OBJECTIVE-Obestatin is a newly discovered peptide encoded by the ghrelin gene whose biological functions are poorly understood. We investigated obestatin effect on survival of ␤-cells and human pancreatic islets and the underlying signaling pathways. RESEARCH DESIGN AND METHODS-␤-Cells and humanislets were used to assess obestatin effect on cell proliferation, survival, apoptosis, intracellular signaling, and gene expression. RESULTS-Obestatinshowed specific binding on HIT-T15 and INS-1E ␤-cells, bound to glucagon-like peptide-1 receptor (GLP-1R), and recognized ghrelin binding sites. Obestatin exerted proliferative, survival, and antiapoptotic effects under serumdeprived conditions and interferon-␥/tumor necrosis factor-␣/ interleukin-1␤ treatment, particularly at pharmacological concentrations. Ghrelin receptor antagonist [D-Lys 3 ]-growth hormone releasing peptide-6 and anti-ghrelin antibody prevented obestatin-induced survival in ␤-cells and human islets. ␤-Cells and islet cells released obestatin, and addition of anti-obestatin antibody reduced their viability. Obestatin increased ␤-cell cAMP and activated extracellular signal-related kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI 3-kinase)/Akt; its antiapoptotic effect was blocked by inhibition of adenylyl cyclase/cAMP/ protein kinase A (PKA), PI 3-kinase/Akt, and ERK1/2 signaling. Moreover, obestatin upregulated GLP-1R mRNA and insulin receptor substrate-2 (IRS-2) expression and phosphorylation. The GLP-1R antagonist exendin-(9-39) reduced obestatin effect on ␤-cell survival. In human islets, obestatin, whose immunoreactivity colocalized with that of ghrelin, promoted cell survival and blocked cytokine-induced apoptosis through cAMP increase and involvement of adenylyl cyclase/cAMP/PKA signaling. Moreover, obestatin 1) induced PI 3-kinase/Akt, ERK1/2, and also cAMP response element-binding protein phosphorylation; 2) stimulated insulin secretion and gene expression; and 3) upregulated GLP-1R, IRS-2, pancreatic and duodenal homeobox-1, and glucokinase mRNA. O bestatin is a 23-amino acid amidated peptide, recently identified as a product of the ghrelin gene (1). It was originally reported to be the ligand for the orphan receptor G-protein-coupled receptor 39 (GPR39); however, several groups were unable to confirm that obestatin has agonist properties on GPR39 or activates specific GPR39 signaling (2-6). Therefore, to date, the receptor for obestatin remains unknown. CONCLUSIONS-TheseObestatin has been reported to reduce food intake, body weight gain, gastric emptying, and jejunal motility (1,7,8). Moreover, it was found to counteract ghrelin stimulatory effects on these end points (1,9) and to inhibit ghrelininduced growth hormone secretion in vivo (9) but not in vitro (10), suggesting that it would serve as a physiological opponent of ghrelin. However, a number of studies failed to confirm obestatin anorexigenic effects (11-14), and besides not being the cognate ligand for GPR39, its biological actions seem to be a controversial issue.Obestatin ...

The metabolic actions of the ghrelin gene-derived peptide obestatin are still unclear. We investigated obestatin effects in vitro, on adipocyte function, and in vivo, on insulin resistance and inflammation in mice fed a high-fat diet (HFD). Obestatin effects on apoptosis, differentiation, lipolysis, and glucose uptake were determined in vitro in mouse 3T3-L1 and in human subcutaneous (hSC) and omental (hOM) adipocytes. In vivo, the influence of obestatin on glucose metabolism was assessed in mice fed an HFD for 8 wk. 3T3-L1, hSC, and hOM preadipocytes and adipocytes secreted obestatin and showed specific binding for the hormone. Obestatin prevented apoptosis in 3T3-L1 preadipocytes by increasing phosphoinositide 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK)1/2 signaling. In both mice and human adipocytes, obestatin inhibited isoproterenol-induced lipolysis, promoted AMP-activated protein kinase phosphorylation, induced adiponectin, and reduced leptin secretion. Obestatin also enhanced glucose uptake in either the absence or presence of insulin, promoted GLUT4 translocation, and increased Akt phosphorylation and sirtuin 1 (SIRT1) protein expression. Inhibition of SIRT1 by small interfering RNA reduced obestatin-induced glucose uptake. In HFD-fed mice, obestatin reduced insulin resistance, increased insulin secretion from pancreatic islets, and reduced adipocyte apoptosis and inflammation in metabolic tissues. These results provide evidence of a novel role for obestatin in adipocyte function and glucose metabolism and suggest potential therapeutic perspectives in insulin resistance and metabolic dysfunctions.

