The supply of branched-chain amino acids and branched-chain keto acids alter lipid metabolism, oxidative stress, and apoptosis in primary bovine hepatocytes

Fatty liver impairs liver function and reduces productivity in dairy cows. Our previous in vivo findings demonstrated that branched-chain amino acids (BCAA) or branched-chain ketoacid (BCKA) improved liver function and lactation performance in dairy cows; however, the underlying mechanisms remain unclear. This study aimed to assess the impact of BCAA or BCKA supplementation on intracellular triglyceride (TG) accumulation, lipid metabolism, antioxidant response, and apoptosis in hepatocytes. Treatments were: control (CON): customized medium with amino acids, volatile fatty acids, fatty acids (FA), glucose, choline, insulin, and albumin concentrations equal to circulating levels in cows 4d postpartum; BCAA: CON + 33% additional BCAA of plasma BCAA 4d postpartum; and BCKA: CON + 33% additional BCKA of plasma BCAA 4d postpartum. Compared to CON, BCAA and BCKA reduced intracellular TG concentration by 32% and 40%, respectively, after 72h. BCAA and BCKA enhanced the uptake of palmitic acid, but upregulated the expression of genes regulating FA oxidation. Although mitochondrial membrane potential was reduced, oxidative protein damage (protein carbonyl levels) was decreased in BCAA- and BCKA-treated hepatocytes without changes in mitochondrial copy number. Additionally, compared to CON, BCAA and BCKA decreased the expression of executioner caspases (caspase 3 and caspase 7) and reduced the portion of hepatocytes with activated caspase 3/7, suggesting reduced apoptosis. These findings suggest that BCAA or BCKA supplementation improves hepatocyte lipid metabolism, antioxidant defenses, and apoptosis regulation, potentially mitigating the adverse effects of fatty liver. These mechanisms likely underlie the previously observed improvements in liver function and lactation performance.