Melanocortin-4 Receptor Knockout Mice Research Articles

Complementary role of peripheral and central autonomic nervous system on insulin-like growth factor-1 activation to prevent fatty liver disease.

Insulin-like growth factor-1 (IGF-1) is involved in the pathology of non-alcoholic fatty liver disease (NAFLD) and ameliorates fatty infiltration in the liver. It is activated by growth hormone (GH); however, the role of GH-IGF-1 axis in NAFLD developmental phase has not been well identified. Therefore, in this study, we focused on the effect of IGF-1 in NAFLD pathology and GH excretion activation from the pituitary gland by peripheral autonomic neural pathways relaying liver-brain-gut pathway and by central neuropeptides. GH and IGF-1 levels were assessed in wild-type and melanocortin-4 receptor knockout mice upon the development of diet-induced NAFLD. The contribution of the peripheral autonomic nervous system connecting the liver-brain-gut axis was assessed by its blockade using capsaicin and that of the central nervous system was assessed by the expression of hypothalamic brain-derived neurotrophic factor (BDNF) and corticotropin-releasing factor (CRH), which activates GH release from the pituitary gland. In the NAFLD mouse models, the levels of GH and IGF-1 increased (p < .05). Further, hepatic fatty infiltration was suppressed even under peripheral autonomic nervous system blockade (p < .001), which inhibited gastric ghrelin expression. In mice with peripheral autonomic nervous blockade, hypothalamic BDNF and CRH were inhibited (p < .05), resulting in GH and IGF-1 excretion, whereas other neuropeptides of somatostatin and cortistatin showed no changes. These complementary effects were canceled in melanocortin-4 receptor knockout mice, which diminished BDNF and CRH release control. Our study demonstrates that the release of IGF-1 by the nervous system is a key factor in maintaining the pathological homeostasis of NAFLD, suggesting its therapeutic potential.

Involvement of the liver-gut peripheral neural axis in nonalcoholic fatty liver disease pathologies via hepatic HTR2A.

ABSTRACTSerotonin (5-HT) is one of the key bioamines of nonalcoholic fatty liver disease (NAFLD). Its mechanism of action in autonomic neural signal pathways remains unexplained; hence, we evaluated the involvement of 5-HT and related signaling pathways via autonomic nerves in NAFLD. Diet-induced NAFLD animal models were developed using wild-type and melanocortin 4 receptor (MC4R) knockout (MC4RKO) mice, and the effects of the autonomic neural axis on NAFLD physiology, 5-HT and its receptors (HTRs), and lipid metabolism-related genes were assessed by applying hepatic nerve blockade. Hepatic neural blockade retarded the progression of NAFLD by reducing 5-HT in the small intestine, hepatic HTR2A and hepatic lipogenic gene expression, and treatment with an HTR2A antagonist reproduced these effects. The effects were milder in MC4RKO mice, and brain 5-HT and HTR2C expression did not correlate with peripheral neural blockade. Our study demonstrates that the autonomic liver-gut neural axis is involved in the etiology of diet-induced NAFLD and that 5-HT and HTR2A are key factors, implying that the modulation of the axis and use of HTR2A antagonists are potentially novel therapeutic strategies for NAFLD treatment. This article has an associated First Person interview with the first author of the paper.

Cardiac Phenotype and Tissue Sodium Content in Adolescents With Defects in the Melanocortin System

Pro-opiomelanocortin (POMC) and the melanocortin-4 receptor (MC4R) play a pivotal role in the leptin-melanocortin pathway. Mutations in these genes lead to monogenic types of obesity due to severe hyperphagia. In addition to dietary-induced obesity, a cardiac phenotype without hypertrophy has been identified in MC4R knockout mice. We aimed to characterize cardiac morphology and function as well as tissue Na+ content in humans with mutations in POMC and MC4R genes. A cohort of 42 patients (5 patients with bi-allelic POMC mutations, 6 heterozygous MC4R mutation carriers, 19 obese controls without known monogenic cause, and 12 normal weight controls) underwent cardiac magnetic resonance (CMR) imaging and 23Na-MRI. Monogenic obese patients with POMC or MC4R mutation respectively had a significantly lower left ventricular mass/body surface area (BSA) than nonmonogenic obese patients. Left ventricular end-diastolic volume/BSA was significantly lower in POMC- and MC4R-deficient patients than in nonmonogenic obese patients. Subcutaneous fat and skin Na+ content was significantly higher in POMC- and MC4R-deficient patients than in nonmonogenic obese patients. In these compartments, the water content was significantly higher in patients with POMC and MC4R mutation than in control groups. Patients with POMC or MC4R mutations carriers had a lack of transition to hypertrophy, significantly lower cardiac muscle mass/BSA, and stored more Na+ within the subcutaneous fat tissue than nonmonogenic obese patients. The results point towards the role of the melanocortin pathway for cardiac function and tissue Na+ storage and the importance of including cardiologic assessments into the diagnostic work-up of these patients.

