OR12-3 Mice with MC4R Site Mutation (F51L) Develop Severe Obesity Independent of G(s)-alpha/cAMP Signaling

The central melanocortin α-MSH acts through MC4R receptors, which are known to activate G(s)α/cAMP signaling, to promote negative energy balance by inhibiting food intake (FI) and stimulating energy expenditure (EE). However, we have shown that mice with central G(s)α deficiency become obese with decreased EE but without hyperphagia, and that MC4R inhibition of food intake in the paraventricular nucleus of the hypothalamus (PVN) is mediated via the G proteins G(q)α/G(11)α. Previous in vitro studies have also shown that several MC4R mutations associated with early-onset human obesity, including F51L, have no effect on G(s)α/cAMP signaling. To determine whether there are G(s)α/cAMP-independent signaling pathways that regulate energy homeostasis through MC4Rs in vivo, we generated mice with an MC4R site mutation at F51L (MC4RF51L) using CRISPR/Cas9 and studied its metabolic consequences. MC4RF51L mice developed severe early-onset obesity with increased fat mass and body length, associated primarily with increased FI. In response to i.p. administered MC3R/MC4R agonist MTII, MC4RF51L mice showed impaired FI inhibition and EE stimulation. When MTII was directly administrated via a cannula unilaterally implanted into the PVN, FI was markedly inhibited in WT mice, but not in MC4RF51L mice, while heart rate was stimulated in both WT and MC4RF51L mice. In contrast, PVN administration of MTII did not affect EE in either WT or MC4RF51L mice. CREB phosphorylation levels in the PVN were similarly elevated in WT and MC4RF51L mice following i.p. injection of MTII, confirming that G(s)α/cAMP signaling pathway is intact in these mutants. In addition, MC4RF51L mice had reduced glucose tolerance and insulin sensitivity secondary to obesity, as glucose intolerance was not seen in non-obese young mutants. MC4RF51L mice had normal body temperature at room temperature (22(o)C) and maintained their temperature when exposed to 6(o)C for 5 hours, consistent with an intact thermogenic response to acute cold conditions. Taken together, our data show that MC4R/G(s)α/cAMP signaling in the PVN is not required for MTII-inhibited FI and that MC4R activation in the PVN has no observable effect on EE. Thus, these results show that additional MC4R pathways are required for normal energy homeostasis and provide support that MC4Rs mediate their actions on food intake in the PVN via G protein pathways other than G(s)α, most likely G(q)α/G(11)α. This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases.