increases body weight in mice, rats, and humans (1-8). ICV injection of antighrelin IgG suppresses starvation-induced feeding (3). The daily pattern of plasma ghrelin levels in normal humans is characterized by a preprandial rise and postprandial fall (9). These findings have suggested that ghrelin plays a physiological role in the meal initiation.The neuropeptide-Y (NPY)-containing neurons localized in the ARC have been implicated in the stimulation of food intake-injection of NPY into the hypothalamus of rats potently stimulates food intake (10), and NPY secretion in the hypothalamus is increased during fasting (11). Regarding a possible link between ghrelin and the NPY neurons in the ARC, it has been shown that GHSR mRNA is expressed in 94% of the NPY neurons in the ARC by double-labeling in situ hybridization histochemistry (12). Systemic or ICV administration of ghrelin causes the ARC neurons to express Fos and Egr-1 (3,13-15) and ϳ90% of these Fos-positive neurons express NPY mRNA (13). Moreover, ghrelin increases the expression of NPY mRNA (3-5), and ICV administration of a NPY Y1 antagonist suppresses the ghrelin-stimulated food intake (3-5,15). These findings suggest that the NPY neurons in the ARC could be an important effector for the orexigenic action of ghrelin.

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