Blocking sphingosine 1-phosphate receptor 2 accelerates hepatocellular carcinoma progression in a mouse model of NASH

The role of sphingosine 1-phosphate (S1P) and its sphingosine-1-phosphate receptors (S1PRs) in non-alcoholic steatohepatitis (NASH) is unclear. We aimed to analyze the role of S1P/S1PRs in a Melanocortin-4 receptor (Mc4r)-deficient NASH murine model using FTY720, the functional antagonist of S1PR1, S1PR3, S1PR4, and S1PR5, and JTE-013, the antagonist of S1PR2. We observed that, compared to that in the control, the mRNA of S1pr1 tended to decrease, whereas those of S1pr2 and S1pr3 significantly increased in Mc4r-knockout (KO) mice subjected to a Western diet (WD). While the fat area did not differ, fibrosis progression differed significantly between control mice and mice in which liver S1PRs were blocked. Lipidomic and metabolomic analysis of liver tissues showed that JTE-013-administered mice showed elevation of S-adenosyl-l-methionine level, which can induce aberrant methylation due to reduction in glycine N-methyltransferase (GNMT) and elevation in diacylglycerol (DG) and triacylglycerol (TG) levels, leading to increased susceptibility to hepatocellular carcinoma (HCC). These phenotypes are similar to those of Gnmt-KO mice, suggesting that blocking the S1P/S1PR2 axis triggers aberrant methylation, which may increase DG and TG, and hepatocarcinogenesis. Our observations that the S1P/S1PR2 axis averts HCC occurrence may assist in HCC prevention in NASH.

Emerging roles of melanocortin receptor accessory proteins (MRAP and MRAP2) in physiology and pathophysiology.

Melanocortin-2 receptor accessory protein (MRAP) has an unusual dual topology and influences the expression, localisation, signalling and internalisation of the melanocortin receptor 2 (MC2); the adrenocorticotropic hormone (ACTH) receptor. Mutations in MRAP are associated with familial glucocorticoid deficiency type-2 and evidence is emerging of the importance of MRAP in adrenal development and ACTH signalling. Human MRAP has two functional splice variants: MRAP-α and MRAP-β, unlike MRAP-β, MRAP-α has little expression in brain but is highly expressed in ovary. MRAP2, identified through whole human genome sequence analysis, has approximately 40% sequence homology to MRAP. MRAP2 facilitates MC2 localisation to the cell surface but not ACTH signalling. MRAP and MRAP2 have been found to regulate the surface expression and signalling of all melanocortin receptors (MC1-5). Additionally, MRAP2 moderates the signalling of the G-protein coupled receptors (GCPRs): orexin, prokineticin and GHSR1a; the ghrelin receptor. Whilst MRAP appears to be mainly involved in glucocorticoid synthesis, an important role is emerging for MRAP2 in regulating appetite and energy homeostasis. Transgenic models indicate the importance of MRAP in adrenal gland formation. Like MC3R and MC4R knockout mice, MRAP2 knockout mice have an obese phenotype. In vitro studies indicate that MRAP2 enhances the MC3 and MC4 response to the agonist αMSH, which, like ACTH, is produced through precursor polypeptide proopiomelanocortin (POMC) cleavage. Analysis of cohorts of individuals with obesity have revealed several MRAP2 genetic variants with loss of function mutations which are causative of monogenic hyperphagic obesity with hyperglycaemia and hypertension. MRAP2 may also be associated with female infertility. This review summarises current knowledge of MRAP and MRAP2, their influence on GPCR signalling, and focusses on pathophysiology, particularly familial glucocorticoid deficiency type-2 and obesity.

Optimized High-Yield Purification of Obesity-Associated Melanocortin 4 Receptor.

