Melanocortin-4 Receptor-deficient Mice Research Articles

Melanocortin-4 receptor in macrophages attenuated angiotensin II-induced abdominal aortic aneurysm in mice

Obesity is recognized as an independent risk factor for abdominal aortic aneurysm (AAA). While mutations in the melanocortin-4 receptor (MC4R) gene is the most common cause of obesity caused by mutations in a single gene, the link between MC4R function and vascular disease has still remained unclear. Here, by using melanocortin-4 receptor (MC4R) deficient mice, we confirmed MC4R deficiency promotes AAA and atherosclerosis. We demonstrated the contribution of two novel factors towards vascular vulnerability in this model: leptin signaling in vascular smooth muscle cells (VSMCs) and loss of MC4R signaling in macrophages. Leptin was shown to promote vascular vulnerability via PI3K-dependent upregulation of Spp1 expression in VSMC. Additionally, Ang II-induced AAA incidence was significantly reduced when MC4R gene expression was myeloid cell-specifically rescued in MC4R deficient (MC4RTB/TB) mice. Ex vivo analysis showed a suppression in NF-κB activity in bone marrow-derived macrophages from LysM(+);MC4RTB/TB mice compared to LysM(−);MC4RTB/TB mice, which exaggerates with endogenous MC4R ligand treatment; α-MSH. These results suggest that MC4R signaling in macrophages attenuates AAA by inhibiting NF-κB activity and subsequent vascular inflammation.

OR26-01 Epinephrine Is Essential for Normal Renal Glucose Reabsorption via the Glucose Transporter GLUT2

Humans and mice with Melanocortin 4 receptor (MC4R) deficiency remain protected from hyperglycemia despite chronic obesity and insulin resistance. We have observed that elevated glycosuria in MC4R deficient mice protects them from hyperglycemia. Moreover, our results indicate that circulating epinephrine may couple MC4R signaling with kidney glucose reabsorption. However, the direct role of epinephrine in regulating kidney glucose reabsorption remains unclear. We hypothesize that epinephrine is essential for maintaining glucose homeostasis via kidney glucose reabsorption. To test this hypothesis, we performed oral glucose tolerance tests (OGTTs) and intraperitoneal insulin tolerance tests (ITTs) in phenylethanolamine-N-methyltransferase (Pnmt) knockout (KO) mice that specifically lack epinephrine but have normal norepinephrine levels. Pnmt KO mice exhibited reduced insulin sensitivity compared to their Wild-Type (WT) littermates (Area under the curve for ITT: 9,700±256 vs. 8,482±417 mg/dL.min, p<0.05). Paradoxically, we observed improved rather than impaired glucose tolerance in Pnmt KO mice compared to their WT controls (Area under the curve for OGTT: 32,546±1,592 vs. 40,058±1,918 mg/dL.min, p<0.05). To ascertain if Pnmt KO mice, like MC4R deficient mice, show elevated glycosuria, we quantified their 24 urine glucose levels after oral glucose (250 mg) challenge. Indeed, Pnmt KO mice demonstrated elevated glycosuria compared to their WT littermates (Urine glucose: Baseline, 24.63±2.2 vs. 11.14±0.82 mg/dl; post glucose challenge: 67.83±5 vs. 16.09±1.13 mg/dl, p<0.001), again validating the phenotype similar to that of MC4R deficient mice. To determine the glucose transporters involved in mediating elevated glycosuria in the Pnmt KO mice, we measured the levels of different renal glucose transporters using western blot. We found that GLUT2 was decreased by ~26% in Pnmt KO mice compared to their WT littermates. Levels of other glucose transporters were not changed, indicating that suppression of renal GLUT2 mediates elevated glycosuria in the epinephrine deficient mice. We validated the direct effect of epinephrine on GLUT2 levels in vitro using mouse primary renal proximal tubule epithelial cells. Indeed, epinephrine selectively increased GLUT2, but did not affect other glucose transporters in the mouse kidney primary cells. Our findings establish the essential role of epinephrine in glucose reabsorption via the renal glucose transporter GLUT2. Therefore, modulating the renal adrenergic system, or, kidney-specific GLUT2 may afford alternative strategies to regulate glycosuria and ultimately mitigate diabetes.

Hyperphagia in male melanocortin 4 receptor deficient mice promotes growth independently of growth hormone.

