Hydroxyacyl coenzyme A dehydrogenase trifunctional multienzyme complex subunit α depletion dysregulates hepatic energy and lipid metabolism in dairy cows hepatocytes: a multiomic integration analysis

Objective To elucidate the species-specific regulatory mechanisms of hydroxyacyl-CoA dehydrogenase alpha subunit (HADHA) in ruminant liver metabolism and to decipher the consequent metabolic-transcriptional network dysregulation using an integrated multiomics approach in a dairy cow hepatocyte model. Methods A loss-of-function study was conducted using transfecting primary hepatocytes isolated from dairy cows with HADHA-targeting small interfering RNA (n = 6). LC-MS+GC-MS metabolomics and transcriptomics were integrated and analyzed via orthogonal partial least squares–discriminant analysis, differential and enrichment analysis, and multiomics network analysis. Results HADHA knockdown induced profound metabolic reprogramming, identifying 692 differentially abundant metabolites and 736 differentially expressed genes. Key dysregulated metabolomic pathways included upregulation of forkhead box O signaling, tricarboxylic acid cycle, and butanoate metabolism and downregulation of glycolysis/gluconeogenesis and ATP-binding cassette transporters. This was evidenced by suppression of primary bile acids and accumulation of glyceroneogenesis markers. Transcriptomic analysis revealed downregulation of insulin response and nuclear factor κ B signaling and upregulation of phosphatidylinositol and phosphatidylinositol 3-kinase–protein kinase B signaling. Multiomics integration confirmed disruption in core pathways, including arginine and proline metabolism, nicotinate and nicotinamide metabolism, and cAMP signaling. Conclusions This study provides a comprehensive map of the HADHA-mediated regulatory network in the ruminant hepatocytes. Its depletion directly suppresses fatty acid oxidation and bile acid synthesis and rewires central carbon and lipid metabolism, leading to a state of metabolic imbalance. Clinical Relevance The elucidated mechanisms establish a foundational basis for future research aimed at developing nutritional or genetic strategies to improve metabolic health in the dairy industry.