M3 muscarinic receptor activation reduces hepatocyte lipid accumulation via CaMKKβ/AMPK pathway

Previously, we reported that hepatic muscarinic receptors modulate both acute and chronic liver injury, however, the role of muscarinic receptors in fatty liver disease is unclear. We observed in patients who underwent weight loss surgery, a decrease in hepatic expression of M3 muscarinic receptors (M3R). We also observed that fat loading of hepatocytes, increased M3R expression. Based on these observations, we tested the hypothesis that M3R regulate hepatocyte lipid accumulation. Incubation of AML12 hepatocytes with 1 mM oleic acid resulted in lipid accumulation that was significantly reduced by co-treatment with a muscarinic agonist (pilocarpine or carbachol), an effect blocked by atropine (a muscarinic antagonist). Similar treatment of Hepa 1-6 cells, a mouse hepatoblastoma cell line, showed comparable results. In both, control and fat-loaded AML12 cells, pilocarpine induced time-dependent AMPKα phosphorylation and significantly up-regulated lipolytic genes (ACOX1, CPT1, and PPARα). Compound C, a selective and reversible AMPK inhibitor, significantly blunted pilocarpine-mediated reduction of lipid accumulation and pilocarpine-mediated up-regulation of lipolytic genes. BAPTA-AM, a calcium chelator, and STO-609, a calcium/calmodulin-dependent protein kinase kinase inhibitor, attenuated agonist-induced AMPKα phosphorylation. Finally, M3R siRNA attenuated agonist-induced AMPKα phosphorylation as well as agonist-mediated reduction of hepatocyte steatosis. In conclusion, this proof-of-concept study demonstrates that M3R has protective effects against hepatocyte lipid accumulation by activating AMPK pathway and is a potential therapeutic target for non-alcoholic fatty liver disease.