RFamide peptides 43RFa and 26RFa have been shown to promote food intake and to exert different peripheral actions through G-protein-coupled receptor 103 (GPR103) binding. Moreover, 26RFa was found to inhibit pancreatic insulin secretion, whereas the role of 43RFa on b-cell function is unknown, as well as the effects of both peptides on b-cell survival. Herein, we investigated the effects of 43RFa and 26RFa on survival and apoptosis of pancreatic b-cells and human pancreatic islets. In addition, we explored the role of these peptides on insulin secretion and the underlying signaling mechanisms. Our results show that in INS-1E b-cells and human pancreatic islets both 43RFa and 26RFa prevented cell death and apoptosis induced by serum starvation, cytokine synergism, and glucolipotoxicity, through phosphatidylinositol 3-kinase/Akt-and extracellular signal-related kinase 1/2-mediated signaling. Moreover, 43RFa promoted, whereas 26RFa inhibited, glucose-and exendin-4-induced insulin secretion, through Ga s and Ga i/o proteins, respectively. Inhibition of GPR103 expression by small interfering RNA blocked 43RFa insulinotropic effect, but not the insulinostatic action of 26RFa. Finally, 43RFa, but not 26RFa, induced cAMP increase and glucose uptake. In conclusion, because of their survival effects along with the effects on insulin secretion, these findings suggest potential for 43RFa and 26RFa as therapeutic targets in the treatment of diabetes.Pancreatic b-cell mass plays an essential role in glucose homeostasis. The reduced capacity of the endocrine pancreas to maintain an adequate insulin secretion, due to decreased b-cell mass and function, underlies both type 1 and type 2 diabetes (1). In type 1 diabetes, immunemediated release of inflammatory cytokines such as tumor necrosis factor-a (TNF-a), interferon-g (IFN-g), and interleukin-1b (IL-1b) has been implicated in b-cell apoptosis (2). In type 2 diabetes, b-cell apoptosis results from the combined action of increased plasma glucose/free fatty acid levels (glucolipotoxicity) (3) and cytokines (4). Therefore, identifying molecules capable of increasing pancreatic b-cell survival may be crucial for the treatment and prevention of diabetes.RFamide-related peptides constitute a family of biologically active peptides terminating in arginine-phenylalanine-amide (Arg-Phe-NH 2 ) at their C-terminus. They include a 26-amino acid RFamide peptide (26RFa), which was isolated

The activation of G protein-coupled receptor 103 (GPR103) by its endogenous peptidic ligands, QRFPs, is involved in the central regulation of feeding by increasing food intake, body weight, and fat mass after intracerebroventricular injection in mice. However, the role of GPR103 in regulating peripheral metabolic pathways has not yet been explored. The present study aimed to investigate the role of GPR103 in adipogenesis and lipid metabolism using 3T3-L1 adipocyte cells. Our results show that differentiated 3T3-L1 cells expressed the GPR103b subtype mRNA and protein, as well as QRFP mRNA. QRFP-43 and -26 induced an increase in triglyceride accumulation of 50 and 41%, respectively, and elicited a dose-dependent increase in fatty acid uptake, by up to approximately 60% at the highest concentration, in 3T3-L1-differentiated cells. QRFP-43 and -26 inhibited isoproterenol (ISO)-induced lipolysis in a dose-dependent manner, with IC(50)s of 2.3 +/- 1.2 and 1.1 +/- 1.0 nm, respectively. The expression of genes involved in lipid uptake (FATP1, CD36, LPL, ACSL1, PPAR-gamma, and C/EBP-alpha), was increased by 2- to 3-fold after treatment with QRFP. The effects of QRFP on ISO-induced lipolysis and fatty acid uptake were abolished when GPR103b was silenced. In a mouse model of diet-induced obesity, the expression of GPR103b in epididymal fat pads was elevated by 16-fold whereas that of QRFP was reduced by 46% compared to lean mice. Furthermore, QRFP was bioactive in omental adipocytes from obese individuals, inhibiting ISO-induced lipolysis in these cells. Our results suggest that GPR103b and QRFP work in an autocrine/paracrine manner to regulate adipogenesis.

