Pharmacological effect of human melanocortin-2 receptor accessory protein 2 variants on hypothalamic melanocortin receptors.

PURPOSE: Melanocortin-3 receptor (MC3R), melanocortin-4 receptor (MC4R), and a recently identified melanocortin-2 receptor accessory protein 2 (MRAP2), are highly expressed in hypothalamus and coordinately regulate energy homeostasis, but the single cellular transcriptome of melanocortin system remains unknown. Several infrequent MRAP2 variants are reported from severe obese human patients but the mechanisms on how they affect melanocortin signaling are unclear. METHODS: First, we performed in silico analysis of mouse hypothalamus RNA sequencing datasets at single-cell resolution from two independent studies. Next, we inspected the three-dimensional conformational alteration of three mutations on MRAP2 protein. Finally, the influence of MRAP2 variants on MC3R and MC4R signaling was analyzed in vitro. RESULTS: (1) We confirmed the actual co-expression of Mrap2 with Mc3r and Mc4r, and demonstrated more broad distribution of Mrap2-positive neuronal populations than Mc3r or Mc4r in mouse hypothalamus. (2) Compared with wild-type MRAP2, MRAP2N88Y, and MRAP2R125C showed impaired α-MSH-induced MC4R or MC3R stimulation. (3) MRAP2N88Yexhibited enhanced interaction with MC4R protein and its own. CONCLUSIONS: This is the first dedicated description of single-cell transcriptome signature of Mrap2, Mc3r, and Mc4r in the central nerve system and the first evidence describing the unique dimer formation, conformational change, and pharmacological effect of MRAP2 mutations on MC3R signaling.

Lateral hypothalamic melanocortin receptor signaling modulates binge-like ethanol drinking in C57BL/6J mice.

Binge ethanol drinking is a highly pervasive and destructive behavior yet the underlying neurobiological mechanisms remain poorly understood. Recent work suggests that overlapping neurobiological mechanisms modulate feeding disorders and excessive ethanol intake, and converging evidence indicates that the melanocortin (MC) system may be a promising candidate. The aims of the present work were to examine how repeated binge-like ethanol drinking, using the 'drinking in the dark' (DID) protocol, impacts key peptides within the MC system and if site-specific manipulation of MC receptor (MCR) signaling modulates binge-like ethanol drinking. Male C57BL/6J mice were exposed to one, three or six cycles of binge-like ethanol, sucrose or water drinking, after which brain tissue was processed via immunohistochemistry (IHC) for analysis of key MC peptides, including alpha-melanocyte stimulating hormone (α-MSH) and agouti-related protein (AgRP). Results indicated that α-MSH expression was selectively decreased, while AgRP expression was selectively increased, within specific hypothalamic subregions following repeated binge-like ethanol drinking. To further explore this relationship, we used site-directed drug delivery techniques to agonize or antagonize MCRs within the lateral hypothalamus (LH). We found that the nonselective MCR agonist melanotan-II (MTII) blunted, while the nonselective MCR antagonist AgRP augmented, binge-like ethanol consumption when delivered into the LH. As these effects were region-specific, the present results suggest that a more thorough understanding of the MC neurocircuitry within the hypothalamus will help provide novel insight into the mechanisms that modulate excessive binge-like ethanol intake and may help uncover new therapeutic targets aimed at treating alcohol abuse disorders.

Inhibiting neuropeptide Y Y1 receptor modulates melanocortin receptor- and NF-κB-mediated feeding behavior in phenylpropanolamine-treated rats.

Neuropeptide Y (NPY) and nuclear factor-kappa B (NF-κB) are involved in regulating anorexia elicited by phenylpropanolamine (PPA), a sympathomimetic drug. This study explored whether NPY Y1 receptor (Y1R) is involved in this process, and a potential role for the proopiomelanocortin system was identified. Rats were given PPA once a day for 4days. Changes in the hypothalamic expression of the NPY, Y1R, NF-κB, and melanocortin receptor 4 (MC4R) levels were assessed and compared. The results indicated that food intake and NPY expression decreased, with the largest reductions observed on Day 2 (approximately 50% and 45%, respectively), whereas NF-κB, MC4R, and Y1R increased, achieving maximums on Day 2 (160%, 200%, and 280%, respectively). To determine the role of Y1R, rats were pretreated with Y1R antisense or a Y1R antagonist via intracerebroventricular injection 1h before the daily PPA dose. Y1R knockdown and inhibition reduced PPA anorexia and partially restored the normal expression of NPY, MC4R, and NF-κB. The data suggest that hypothalamic Y1R participates in the appetite-suppression from PPA by regulating MC4R and NF-κB. The results of this study increase our understanding of the molecular mechanisms in PPA-induced anorexia.