Obesity has emerged as a global public health challenge associated with increased risk of hyperlipidemia and hypertension. It contributes to high sympathetic activity and increased catecholamine levels. The hypothalamic melanocortin system is known to regulate the energy homeostasis. The role of melanocortin 4 receptor (MC4R) has been demonstrated pharmacologically and in animal studies, which showed that severe obesity in MC4R knockout mice was caused by increased food intake and decreased energy consumption. Over 70 multiple different mis- -sense and nonsense mutations in hMC4R have been found at a high frequency of 2-8% in severe early onset or hereditary obesity. The single amino acid variation (D90N) located in the second transmembrane domain (TM2) of MC4R results in accelerated growth and childhood onset obesity. Interestingly, the functional characterization of D90N hMC4R mutant TM2 (m-hMC4R-TM2) revealed normal cell surface expression and binding with agonist similar to the hMC4R wild-type TM2 (wt-hMC4R-TM2) but loss of signal transduction mediated via Gs/adenylyl cyclase activation. It is essential to delineate the three-dimensional structure of MC4Rs in order to elucidate their functional aspects. In this study, we demonstrate the optimized expression and isolation of wt/m-hMC4R-TM2 proteins under different chemical cleavage reaction times and purification procedures via SDS precipitation. The solid-state NMR spectroscopy was carried out to study the structure of wt/m-hMC4R- TM2 protein in the anisotropic phospholipid bicelles. The KSI-wt/m-hMC4R-TM2 fusion proteins developed in cell culture with LB medium. In order to isolate the expressed fusion protein from the cell, ultrasonication, Ni-NTA affinity chromatography, dialysis, and lyophilization techniques were used. Then, to obtain a protein with higher purity and higher yield, the CNBr chemical cleavage time was subdivided into 30 minutes, 1 h, 2 h, 3 h, and 4 h. Purification process was performed using FPLC, and 100 mM KCl and dialysis were used to remove the SDS. CD spectrometer, MALDI-TOF, solution-state NMR, and solid-state NMR were used to confirmed purity and structure of the wt/m-hMC4R-TM2. The precipitation method was used to remove the SDS bound to proteins as KCl-SDS. We optimized the 2 h cleavage reaction times for both wt-hMC4R-TM2 and m-hMC4R-TM2 depending on the purity based on mass spectra and 1H-15N HSQC spectra and the yield after final purification. The 1D 1H-15N CP (Cross polarization) solid-state NMR spectra suggest that the wt/m-hMC4R- TM2 undergo rotational diffusion around a perpendicular axis along the bilayer normal. We expressed wt/m-hMC4R-TM2 in E.coli and optimized the isolation and purification process, especially CNBr chemical cleavage time. The efficiency of KCl-SDS precipitation was confirmed via MALDI-TOF MS and the pure proteins obtained using this method were characterized by CD spectroscopy and solution-state NMR. The results of 1H-15N HSQC spectra in solution- state NMR also show the probability for structural studies. The 1D 1H-15N CP solid-state NMR spectra indicate that most of the residues in both the wt/m-hMC4R-TM2 peptides are integrated into the membrane.

MON-639 Melanocortin 4 Receptor Contributes to Glucose Homeostasis by Regulating Kidney Glucose Reabsorption via the Glucose Transporter GLUT2

Melanocortin 4 receptor (MC4R) is essential for normal body weight and food intake. Deficiency of MC4R causes obesity in humans and mice. While the function of MC4R is well established in appetite regulation, its direct role in glucose homeostasis is unclear. Humans and mice with MC4R deficiency exhibit hyperinsulinemia and insulin resistance; however, they remain protected from fasting hyperglycemia/diabetes. To determine the role of MC4R in glucose homeostasis, we performed oral glucose and intra-peritoneal insulin tolerance tests (OGTT / ITT) in male and female Mc4r knockout (KO) and wild type (WT) mice. Remarkably, Mc4r KO mice exhibited improved glucose tolerance compared to WT mice (Area under the curve for OGTT, male: 29,125±2,028 vs. 38,493±1,161 mg/dL.min; female: 36,322±1,100 vs. 49,539±1,911 mg/dL.min, p<0.0001). The improvement in glucose tolerance was despite insulin resistance in Mc4r KO mice (Plasma insulin, male: 9.9±1.7 vs. 0.7±0.1 ng/mL, female: 6.2±2.0 vs. 1.1±0.3 ng/mL, p<0.05; Area under the curve for ITT, male: 13,174±1,073 vs. 8,132±255 mg/dL.min; female: 13,927±1,253 vs. 7,506±267 mg/dL.min, p<0.01). Based on our previous findings from POMC deficient mice, we hypothesized that the improved glucose tolerance in the Mc4r KO mice is due to their elevated glycosuria (excretion of glucose in urine). To test this hypothesis, we challenged Mc4r KO and WT mice with oral glucose (250 mg) and collected their 24h urine to evaluate glycosuria. Indeed, the KO mice demonstrated elevated glycosuria compared to their WT littermates (Urine glucose, male: 284±48 vs. 0.4±0.03 mg/24h, female: 63.4±14 vs. 1±0.6 mg/24h, p<0.002). To assess molecular mechanisms underlying elevated glycosuria in Mc4r KO mice, we measured the gene expression and levels of the kidney glucose transporters GLUT1, GLUT2, SGLT1 and SGLT2. Glut2 mRNA was reduced by ~ 40% and the protein level was decreased by ~ 20% in Mc4r KO mice compared to their WT littermates. The other glucose transporters remained unchanged. Altogether, our study demonstrates that MC4R contributes to glucose homeostasis by regulating kidney glucose reabsorption via GLUT2. These findings may explain why MC4R deficient mice or humans remain protected from diabetes despite their longstanding obesity and insulin resistance.

