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Specific interactions of human melanocortin-4 receptor (hMC4R) with its non-peptide and peptide agonists were studied using alanine-scanning mutagenesis. The binding affinities and potencies of two synthetic small-molecule agonists (THIQ, MB243) were strongly affected by substitutions in transmembrane α-helices (TM) 2, 3, 6, and 7 (residues Glu 100 Asp 122 , Asp 126 , Phe 261 , His 264 , Leu 265 , and Leu 288 ). In addition, I129A mutation primarily affected binding and potency of THIQ, while F262A, W258A, Y268A mutations impaired interactions with MB243. By contrast, binding affinity and potency of the linear peptide agonist NDP-MSH were substantially reduced only in D126A and H264A mutants. 3D models of receptor-ligand complexes with their agonists were generated by distance geometry using the experimental, homology-based, and other structural constraints, including interhelical H-bonds and two disulfide bridges (Cys 40 -Cys 279 , Cys 271 -Cys 277 ) of hMC4R. In the models, all pharmacophore elements of small-molecule agonists are spatially overlapped with the corresponding key residues (His 6 , D-Phe 7 , Arg 8 and Trp 9 ) of the linear peptide: their charged amine groups interact with acidic residues from TM2 and TM3, similar to His 6 and Arg 6 of NDP-MSH; their substituted piperidines mimic Trp 9 of the peptide and interact with TM5 and TM6; while the D-Phe aromatic rings of all three agonists contact with Leu 133 , Trp 258 , and Phe 261 residues.Melanotropins, which include melanocyte-stimulating hormones (α-, β-, and γ-MSH) and adrenocorticotropic hormone (ACTH), are the products of proteolytic cleavage of the 31-36 kDa precursor, pro-opiomelanocortin (1). α-MSH (Ac-Ser 1 -Tyr 2 -Ser 3 -Met 4 -Glu 5 -His 6 -Phe 7 -Arg 8 -Trp 9 -Gly 10 -Lys 11 -Pro 12 -Val 13 -NH 2 ) shares with all melanotropins the central core tetrapeptide 'His 6 -Phe 7 -Arg 8 -Trp 9 ', which is essential for its biological activity (2). These neuropeptides exert their function through five subtypes of melanocortin receptors (MCRs), which have been cloned and characterized (1,3). MCRs belong to the G protein-coupled receptor (GPCR) superfamily (4) and are positively coupled to cAMP-generation by adenylate cyclase via the stimulatory Gs-proteins. They are involved in regulation of multiple physiological functions, such as pigmentation (MC1R), adrenal cortical steroidogenesis † This work was supported by NIH grants DK054032 (I. Table of receptor type-specific distance constraints for the distance geometry refinement of the model of the active conformation of MC4R. This material is available free of charge via the Internet at http://pubs.acs.org.G NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 September 8. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript (MC2R), exocrine secretion (MC5R), energy homeostasis, penile erection (MC3R and MC4R) and many others (1,5,6).The MC4R subtype is regarded as a potential drug target, because it is involved in feeding and sexual ...

Although ghrelin has been demonstrated to stimulate energy intake and storage through a central mechanism, its effect on hepatic lipid metabolism remains largely uncharacterized. Ghrelin receptor antagonism or gene deletion significantly decreased obesity-associated hepatic steatosis by suppression of de novo lipogenesis, whereas exogenous ghrelin stimulated lipogenesis, leading to hepatic lipid accumulation in mice. The effects of ghrelin were mediated by direct activation of its receptor on hepatocytes. Cultured hepatocytes responded to ghrelin with increased lipid content and expression of lipogenesis-related genes. Ghrelin increased phosphorylation of S6, the downstream target of mammalian target of rapamycin (mTOR) signaling in cultured hepatocytes, whereas ghrelin receptor antagonism reduced hepatic phosphorylation of S6 in db/db mice. Inhibition of mTOR signaling by rapamycin markedly attenuated ghrelin-induced up-regulation of lipogenesis in hepatocytes, whereas activation of hepatic mTOR signaling by deletion of TSC1 increased hepatic lipogenesis. By interacting with peroxisome proliferator-activated receptor-γ (PPARγ), mTOR mediates the ghrelin-induced up-regulation of lipogenesis in hepatocytes. The stimulatory effect of ghrelin on hepatic lipogenesis was significantly attenuated by PPARγ antagonism in cultured hepatocytes and in PPARγ gene-deficient mice. Our study indicates that ghrelin activates its receptor on hepatocytes to promote lipogenesis via a mechanism involving the mTOR-PPARγ signaling pathway.NAFLD | gastric hormone | growth hormone secretagogue receptor | GHSR T riglyceride deposition in the liver, which is strongly associated with obesity, is the initial event in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Over time, hepatic steatosis may progress to steatohepatitis, cirrhosis, and primary hepatocellular carcinoma (1). The current therapeutic strategy for NAFLD has been focused on reversal of hepatic steatosis, primarily through weight reduction. Treatment is often ineffective because of the difficulty in achieving sustained weight loss. Alternative approaches are needed but are limited by incomplete understanding of the mechanisms controlling the development of steatosis. Gastric hormones may be involved in regulation of lipid metabolism. Studies in both animals and humans demonstrate that ghrelin, a 28-aa peptide hormone secreted by X/A-like endocrine cells in the gastric fundus (2, 3), stimulates lipid accumulation in adipose tissue (4). Chronic infusion of ghrelin increases both adipose and hepatic lipid storage (5). Genetic disruption of either ghrelin or ghrelin receptor genes renders mice resistant to obesity and to the development of hepatic steatosis (6). Interestingly, the anabolic effect of ghrelin appears to be independent of its hyperphagic action. Chronic third intracerebroventricular infusion of ghrelin in diet-induced obese rats increases adiposity and gene expression of lipogenic enzymes in white adipose tissue while food intake remains unchanged (7)....

Ghrelin, a gastric peptide hormone, has been reported to regulate GH secretion and energy homeostasis. Here, we examined the effect of des-acyl ghrelin driven from the fatty acid-binding protein-4 (FABP4) promoter on adiposity and glucose metabolism. A high level of expression of des-acyl ghrelin (692 +/- 293 fmol/g fat) in adipose tissue was detected in FABP4-ghrelin transgenic mice, but not in wild-type littermates. Circulating des-acyl ghrelin was significantly higher in FABP4-ghrelin transgenic mice (8409 +/- 3390 pm) compared with wild-type mice (513 +/- 58 pm). No significant change was observed for plasma acylated ghrelin and obestatin. Epididymal and perirenal fat masses decreased 35 +/- 9 and 52 +/- 9%, respectively, in FABP4-ghrelin transgenic mice. FABP4-ghrelin transgenic mice are resistant to obesity induced by high-fat diet. Brown fat mass was not affected by overexpression of ghrelin in adipose tissue. Glucose tolerance tests showed glucose levels to be significantly lower in FABP4-ghrelin transgenic mice than in controls after glucose administration. Insulin sensitivity testing showed that FABP4-ghrelin transgenic mice had a 28 +/- 5% greater hypoglycemic response to insulin. Our study demonstrates that overexpression of ghrelin from the FABP4 promoter impairs the development of white adipose tissues, and alters glucose tolerance and insulin sensitivity in mice.