Loss of function of the melanocortin 4 receptor (MC4R) results in hyperphagia, obesity and increased growth. Despite knowing that MC4Rs control food intake, we are yet to understand why defects in the function of the MC4R receptor contribute to rapid linear growth. We show that hyperphagia following germline loss of MC4R in male mice promotes growth while suppressing the growth hormone-insulin-like growth factor-1 (GH-IGF-1) axis. We propose that hyperinsulinaemia promotes growth while suppressing the GH-IGF-1 axis. It is argued that physiological responses essential to maintain energy flux override conventional mechanisms of pubertal growth to promote the storage of excess energy while ensuring growth. Defects in melanocortin-4-receptor (MC4R) signalling result in hyperphagia, obesity and increased growth. Clinical observations suggest that loss of MC4R function may enhance growth hormone (GH)-mediated growth, although this remains untested. Using male mice with germline loss of the MC4R, we assessed pulsatile GH release and insulin-like growth factor-1 (IGF-1) production and/or release relative to pubertal growth. We demonstrate early-onset suppression of GH release in rapidly growing MC4R deficient (MC4RKO) mice, confirming that increased linear growth in MC4RKO mice does not occur in response to enhanced activation of the GH-IGF-1 axis. The progressive suppression of GH release in MC4RKO mice occurred alongside increased adiposity and the progressive worsening of hyperphagia-associated hyperinsulinaemia. We next prevented hyperphagia in MC4RKO mice through restricting calorie intake in these mice to match that of wild-type (WT) littermates. Pair feeding of MC4RKO mice did not prevent increased adiposity, but attenuated hyperinsulinaemia, recovered GH release, and normalized linear growth rate to that seen in pair-fed WT littermate controls. We conclude that the suppression of GH release in MC4RKO mice occurs independently of increased adipose mass, and is a consequence of hyperphagia-associated hyperinsulinaemia. It is proposed that physiological responses essential to maintain energy flux (hyperinsulinaemia and the suppression of GH release) override conventional mechanisms of pubertal growth to promote the storage of excess energy while ensuring growth. Implications of these findings are likely to extend beyond individuals with defects in MC4R signalling, encompassing physiological changes central to mechanisms of growth and energy homeostasis universal to hyperphagia-associated childhood-onset obesity.

Melanocortin 4 Receptors Reciprocally Regulate Sympathetic and Parasympathetic Preganglionic Neurons

Melanocortin 4 receptors (MC4Rs) in the central nervous system are key regulators of energy and glucose homeostasis. Notably, obese patients with MC4R mutations are hyperinsulinemic and resistant to obesity-induced hypertension. Although these effects are probably dependent upon the activity of the autonomic nervous system, the cellular effects of MC4Rs on parasympathetic and sympathetic neurons remain undefined. Here, we show that MC4R agonists inhibit parasympathetic preganglionic neurons in the brainstem. In contrast, MC4R agonists activate sympathetic preganglionic neurons in the spinal cord. Deletion of MC4Rs in cholinergic neurons resulted in elevated levels of insulin. Furthermore, re-expression of MC4Rs specifically in cholinergic neurons (including sympathetic preganglionic neurons) restores obesity-associated hypertension in MC4R null mice. These findings provide a cellular correlate of the autonomic side effects associated with MC4R agonists and demonstrate a role for MC4Rs expressed in cholinergic neurons in the regulation of insulin levels and in the development of obesity-induced hypertension.

Role of Melanocortin 4 Receptor (Mc4R) in Obesity

Obesity is increasing worldwide and closely associated with diabetes, abnormal lipid levels, hypertension and other diseases. Melanocortin 4 Receptor (Mc4R) deficiency is the most common monogenic form of obesity. It is estimated that 5% of extremely obese patients have loss‐of‐function mutations of Mc4R. The goal of this project was to determine how Mc4R affects energy homeostasis and diseases associated with obesity. It was hypothesized that a disruption of Mc4R signaling leads to obesity and its associated diseases. Metabolic parameters were compared between normal and Mc4R KO mice. Both genotypes were weaned on standard rodent chow. Food intake was weighed and body composition measured using Nuclear Magnetic Resonance, NMR. At 6 weeks, mice were euthanized and blood was harvested and centrifuged for serum chemistry analysis. Data were analyzed using t test or ANOVA. We found a significant increase in food intake and body weight, in Mc4R deficient mice. There was a mild difference in serum lipid levels suggesting that this model of obesity has a greater adverse impact on glucose homeostasis than lipid metabolism.

