KN21, a direct AMPK activator, alleviates hepatic steatosis and fibrosis by modulating long-chain TAG and pyrimidine metabolism in a MASH mouse model

Metabolic dysfunction-associated steatohepatitis (MASH) is a severe form of metabolic dysfunction-associated steatotic liver disease (MASLD) that is characterized by hepatic steatosis, inflammation, and fibrosis and can progress to cirrhosis and hepatocellular carcinoma. AMP-activated protein kinase (AMPK) is a critical regulator of cellular energy homeostasis and has emerged as a promising therapeutic target for MASH. This study aimed to evaluate the mechanism of action of KN21, a novel direct AMPK activator, in a diet-induced MASH mouse model to support its therapeutic potential. C57BL6J mice were fed a choline-deficient, L-amino acid-defined high-fat diet (CDAHFD) to induce MASH, followed by treatment with KN21. Metabolomic and lipidomic profiling were performed using liquid chromatography-mass spectrometry. Histological, biochemical, and molecular analyses were conducted to assess hepatic steatosis and fibrosis. KN21 treatment significantly promoted hepatic AMPK phosphorylation, reduced the liver-to-body weight ratio, mitigated hepatic steatosis, and decreased fibrosis in CDAHFD-fed mice. Lipidomic analysis revealed that KN21 reduced long-chain triacylglycerol (TAG) accumulation, whereas metabolomic analysis revealed that the abundance of pyrimidine metabolites, especially uridine and uracil, was restored in CDAHFD-fed mice. These changes were strongly correlated with reduced fibrotic marker levels, suggesting that KN21 attenuates both lipid accumulation and fibrosis, which are associated with TAG and pyrimidine metabolism and homeostasis. KN21 effectively reduced hepatic steatosis and fibrosis in a diet-induced MASH mouse model through AMPK activation, accompanied by modulation and restoration of long-chain TAG and pyrimidine metabolism. These findings highlight its potential as a promising therapeutic strategy for MASH and provide new insights into the metabolic mechanisms underlying AMPK-mediated hepatoprotection.