Branched‐Chain Amino and Keto Acids Reduce Hepatocyte Lipid Droplet Size and Number via Distinct Proteomic Pathways

Branched-chain amino acids (BCAA) and their corresponding keto acids (BCKA) have been associated with changes in hepatic lipid metabolism and the resulting alterations in intracellular triglyceride concentrations. In this study, we utilized a previously established hepatocyte model to investigate the impact of BCAA and BCKA supplementation on triglyceride accumulation and proteomic responses when exposed to physiologically high levels of fatty acids (FA). Confocal imaging revealed that BCAA and BCKA effectively reduced intracellular lipid droplet size and number. Principal component analysis and hierarchical clustering suggest distinct proteomic profiles across treatment groups, with a total of 299 differentially abundant proteins. Gene Ontology and KEGG pathway enrichment analysis revealed distinct effects on hepatic metabolism. Compared to control, BCAA supplementation upregulated proteins in the TCA cycle, oxidative phosphorylation, and thermogenesis, supporting an elevated protein synthesis and energy metabolism. In contrast, BCKA treatment downregulated various proteins in the energy metabolism pathways, including the TCA cycle and oxidative phosphorylation, reflecting a potential feedback mechanism to limit oxidative stress. Increased protein abundance of mitochondrial electron transport chain (ETC) complexes was observed in treated hepatocytes. These results underscore the potential of BCAA and BCKA to regulate hepatic lipid homeostasis in dairy cows when metabolically challenged during early postpartum.