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.