SUN-672 SGLT2 Inhibitor Reduces Hyperinsulinemia and Restores Pulsatile Growth Hormone Secretion in Obese MC4RKO Mice

Insulin and growth hormone (GH) are crucial counter-regulatory hormones in regulating glucose and lipid metabolisms. Insulin promotes fat storage, while GH promotes lipolysis and fat oxidation. In obese individuals, reduced GH secretion (hyposomatotropism) and increased insulin secretion (hyperinsulinemia) co-exist. The imbalance of these two hormones exacerbates fat accumulation. Therapeutic approaches to correct such hormonal imbalance in obesity are limited. The sodium/glucose cotransporter 2 inhibitor (SGLT2i), which promotes urinary glucose excretion, is a novel drug for overt type 2 diabetes (T2D). However, little is known about its efficacy in obese individuals without T2D in the clinic, in particular with hormonal imbalance. By applying SGLT2i (dapagliflozin, 1 mg/kg/d for 10 weeks) to a hyperphagic obese melanocortin 4 receptor knockout (MC4RKO) mouse model, we observed a significant reduction of hyperinsulinemia (fasting: 1.36±0.19 vs. 4.93±1.04 ng/ml, p<0.01; fed: 9.50±3.37 vs. 31.11±5.85 ng/ml, p<0.05, n=8) and restored pulsatile GH secretion without changing secretion pattern (pulsatile GH: 185.3±18.37 vs. 56.28±13.22 ng/ml per 6h, p<0.001; GH mass per secretion pulse: 50.31±8.20 vs. 15.55±3.18 ng/ml, p<0.01; number of secretory pulse per 6h: 3.71±0.29 vs. 3.57±0.43, p=0.78, n=8) as early as 4 weeks after the initiation of the treatment. Lipolysis and lipid oxidation-related gene expression levels were increased by SGLT2i treatment, whereas lipogenesis and inflammation gene expression levels were reduced, leading to decreased whole-body fat mass. Following the treatment, glucose tolerance and insulin sensitivity were both improved. Although a null effect was observed in food intake and daily activity, the treatment significantly promoted lipid usage and shifted energy metabolism towards negative energy balance. In conclusion, 10-week SGLT2i treatment improved glucose and lipid metabolisms in the hyperphagic obese MC4RKO mice. Such improvement occurs alongside reduced hyperinsulinemia and restored pulsatile GH secretion. This work provides insights for the potential use of SGLT2i in obese individuals prior to overt T2D. The final version of this work is published (1).Acknowledgements: grant (NHMRC, University of Queensland) and scholarship (CSC and UQ International scholarship)Reference: (1) Huang, Zhengxiang, et al. “Dapagliflozin restores insulin and growth hormone secretion in obese mice.” Journal of Endocrinology 245.1 (2020): 1-12.Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

2009-P: Molecular Mechanisms of Obesity-Associated Vascular Vulnerability via Signals of Appetite Regulation

