Peripheral Melanocortin 3 Receptor (MC3R) Regulates Hepatic Autophagy in Obesity

We have previously reported homozygosity for two MC3R SNPs (C17A+G241A) that partially inactive MC3R increases adiposity in humans and mouse models. However, the precise mechanisms by which hypoactive MC3R alters metabolic homeostasis remain unclear. Given the essential role of hepatic autophagy in cellular mechanisms of obesity, we hypothesized that diminished peripheral MC3R activity leads to hepatic lipid accumulation by interfering with autophagy, leading to steatosis. We used knock-in mouse models that carry either homozygous human “wild type” (MC3RhWT/hWT) or “double mutant” MC3R (C17A+G241A, MC3RhDM/hDM). We studied hepatic autophagic pathways with biochemical/imaging analysis in wild type Mc3r (C57/BL6) and MC3RhWT/hWT as controls (WT mice), and in MC3RhDM/hDM, and mice lacking Mc3r (Mc3r-/-) which each have greater fat mass and hepatic triglycerides. We first demonstrated the MC3R specific agonist D-trp-γ-MSH induced LC3II (a marker of autophagosome formation) in hepatocytes from WT, but not Mc3r-/- or MC3RhDM/hDM mice. Interestingly D-trp-γ-MSH also induced TFEB (a master regulator of lysosomal biogenesis) translocation from cytosol to nucleus in TFEB-overexpressed NIH-3T3 cells, indicating MC3R may regulate autophagy by affecting TFEB signaling. Overnight fasting induced liver LC3II expression in WT mice, but not in MC3RhDM/hDM or Mc3r-/-; rather, basal LC3II was upregulated even in the fed state for the latter two genotypes. Hepatocytes from MC3RhDM/hDM or Mc3r-/- failed to increase autophagic flux with starvation or with the presence of chloroquine, suggesting MC3R may play a role in the regulation of autolysosomal degradation under the control of TFEB. Electron microscopy analysis of liver sections further demonstrated MC3RhDM/hDM mice exhibited fewer lipid droplet-associated autophagosomes compared to MC3RhWT/hWT mice. These data suggest MC3R plays an important peripheral role for the regulation of hepatic autophagy that is targeted to lipids.