Molecular cloning and characterization of hepatocyte nuclear factor 4 alpha and its role in docosahexaenoic acid-induced fatty acid β-oxidation in tiger puffer (Takifugu rubripes) hepatocytes

Hepatocyte nuclear factor 4 alpha (HNF4α) plays a critical role in hepatic lipid metabolism in mammals, particularly with respect to fatty acid β-oxidation (FAO). However, its gene structure and biological function in fish remain largely unknown. In this study, the hnf4α gene, which encodes a peptide consisting of 454 Amino acids, was characterized from the tiger puffer (Takifugu rubripes). As a member of the Nuclear Receptor Superfamily, the Hnf4α protein contains a conserved deoxyribonucleic acid-binding domain at the N-terminus and a C-terminal multifunctional hydrophobic ligand-binding domain, which binds fatty acids. Amino acid sequence alignment, gene structure comparison, and phylogenetic analysis indicated that tiger puffer Hnf4α is highly conserved relative to mammals and Other teleosts, suggesting evolutionarily conserved functions. Tissue distribution analysis indicated that hnf4α transcript levels were highest in the intestine, followed by the liver and kidney. To explore whether HNF4α participates in regulating hepatic FAO in tiger puffer, a lipid accumulation model was first established in hepatocytes using 200 μM oleic acid (OA). Subsequent treatment with 100 μM docosahexaenoic acid (DHA) visually diminished OA-induced lipid droplets accumulation, accompanied by a significant reduction in triglyceride content and upregulation of hnf4α as well as several FAO-related genes, including Peroxisome Proliferator-activated Receptor alpha (PPARα), Peroxisome Proliferator-activated Receptor γ coactivator 1 alpha (pgc1α), and carnitine palmitoyltransferase 1ab (cpt1ab). Taken together, these findings offer initial evidence that Hnf4α may be involved in the DHA-induced promotion of FAO in tiger puffer hepatocytes.