Background: Obesity is a risk factor of hypertension and abdominal aortic aneurysm (AAA). Melanocortin 4 receptor (MC4R) is activated by α-melanocyte-stimulating hormone (α-MSH) to mediate most of the anorectic effects of leptin. In humans, deficiency or mutation of MC4R is the most common monogenic form of obesity. However, it remains unknown whether MC4R signaling affects the development of obesity-associated hypertension, vascular vulnerability and aortic aneurysm. Methods and Results: MC4R-knockout (KO) mice fed a high-fat/high-sucrose diet (HF/HS) for 20 weeks displayed obesity associated with hyperleptinemia compared to HF/HS-fed wild type (WT) mice. MC4R-KO mice exhibited hypertension, increase of medial thickness and elastic lamina destruction, and elevation of inflammatory and fibrosis-related genes including TGF-β and osteopontin (OPN) in aortae. MC4R-KO mice showed higher incidence of angiotensin II (Ang II, 500 ng/kg/min)-induced AAA (73%) than WT mice (8%). MC4R-KO mice with ob/ob background exhibited lower incidence of Ang II-induced AAA (50%) than MC4R-KO mice, but higher than ob/ob mice (12.5%). Treatment of OPN-blocking antibody decreased Ang II-induced AAA incidence in HF/HS-fed MC4R-KO mice. In vascular smooth muscle cells (VSMCs), leptin (100 ng/ml) significantly upregulated OPN gene expression. α-MSH suppressed AngII-induced TGF-β gene expression in VSMCs, and LPS/IFN-γ-induced iNOS gene expression in peritoneal macrophages. However, these effects of α-MSH were abolished in primary macrophages from MC4R-KO mice and RAW264.7 cells pretreated with MC4R antagonist HS024. Conclusion: Our observation indicates that deficiency of MC4R promotes the vascular vulnerability in mice. It suggests that, not only hyperleptinemia, but also impairment of α-MSH-MC4R signaling in VSMCs and macrophages, may also promote the vascular vulnerability. Disclosure K. Mori: None. K. Tsuchiya: None. S. Nakamura: None. K. Shiba: None. Y. Miyachi: None. Y. Ogawa: None. K. Kitamura: None.

Structure⁻Activity Relationships of the Tetrapeptide Ac-His-Arg-(pI)DPhe-Tic-NH2 at the Mouse Melanocortin Receptors: Modification at the (pI)DPhe Position Leads to mMC3R Versus mMC4R Selective Ligands.

The five melanocortin receptors (MC1R–MC5R) are involved in numerous biological pathways, including steroidogenesis, pigmentation, and food intake. In particular, MC3R and MC4R knockout mice suggest that the MC3R and MC4R regulate energy homeostasis in a non-redundant manner. While MC4R-selective agonists have been utilized as appetite modulating agents, the lack of MC3R-selective agonists has impeded progress in modulating this receptor in vivo. In this study, the (pI)DPhe position of the tetrapeptide Ac-His-Arg-(pI)DPhe-Tic-NH2 (an MC3R agonist/MC4R antagonist ligand) was investigated with a library of 12 compounds. The compounds in this library were found to have higher agonist efficacy and potency at the mouse (m) MC3R compared to the MC4R, indicating that the Arg-DPhe motif preferentially activates the mMC3R over the mMC4R. This observation may be used in the design of new MC3R-selective ligands, leading to novel probe and therapeutic lead compounds that will be useful for treating metabolic disorders.

Deficiency of Melanocortin 4 Receptor Promotes Vascular Vulnerability in Mice

Background: Obesity is known as a risk factor of hypertension and abdominal aortic aneurysm (AAA) in humans. Melanocortin 4 receptor (MC4R) is primarily expressed in brain, and mediates most of the anorectic effects of leptin; in humans, deficiency or mutation of MC4R is the most common monogenic form of obesity. However, it remains unknown whether MC4R signaling affects the development of obesity-associated hypertension, vascular vulnerability and aortic aneurysm. Methods and Results: MC4R-knockout (KO) mice fed a high-fat/high-sucrose diet (HF/HS) for 20 weeks displayed obesity and insulin resistance associated with hyperleptinemia compared to HF/HS-fed wild type mice. HF/HS-fed MC4R-KO mice exhibited hypertension, and showed increase of medial thickness and elastic lamina destruction in aortae. In aortae of HF/HS-fed MC4R-KO mice, genes of MCP-1, TNF-α, and F4/80 were markedly upregulated relative to HF/HS-fed wild type mice. Following the administration of angiotensin II (Ang II, 500 ng/kg/min), HF/HS-fed MC4R-KO mice showed significantly lower survival rate and higher incidence of AAA than HF/HS-fed wild type mice, with increased expression of F4/80- and osteopontin-positive cells in aortae. Furthermore, HF/HS-fed ob/ob mice exhibited significantly lower incidence of Ang II-induced AAA with a trend of higher survival rate than HF/HS-fed MC4R-KO mice. Leptin (100 ng/ml) significantly upregulated osteopontin gene expression in primary cultured vascular smooth muscle cells (VSMCs), but not in primary cultured macrophages. Conclusion: Our observation clearly demonstrates that deficiency of melanocortin 4 receptor promotes the development of hypertension, vascular vulnerability, and Ang II-induced AAA in mice. It also suggests that hyperleptinemia caused by central leptin resistance could contribute to the development of these vascular phenotypes in MC4R-KO mice, at least partly via induction of osteopontin in VSMCs. Disclosure K. Mori: None. K. Tsuchiya: None. S. Nakamura: None. Y. Miyachi: None. K. Shiba: None. Y. Ogawa: Research Support; Self; AstraZeneca. Advisory Panel; Self; Gilead Sciences, Inc.. Research Support; Self; Japan Agency for Medical Research and Development, Japan Society for the Promotion of Science, Mitsubishi Tanabe Pharma Corporation, Mochida Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd.. K. Kitamura: None.