Des-acyl ghrelin, although devoid of binding to ghrelin receptor (GRLN), exerts many biological effects, including regulation of glucose and lipid metabolism. Indeed, des-acyl ghrelin promotes pancreatic β-cell and human islet cell survival and prevents diabetes in streptozotocin (STZ) treated rats. We investigated whether des-acyl ghrelin fragments excluding serine 3 , which is essential for binding to GRLN, would display similar actions. Among the different compounds tested, des-acyl ghrelin (6)(7)(8)(9)(10)(11)(12)(13) and des-acyl ghrelin (6)(7)(8)(9)(10)(11)(12)(13) with alanine substitutions or cyclization, but not with d-amino acid substitutions, showed the best survival effect, similar to des-acyl ghrelin. Des-acyl ghrelin (6)(7)(8)(9)(10)(11)(12)(13) even prevented diabetes in STZ-treated rats and protected human circulating angiogenic cells from oxidative stress and senescence, similar to des-acyl ghrelin. These results suggest that not only fulllength des-acyl ghrelin but also short des-acyl ghrelin fragments have clear beneficial effects on several tissues in vitro and in vivo. Ghrelin is a 28 amino acid peptide mainly produced by the stomach but also in other tissues such as the gastrointestinal tract and the pancreas.(1, 2) Ghrelin potently stimulates growth hormone (GH) release from the pituitary and exerts orexigenic activities at the central level. (3) These neuroendocrine actions require acylation on the third serine residue by ghrelin O-acyl transferase (GOAT), and ghrelin acylaton is essential for binding to its receptor, the GH secretagogue receptor type 1a (GHS-R1a), lately designated GRLN.(1, 4-8) Besides the hypothalamus-pituitary and other central areas, GRLN is distributed in peripheral tissues, including the endocrine pancreas and adipose tissue.(2, 3) Consistently, ghrelin elicits many peripheral actions, including regulation of pancreatic β-cell function and influence on glucose and lipid metabolism.(9-12) Mice deleted for both ghrelin and GRLN genes show improved glucose tolerance and insulin secretion and sensitivity under high-fat diet treatment. In obese, leptindeficient (ob/ob) mice, ablation of ghrelin was found to increase insulin release and to reduce hyperglycemia, suggesting negative effects of ghrelin on insulin secretion and glucose metabolism.(13, 14) Notably, ghrelin infusion in humans induces acute insulin resistance and lipolysis, and ghrelin levels are strongly increased in insulin-resistant obese individuals, suggesting that ghrelin may contribute to insulin resistance in obesity.(15, 16) At variance with ghrelin, des-acyl ghrelin is devoid of endocrine activities and GRLN binding. However, des-acyl ghrelin is the most abundant circulating form of ghrelin, exerting a variety of effects, including positive actions on glucose and lipid metabolism.(3, 9, 17) Indeed, des-acyl ghrelin has been shown to modulate the expression of metabolic genes in GRLN-deleted mice tissues, to inhibit lipolysis in adipocytes, and to counteract the ghrelin diabetogenic actions in ...