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Insulin plays a pivotal role in the regulation of lipid metabolism.However, the mechanism of insulin action on lipid metabolism is still completely unclear in dairy cows.Hepatocytes were isolated from newborn female cows and co-cultured in vitro with various concentrations of insulin in the presence or absence of AICAR (an AMPKα activator).The results showed that insulin increased the phosphorylation of insulin receptor and decreased phosphorylation of AMP-activated protein kinase alpha (AMPKα).Moreover, insulin increased the expression and transcriptional activity of the carbohydrate responsive element-binding protein (ChREBP) and sterol regulatory element-binding protein (SREBP) ,resulted in the upregulation of lipogenic genes and decreased peroxisome proliferators-activated receptor-α (PPARα), resulted in the downregulation of lipid oxidation genes.While contrast results were observed after hepatocytes were treated with AICAR.In addition, the triglyceride (TG) content was significantly increased in insulin-treated groups.Collectively, these results indicated that insulin suppressed the AMPKα signaling pathway through insulin receptor to mediate a decreased lipid oxidation and an increased lipid synthesis in bovine hepatocytes, which elucidates the pathological mechanism of lipid metabolism disorder induced by hyperinsulinemia which occurs in insulin resistance of dairy cows with negative energy balance (NEB).