Promoter demethylation and protein O-GlcNAcylation-mediated enhancement of fatty acid synthase contributes to hepatic steatosis and inflammation in MASLD.

Dysregulated lipid metabolism in hepatocytes heightens the risk of metabolic dysfunction-associated steatotic liver disease (MASLD). Fatty acid synthase (FAS), one of the key enzymes regulating lipid production in the liver, is upregulated in MASLD patients, making it a prime target for treatment. However, the regulatory mechanisms governing FAS expression and its post-translational modification in MASLD, as well as their potential contribution to hepatic inflammation, remain incompletely understood. In this study, we find that ten-eleven translocation 2 (TET2), thymine DNA glycosylase (TDG), FAS, and glutamine synthetase (GS) are upregulated in lipid mixture- or high-fat diet-induced hepatic steatosis, both in vitro and in vivo. The lipid mixture increases FAS and GS expression through TDG-mediated promoter demethylation. It also promotes hepatic lipid droplet accumulation and inflammation through TDG, FAS, and GS. Additionally, GS is essential for lipid mixture-induced O-linked N-acetylglucosaminylation (O-GlcNAcylation) of FAS, which enhances its stability in hepatocytes. These findings demonstrate that upregulation of FAS through TDG-mediated promoter demethylation and GS-mediated O-GlcNAcylation accelerates hepatic steatosis and inflammation in MASLD, providing mechanistic insights and highlighting these regulatory pathways as potential targets for therapeutic intervention.