Choline attenuates NEFA-induced hepatic steatosis via GNMT regulation in hepatocytes.

To elucidate the molecular mechanisms by which choline regulates hepatic lipid metabolism under negative energy balance conditions, we established non-esterified fatty acid (NEFA)-induced hepatic steatosis models in both calf primary hepatocytes and human LO2 hepatocytes. Choline supplementation significantly reduced intracellular triglyceride accumulation and cytotoxicity induced by NEFA exposure. Transcriptomic profiling identified glycine N-methyltransferase (GNMT) as a key differentially expressed gene. Subsequent experiments confirmed that choline upregulated GNMT expression at both the mRNA and protein levels in a concentration-dependent manner. Knockdown of GNMT reversed the beneficial effects of choline on genes related to lipid synthesis (FAS, ACC), fatty acid oxidation (CPT1), lipoprotein assembly (ApoB100, MTTP), and bile acid metabolism (CYP7A1, CYP27A1, BSEP). Furthermore, inhibition of AMP-activated protein kinase (AMPK) reduced GNMT protein expression and elevated Myc, a negative transcriptional regulator of GNMT, suggesting that choline may regulate GNMT through the AMPK/Myc axis. Collectively, our findings demonstrate that choline alleviates NEFA-induced lipid accumulation and hepatocellular damage by modulating lipid and bile acid metabolism through GNMT, with the AMPK/Myc/GNMT signaling axis playing a pivotal regulatory role. These results provide mechanistic insights into the hepatic protective effects of choline and suggest GNMT as a potential therapeutic target for metabolic disorders in dairy cows and beyond.