Mitochondrial uncoupling in the melanocortin system differentially regulates NPY and POMC neurons to promote weight-loss

ObjectiveThe mitochondrial uncoupling agent 2,4-dinitrophenol (DNP), historically used as a treatment for obesity, is known to cross the blood-brain-barrier, but its effects on central neural circuits controlling body weight are largely unknown. As hypothalamic melanocortin neuropeptide Y/agouti-related protein (NPY/AgRP) and pro-opiomelanocortin (POMC) neurons represent key central regulators of food intake and energy expenditure we investigated the effects of DNP on these neurons, food intake and energy expenditure. MethodC57BL/6 and melanocortin-4 receptor (MC4R) knock-out mice were administered DNP intracerebroventricularly (ICV) and the metabolic changes were characterized. The specific role of NPY and POMC neurons and the ionic mechanisms mediating the effects of uncoupling were examined with in vitro electrophysiology performed on NPY hrGFP or POMC eGFP mice. ResultsHere we show DNP-induced differential effects on melanocortin neurons including inhibiting orexigenic NPY and activating anorexigenic POMC neurons through independent ionic mechanisms coupled to mitochondrial function, consistent with an anorexigenic central effect. Central administration of DNP induced weight-loss, increased BAT thermogenesis and browning of white adipose tissue, and decreased food intake, effects that were absent in MC4R knock-out mice and blocked by the MC4R antagonist, AgRP. ConclusionThese data show a novel central anti-obesity mechanism of action of DNP and highlight the potential for selective melanocortin mitochondrial uncoupling to target metabolic disorders.

Evaluation of a melanocortin-4 receptor (MC4R) agonist (Setmelanotide) in MC4R deficiency

ObjectivePro-opiomelanocortin (POMC)-derived peptides act on neurons expressing the Melanocortin 4 receptor (MC4R) to reduce body weight. Setmelanotide is a highly potent MC4R agonist that leads to weight loss in diet-induced obese animals and in obese individuals with complete POMC deficiency. While POMC deficiency is very rare, 1–5% of severely obese individuals harbor heterozygous mutations in MC4R. We sought to assess the efficacy of Setmelanotide in human MC4R deficiency. MethodsWe studied the effects of Setmelanotide on mutant MC4Rs in cells and the weight loss response to Setmelanotide administration in rodent studies and a human clinical trial. We annotated the functional status of 369 published MC4R variants. ResultsIn cells, we showed that Setmelanotide is significantly more potent at MC4R than the endogenous ligand alpha-melanocyte stimulating hormone and can disproportionally rescue signaling by a subset of severely impaired MC4R mutants. Wild-type rodents appear more sensitive to Setmelanotide when compared to MC4R heterozygous deficient mice, while MC4R knockout mice fail to respond. In a 28-day Phase 1b clinical trial, Setmelanotide led to weight loss in obese MC4R variant carriers. Patients with POMC defects upstream of MC4R show significantly more weight loss with Setmelanotide than MC4R deficient patients or obese controls. ConclusionsSetmelanotide led to weight loss in obese people with MC4R deficiency; however, further studies are justified to establish whether Setmelanotide can elicit clinically meaningful weight loss in a subset of the MC4R deficient obese population.

Abstract 14729: Weight Loss Reduces Atrial Fibrillation Inducibility and Burden in Severe Obesity Induced by Either High-Fat Diet or Genetic Hyperphagia in Mice

