Dairy cows with type Ⅱ ketosis displayed hepatic fat accumulation and hyperinsulinemia, while the underlying mechanism was still completely unclear.This study was aimed to investigate the regulation and exact signaling pathway of hyperinsulinemia on the hepatic fat accumulation.In vitro cultured cow hepatocytes were treated with 0, 1, 10, 100, or 1 000 nmol/L of insulin in presence or absence of AICAR (an AMPKα activator).The results showed that insulin decreased phosphorylation of AMP-activated protein kinase alpha (AMPKα).The inactivation of AMPKα increased the protein and gene expression levels and transcriptional activity of the carbohydrate responsive element-binding protein (ChREBP) and sterol regulatory element-binding protein (SREBP), resulted in the up-regulation of lipogenic genes.Furthermore, he inactivation of AMPKα decreased the protein and gene expression levels expression and transcriptional activity of peroxisome proliferators-activated receptor-α (PPARα), resulted in the downregulation of fat oxidation genes.In addition, insulin decreased the very low-density lipoprotein (VLDL) assembly.Consequently, triglyceride content was significantly increased in insulin treatment hepatocytes.Activation of AMPKα induced by AICAR could revise the effect of insulin on PPARα,SREBP-1c and ChREBP.The results illustrated that insulin suppresses the AMPKα signaling pathway and then decreases oxidation of lipid and VLDL assembly, and increases synthesis of lipid in cow hepatocytes, finally inducing TG accumulation.This mechanism could explain the causal relationship between hepatic fat accumulation and hyperinsulinemia in dairy cows with type Ⅱ ketosis.