The hepatic expression status of hepatocyte nuclear factor 4α in subclinical and clinical ketotic dairy cows and its impact on lipid metabolism in hepatocytes.

Due to sustained lipolysis, excessive nonesterified fatty acid (NEFA) concentrations are a common pathological factor of ketosis in peripartal cows. The NEFA provide an energy source to the liver during the transition period, but when in excess they can induce hepatic steatosis and liver dysfunction. In nonruminants, hepatocyte nuclear factor 4α (HNF4α) is involved in lipid metabolism processes in the liver. However, it is unclear whether HNF4α regulates hepatic lipid metabolism and function in cows with subclinical (SCK) and clinical ketosis (CK). Thus, the objective of this study was to investigate the expression status of the HNF4α in the liver of SCK and CK cows and its effects on lipid metabolism and cell function in hepatocytes. Liver and blood samples were collected from control cows (n = 15), cows with SCK (n = 15) and cows with CK (n = 15). To determine the effects of NEFA on lipid metabolism and cell function in vitro, hepatocytes isolated from calves were (1) treated with 0, 0.3, 0.6, or 1.2 mM NEFA for 12 h; (2) transfected with small interfering RNA for control (si-control) or si-HNF4A, or pCMV-EGFP or pCMV-EGFP-HNF4A for 48 h; (3) transfected with si-control or si-HNF4A, or pCMV-EGFP or pCMV-EGFP-HNF4A for 36 h, and then treated with 1.2 mM NEFA for 12 h. Results revealed that CK cows had hepatic steatosis and liver dysfunction. The protein abundance of HNF4α and its downstream targets carboxylesterase 1 (CES1) and CES2 as well as the mRNA abundance of CES1 and CES2 were upregulated in the liver of cows with SCK, but downregulated in CK cows. A dose of 1.2 mM NEFA decreased protein abundance of HNF4α, CES1, and CES2, and induced lipid accumulation and hepatocyte dysfunction. Knockdown of HNF4A decreased protein abundance of HNF4α, CES1, and CES2 and increased activities of glutamate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase in supernatant as well as decreased cell viability. The mRNA abundance of HNF4A, CES1, and CES2 was downregulated by si-HNF4A. Genes involved in fatty acid oxidation (FAO), including peroxisome proliferator-activated receptor-α (PPARA), carnitine palmitoyltransferase 1A (CPT1A), and pyruvate dehydrogenase kinase 4 (PDK4), were also downregulated due to transfection with si-HNF4A, while lipid accumulation increased. Overexpression of HNF4A increased protein abundance of HNF4α, CES1, and CES2 and upregulated mRNA abundance of HNF4A, CES1, CES2, PPARA, CPT1A, and PDK4 in hepatocytes while reducing lipid accumulation. In the presence of 1.2 mM NEFA, knockdown of HNF4A decreased whereas overexpression of HNF4A increased protein abundance of HNF4α, CES1, and CES2. Knockdown of HNF4A downregulated whereas overexpression of HNF4A upregulated mRNA abundance of CES1, CES2, PPARA, CPT1A, and PDK4. Furthermore, knockdown of HNF4A aggravated but overexpression of HNF4A alleviated NEFA-induced lipid accumulation and hepatocyte dysfunction. Overall, HNF4α accelerates the metabolism of NEFA by activating FAO, thereby alleviating steatosis, improving hepatocyte function, and inhibiting the occurrence and development of ketosis.