The orally active melanocortin-4 receptor antagonist BL-6020/979: a promising candidate for the treatment of cancer cachexia

BackgroundUnder physiological conditions, the melanocortin system is a crucial part of the complex network regulating food intake and energy expenditure. In pathological states, like cachexia, these two parameters are deregulated, i.e., food intake is decreased and energy expenditure is increased—a vicious combination leading to catabolism. Agouti-related protein (AgRP), the endogenous antagonist at the melanocortin-4 receptor (MC-4R), was found to increase food intake and to reduce energy expenditure. This qualifies MC-4R blockade as an attractive mode of action for the treatment of cachexia. Based on this rationale, a novel series of small-molecule MC-4R antagonists was designed, from which the orally active compound BL-6020/979 (formerly known as SNT207979) emerged as the first promising development candidate showing encouraging pre-clinical efficacy and safety properties which are presented here.Methods and resultsBL-6020/979 is an orally available, selective and potent MC-4R antagonist with a drug-like profile. It increased food intake and decreased energy expenditure in healthy wild-type but not in MC-4R deficient mice. More importantly, it ameliorated cachexia-like symptoms in the murine C26 adenocarcinoma model; with an effect on body mass and body composition and on the expression of catabolic genes. Moreover, BL-6020/979 showed antidepressant-like properties in the chronic mild stress model in rats and exhibits a favorable safety profile.ConclusionThe properties of BL-6020/979 demonstrated in animal models and presented here make it a promising candidate suitable for further development towards a first-in-class treatment option for cachexia that potentially opens up the opportunity to treat two hallmarks of the disease, i.e., decreased food intake and increased energy expenditure, with one drug.

Impact of obesity on renal structure and function in the presence and absence of hypertension: evidence from melanocortin-4 receptor-deficient mice

The purpose of this study was to determine the long-term impact of obesity and related metabolic abnormalities in the absence and presence of hypertension on renal injury and salt-sensitivity of blood pressure. Markers of renal injury and blood pressure salt sensitivity were assessed in 52- to 55-wk-old normotensive melanocortin-4 receptor-deficient (MC4R-/-) mice and lean C57BL/6J wild-type (WT) mice and in 22-wk-old MC4R-/- and WT mice made hypertensive by N(G)-nitro-L-arginine methyl ester (L-NAME) in the drinking water for 8 wk. Old MC4R-/- mice were 60% heavier, hyperinsulinemic, and hyperleptinemic but had similar mean arterial pressure (MAP) as WT mice (115 +/- 2 and 117 +/- 2 mmHg) on normal salt diet (0.4% NaCl). A high-salt diet (4.0% NaCl) for 12 days did not raise MAP in obese or lean mice [DeltaMAP: MC4R (-/-) 4 +/- 2 mmHg; WT, 2 +/- 1 mmHg]. Obese MC4R-/- mice had 23% greater glomerular tuft area and moderately increased GFR compared with WT mice. Bowman's space, total glomerular area, mesangial matrix, urinary albumin excretion (UAE), renal TGF-beta and collagen expression were not significantly different between old MC4R-/- and WT mice. Renal lipid content was greater but renal macrophage count was markedly lower in MC4R-/- than WT mice. Mild increases in MAP during L-NAME treatment (approximately 16 mmHg) caused small, but greater, elevations in UAE, renal TGF-beta content, and macrophage infiltration in MC4R-/- compared with WT mice without significant changes in glomerular structure. Thus despite long-term obesity and multiple metabolic abnormalities, MC4R-/- mice have no evidence of renal injury or salt-sensitivity of blood pressure. These observations suggest that elevations in blood pressure may be necessary for obesity and related metabolic abnormalities to cause major renal injury or that MC4R-/- mice are protected from renal injury by mechanisms that are still unclear.

Structure-Activity Relationships of β -MSH Derived Melanocortin-4 Receptor Peptide Agonists

The recent emergence of obesity as a major health threat in the industrialized world has intensified the search for novel and effective pharmacologic treatment. The proopiomelanocortin (POMC)-melanocortin 4 receptor (MC4R) axis has been shown to regulate food intake and energy homeostasis and is considered among the most promising antiobesity targets. Our initial efforts in this area have focused on affinity and selectivity directed optimization of the native beta-MSH(5-22) sequence and resulted in the discovery of a potent MC4R agonist: Ac-Tyr-Arg-[Cys-Glu-His-D-Phe-Arg-Trp-Cys]-NH(2) (10). Subcutaneous administration of this peptide produced an excellent in vivo efficacy in reducing food intake and increasing fat metabolism. Additionally, suppression of food intake was observed in wild type but not in MC4R deficient mice, suggesting that the effects observed in the wild type mice were mediated through MC4R signaling. Subsequent optimization efforts led to the identification of a novel series of disulfide constrained hexapeptides as exemplified by Ac-[hCys-His-D-Phe-Arg-Trp-Cys]-NH(2) (100). These cyclic hexapeptides showed a further improved potency in binding MC4R and an enhanced selectivity over MC1R. At a dose of 0.07 mg/kg analog 102 reduced food intake by 38% and increased fat utilization by 58% in rats. These cyclic peptides provide novel and enhanced reagents for the elucidation of melanocortin receptors biology and may find applications in the treatment of obesity and related metabolic disorders.