Introduction: Atrial fibrillation (AF), the most common sustained arrhythmia worldwide, is associated with increased morbidity and mortality. Obesity is increasingly recognized as an important risk factor to develop AF and heart disease with a diet rich in fats leading to morbid obesity. Melanocortin-4 receptor (MC4R) gene is a critical regulator of energy homeostasis, and homozygous loss-of-function mutations cause hyperphagia and morbid obesity. Hypothesis: We hypothesized that obesity and its comorbidities can create a profibrillatory substrate for AF in high fat diet-induced obese (DIO) and MC4R knock-out (MC4R-KO) mice, and that this substrate can be reversed by weight loss. Methods: Transesophageal rapid pacing was performed using atrial burst pacing (cycle length: 50-15 ms, for 15 s) to determine AF inducibility (% of mice that develop AF) and AF burden (number of AF episodes and total AF duration/mouse) in lightly anesthesized normotensive mice (C57bl6 mice [LEAN], DIO, and MC4R-KO), with continuous ECG monitoring. Transthoracic echocardiography was performed to assess right (R) and left (L) atrial appendage (AA) sizes. Results: Atrial burst pacing induced AF in 91% of DIO and 100% of MC4R-KO vs. 50% of LEAN (P&lt;0.01, N=8 mice/group). Compared to LEAN, both DIO and MC4R-KO exhibited greater number of inducible AF episodes (0.7±0.2 vs. 1.8±0.2 vs. 1.8±0.1, P&lt;0.01, N=15 mice/group) with longer duration (17.9±3 vs. 196±22 vs. 244±34 s, P&lt;0.0001, N=15 mice/group; Figure). Both DIO and MC4R-KO had greater LAA volumes as compared with LEAN (5.2±0.2 vs. 6.7±0.4 vs. 4.1±0.1 μl, P&lt;0.01, N=8 mice/group). RAA volume was similar across groups. After 20% weight loss, both AF burden and LAA volumes were significantly reduced to those seen in LEAN (22±5 s, 4.5±0.1 μl, P&lt;0.001, N=8 mice/group). Conclusions: High-fat diet or genetic hyperphagia-induced obesity increases LAA volume and creates a profibrillatory substrate for AF that can be reversed with weight loss.

Melanocortin receptor accessory proteins in adrenal disease and obesity.

Melanocortin receptor accessory proteins (MRAPs) are regulators of the melanocortin receptor family. MRAP is an essential accessory factor for the functional expression of the MC2R/ACTH receptor. The importance of MRAP in adrenal gland physiology is demonstrated by the clinical condition familial glucocorticoid deficiency type 2. The role of its paralog melanocortin-2-receptor accessory protein 2 (MRAP2), which is predominantly expressed in the hypothalamus including the paraventricular nucleus, has recently been linked to mammalian obesity. Whole body deletion and targeted brain specific deletion of the Mrap2 gene result in severe obesity in mice. Interestingly, Mrap2 complete knockout (KO) mice have increased body weight without detectable changes to food intake or energy expenditure. Rare heterozygous variants of MRAP2 have been found in humans with severe, early-onset obesity. In vitro data have shown that Mrap2 interaction with the melanocortin-4-receptor (Mc4r) affects receptor signaling. However, the mechanism by which Mrap2 regulates body weight in vivo is not fully understood and differences between the phenotypes of Mrap2 and Mc4r KO mice may point toward Mc4r independent mechanisms.

Obese carboxypeptidase E knockout mice exhibit multiple defects in peptide hormone processing contributing to low bone mineral density.

Carboxypeptidase E (CPE) is a prohormone/proneuropeptide processing enzyme, and mice bearing CPE mutations exhibit an obese and diabetic phenotype. Studies on CPE knockout (KO) mice revealed poor prohormone processing, resulting in deficiencies in peptide hormones/neuropeptides such as insulin, gonadotropin-releasing hormone, and cocaine- and amphetamine-regulated transcript (CART). Here, we show that CPE KO mice, an obese animal model, have low bone mineral density (BMD) accompanied by elevated plasma CTX-1 (carboxy-terminal collagen crosslinks), and osteocalcin, indicators of increased bone turnover. Receptor activator for NF-kappaB ligand (RANKL) expression was elevated approximately 2-fold relative to osteoprotegerin in the femur of KO animals, suggesting increased osteoclastic activity in the KO mice. In the hypothalamus, mature CART, a peptide involved in eating behavior and implicated in bone metabolism, was undetectable. The melanocortin and neuropeptide Y (NPY) systems in the hypothalamus have also been implicated in bone remodeling, since MC4R KO and NPY KO mice have increased BMD. However, reduction of alpha-MSH, the primary ligand of MC4R by up to 94% and the lack of detectable NPY in the hypothalamus of CPE KO do not recapitulate the single-gene KO phenotypes. This study highlights the complex physiological interplay between peptides involved in energy metabolism and bone formation and furthermore suggests the possibility that patients, bearing CPE and CART mutations leading to inactive forms of these molecules, may be at a higher risk of developing osteoporosis.

