Food demand and meal size in mice with single or combined disruption of melanocortin type 3 and 4 receptors.

Mice with homozygous genetic disruption of the melanocortin-4 receptor gene (MC4R-/-) are known to be hyperphagic and become obese, while those with disruption of the melanocortin-3 receptor gene (MC3R-/-) do not become markedly obese. The contribution of MC3R signaling in energy homeostasis remains little studied. In the present work, we compare MC3R-/- mice with wild-type (WT), MC4R-/-, and mice bearing disruption of both genes (double knockout, DKO) on select feeding and neuroanatomical dimensions. DKO mice were significantly more obese than MC4R-/-, whereas MC3R-/- weighed the same as WT. In a food demand protocol, DKO and MC4R-/- were hyperphagic at low unit costs for food, due primarily to increased meal size. However, at higher costs, their intake dropped below that of WT and MC3R-/-, indicating increased elasticity of food demand. To determine whether this higher elasticity was due to either the genotype or to the obese phenotype, the same food demand protocol was conducted in dietary obese C57BL6 mice. They showed similar elasticity to lean mice, suggesting that the effect is of genotypic origin. To assess whether the increased meal size in MC4R-/- and DKO might be due to reduced CCK signaling, we examined the acute anorectic effect of peripherally administered CCK and subsequently the induction of c-Fos immunoreactivity in select brain regions. The anorectic effect of CCK was comparable in MC4R-/-, DKO, and WT, but it was unexpectedly absent in MC3R-/-. CCK-induced c-Fos was lower in the paraventricular nucleus in MC3R-/- than the other genotypes. These data are discussed in terms of demand functions for food intake, MC receptors involved in feeding, and their relation to actions of gut hormones, such as CCK, and to 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.

Ac-Trp-DPhe(p-I)-Arg-Trp-NH2, a 250-Fold Selective Melanocortin-4 Receptor (MC4R) Antagonist over the Melanocortin-3 Receptor (MC3R), Affects Energy Homeostasis in Male and Female Mice Differently.

The melanocortin-4 receptor (MC4R) has been indicated as a therapeutic target for metabolic disorders such as anorexia, cachexia, and obesity. The current study investigates the in vivo effects on energy homeostasis of a 15 nM MC4R antagonist SKY2-23-7, Ac-Trp-DPhe(p-I)-Arg-Trp-NH2, that is a 3700 nM melanocortin-3 receptor (MC3R) antagonist with minimal MC3R and MC4R agonist activity. When monitoring both male and female mice in TSE metabolic cages, sex-specific responses were observed in food intake, respiratory exchange ratio (RER), and energy expenditure. A 7.5 nmol dose of SKY2-23-7 increased food intake, increased RER, and trended toward decreasing energy expenditure in male mice. However, this compound had minimal effect on female mice's food intake and RER at the 7.5 nmol dose. A 2.5 nmol dose of SKY2-23-7 significantly increased female food intake, RER, and energy expenditure while having a minimal effect on male mice at this dose. The observed sex differences of SKY2-23-7 administration result in the discovery of a novel chemical probe for elucidating the molecular mechanisms of the sexual dimorphism present within the melanocortin pathway. To further explore the melanocortin sexual dimorphism, hypothalamic gene expression was examined. The mRNA expression of the MC3R and proopiomelanocortin (POMC) were not significantly different between sexes. However, the expression of agouti-related peptide (AGRP) was significantly higher in female mice which may be a possible mechanism for the sex-specific effects observed with SKY2-23-7.

Effect of MTII on food intake and brain c-Fos in melanocortin-3, melanocortin-4, and double MC3 and MC4 receptor knockout mice.

Mice with genomic knockout of either melanocortin type 3 receptors (MC3R-/-), type 4 receptors (MC4R-/-) or knockout of both (double knockout, DKO) were tested for their anorectic response to the mixed MC3/4R agonist, MTII, injected into the anterior cerebral ventricle. Wild type (WT) mice showed a strong anorexia and, as expected, DKO were completely unresponsive to MTII. In contrast, both MC3R-/- and MC4R-/- showed a partial anorectic response. Induction of c-Fos immunoreactivity by MTII was examined in brain regions including paraventricular hypothalamus (PVN) and area postrema (AP). Compared with WT, MC4R-/- showed no activation in AP but showed normal activation in PVN, whereas MC3R-/- showed reduced activation in PVN but not in AP. RT-PCR analysis showed that hypothalamic mRNA for MC3R in MC4R-/- and for MC4R in MC3R-/- was unaltered from WT levels. These data suggest that both receptor subtypes are involved in the behavioral action of MTII, and that the critical receptors are in different brain regions.