Design, synthesis, and biological evaluation of a new class of small molecule peptide mimetics targeting the melanocortin receptors.

A new bicyclic template has been developed for the synthesis of peptide mimetics. Straightforward synthetic steps, starting from amino acids, allow the facile construction of a wide range of analogs. This system was designed to target the melanocortin receptors (MCRs), with functional group selection based on a known pharmacophore and guidance from molecular modeling to rationally identify positional and stereochemical isomers likely to be active. The functions of hMCRs are critical to myriad biological activities, including pigmentation, steroidogenesis, energy homeostasis, erectile activity, and inflammation. These G-protein-coupled receptors (GPCRs) are targets for drug discovery in a number of areas, including cancer, pain, and obesity therapeutics. All compounds from this series tested to date are antagonists which bind with high affinity. Importantly, many are highly selective for a particular MCR subtype, including some of the first completely hMC5R-selective antagonists reported.

Mode of leptin action in chicken hypothalamus.

While there have been many studies in various species examining the mode of central leptin action on food intake, there is however a paucity of data in birds. We have, therefore, addressed this issue in broiler chickens because this strain was selected for high growth rate, hence high food intake. Continuous infusion of recombinant chicken leptin (8 microg/kg/h) during 6 h at a constant rate of 3 ml/h resulted in a significant reduction (49-57%) of food intake in 3-week-old broiler chickens (P < 0.05). The effect of leptin within the central nervous system (CNS) was mediated via selective hypothalamic neuropeptides. Leptin significantly decreased the expression of its receptor (Ob-R), neuropeptide Y (NPY), orexin (ORX), and orexin receptor (ORXR) (P < 0.05), but not that of agouti-related protein (AgRP) (anabolic/orexigenic effectors) in chicken hypothalamus. However, the catabolic/anorexigenic neuropeptides namely proopiomelanocortin (POMC) and corticotropin-releasing hormone (CRH) mRNA levels remained unchanged after leptin treatment. Despite the absence of leptin effect on AgRP (the antagonist of melanocortin receptor MCR) and POMC (the precursor of alpha-melanocyte stimulating hormone which is a potent agonist for MCR), leptin significantly decreased the expression of MCR-4/5 gene in chicken hypothalamus (P < 0.05) suggesting that leptin acts directly (as ligand) or indirectly (via other ligands) on MCRs to regulate food intake in birds. Additionally, leptin down-regulated the expression of fatty acid synthase (FAS) gene in chicken hypothalamus, indicating an additional pathway of leptin action on food intake such as described for FAS inhibitors. These findings provide new insight into the mechanism of leptin control of food intake in chickens.

Feeding responses to a melanocortin agonist and antagonist in obesity induced by a palatable high-fat diet.

Hypothalamic melanocortins are critical for the control of food intake, and alterations in POMC mRNA have been described in genetic models of obesity. However, the time course of changes in brain transmitters over the development of dietary obesity is less clear. Therefore, we examined the effect of diet-induced obesity on hypothalamic alpha-MSH content and feeding responsiveness to synthetic melanocortins. Male Sprague-Dawley rats fed a high-fat cafeteria diet (30% fat) or chow (5% fat) for 4 or 12 weeks were implanted with intracerebroventricular cannulae and feeding responses to the MC3/4R agonist MTII (0.5 nmol) and the selective MC4R antagonist HS014 (0.8 nmol) were determined. MTII had a long-lasting inhibitory effect on food intake. Chronically overfed animals had a significantly exaggerated inhibitory feeding response 15 and 24 h after MTII injection and lost more body weight (15 +/- 3 g) compared to control rats (4 +/- 4 g; P < 0.05). Daytime administration of HS014 significantly increased food intake in all rats to the same extent (P < 0.05). No change in hypothalamic alpha-MSH content was observed after 2 or 12 weeks of high-fat diet. The observation of increased responsiveness to the melanocortin agonist, in the face of a high-fat diet, suggests melanocortin analogues may have potential for the pharmacological treatment of obesity.