Analysis of the therapeutic functions of novel melanocortin receptor agonists in MC3R- and MC4R-deficient C57BL/6J mice

Melanocortin receptor agonists act in the brain to regulate food intake and body weight and, independently of these actions, affect insulin sensitivity. These experiments investigated the function of novel non-selective melanocortin receptor agonists (BIM-22493, BIM-22511) that cross the blood–brain barrier when administered peripherally. Treatment of diet induced obese C57BL/6J (B6) mice with melanocortin agonists administered peripherally improved obesity, hyperinsulinemia (∼50%) and fatty liver disease. Specificity of function was determined using B6 melanocortin-3 and melanocortin-4 receptor knockout mice (MC3RKO, MC4RKO). Chow fed MC4RKO but not MC3RKO used for these tests exhibited obesity, hyperinsulinemia and severe hepatosteatosis associated with increased expression of insulin-stimulated genes involved in lipogenesis. Reduced food intake associated with acute BIM-22493 treatment, and weight loss associated with 14 days of treatment with BIM-22511, required functional MC4R but not MC3R. However, while 14 days of treatment with BIM-22511 did not affect body weight and even increased cumulative food intake in MC4RKO, a significant reduction (∼50%) in fasting insulin was still observed. Despite lowering insulin, chronic treatment with BIM-22511 did not improve hepatosteatosis in MC4RKO, and did not affect hepatic lipogenic gene expression. Together, these results demonstrate that peripherally administered melanocortin receptor agonists regulate body weight, liver metabolism and glucose homeostasis through independent pathways. MC4R are necessary for melanocortin agonist-induced weight loss and improvements in liver metabolism, but are not required for improvements in hyperinsulinemia. Agonists with activity at MC4R improve glucose homeostasis at least partially by causing weight loss, however other melanocortin receptors may have potential for treating aberrations in glucose homeostasis associated with obesity.

Fos expression in brain of MC4R, MC3R, and double receptor knockout mice in response to CCK, BN or MTII

Fos expression in brain of MC4R, MC3R, and double receptor knockout mice in response to CCK, BN or MTII

Pro-opiomelanocortin (POMC) deficiency and peripheral melanocortins in obesity.

Melanocortin peptides, derived from pro-opiomelanocortin (POMC), appear to play a significant role in appetite and body weight regulation. Expression of the Pomc gene in the central nervous system results in the production of melanocortin peptides, which bind to the melanocortin-4 receptor (MC4-R) and inhibit food intake. MC4-R knockout mice exhibit adult-onset obesity, whereas MC4-R agonists suppress food intake in several models of obesity. Recently, Pomc knockout mice were generated and shown to develop hyperphagia and obesity with a time-course and severity comparable to MC4-R knockout mice, whereas daily administration of a stable alpha-melanocyte stimulating hormone analogue reversed this effect. These data clearly implicate POMC peptides and melanocortin receptors in the pathophysiology of obesity and provide important new tools for their development as therapeutic targets in obesity.

Voluntary exercise prevents the obese and diabetic metabolic syndrome of the melanocortin‐4 receptor knockout mouse

Exercise is a mechanism for maintenance of body weight in humans. Morbidly obese human patients have been shown to possess single nucleotide polymorphisms in the melanocortin-4 receptor (MC4R). MC4R knockout mice have been well characterized as a genetic model that possesses phenotypic metabolic disorders, including obesity, hyperphagia, hyperinsulinemia, and hyperleptinemia, similar to those observed in humans possessing dysfunctional hMC4Rs. Using this model, we examined the effect of voluntary exercise of MC4R knockout mice that were allowed access to a running wheel for a duration of 8 wk. Physiological parameters that were measured included body weight, body composition of fat and lean mass, food consumption, body length, and blood levels of cholesterol and nonfasted glucose, insulin, and leptin. At the termination of the experiment, hypothalamic mRNA expression levels of neuropeptide Y (NPY), agouti-related protein (AGRP), proopiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART), orexin, brain-derived neurotropic factor (BDNF), phosphatase with tensin homology (Pten), melanocortin-3 receptor (MC3R), and NPY-Y1R were determined. In addition, islet cell distribution and function in the pancreas were examined. In the exercising MC4R knockout mice, the pancreatic islet cell morphology and other physiological parameters resembled those observed in the wild-type littermate controls. Gene expression profiles identified exercise as having a significant effect on hypothalamic POMC, orexin, and MC3R levels. Genotype had a significant effect on AGRP, POMC, CART, and NPY-Y1R, with an exercise and genotype interaction effect on NPY gene expression. These data support the hypothesis that voluntary exercise can prevent the genetic predisposition of melanocortin-4 receptor-associated obesity and diabetes.