Empagliflozin Downregulates AMP‐Activated Protein Kinaseα O‐GlcNAcylation to Ameliorate Hepatic Steatosis

The efficacy of the SGLT2 inhibitor empagliflozin (EMPA) in mitigating hepatic steatosis in patients with type 2 diabetes mellitus and metabolic dysfunction-associated steatotic liver disease (MASLD) has been previously demonstrated. However, the underlying mechanisms remain unclear. In this study, we investigated the role of EMPA in alleviating hepatic steatosis through the modulation of O-GlcNAcylation. High-glucose (HG)-induced alpha mouse liver 12 (AML12) cells, mouse primary hepatocytes (MPHs), and murine MASLD models (high-fat diet-fed and ob/ob mice) were used to examine the effects of EMPA. Protein O-GlcNAcylation, lipid accumulation, and AMP-activated protein kinase α (AMPKα) regulation were evaluated using Western blotting, immunostaining, and siRNA knockdown. Our findings showed that protein O-GlcNAcylation levels were elevated in both in vitro and in vivo models. EMPA treatment reduced O-GlcNAcylation and ameliorated lipid accumulation in HG-induced AML12 cells, MPHs, and MASLD models. Knockdown of O-GlcNAc transferase (OGT) decreased O-GlcNAcylation levels and lipid accumulation in HG-induced AML12 cells. Additionally, OGT knockdown altered both O-GlcNAcylated and phosphorylated AMPKα levels. In these models, EMPA administration decreased O-GlcNAcylated AMPKα while increasing phosphorylated AMPKα. This study further identified serine 344, threonine 447, and serine 501 as critical O-GlcNAcylation sites on AMPKα2. Mutation of these residues to alanine in AMPKα2 attenuated lipid accumulation in AML12 cells, with no additional improvement observed following EMPA treatment. In summary, EMPA effectively improves hepatic steatosis by modulating the O-GlcNAcylation states of AMPKα. Identification of specific O-GlcNAcylation sites on AMPKα2 highlights their importance in the therapeutic mechanism of EMPA in improving hepatic steatosis.