β-Hydroxybutyrate upregulates hepatic histone β-hydroxybutyrylation modification, promotes the expression of PPARα, and alleviates the hepatic steatosis in MASLD

Abstract Background Metabolic dysfunction-associated steatotic liver disease (MASLD) stands as the most widespread chronic liver disorder globally. Histone β-hydroxybutyrylation (Kbhb)—a novel post-translational modification of histones driven by β-hydroxybutyrate (BHB)—has recently been recognized as a key epigenetic modulator. Our study aimed to explore how BHB influences the expression of hepatic lipid metabolism-associated genes in MASLD, and to determine whether histone Kbhb acts as the mechanistic mediator underlying these effects. Methods For in vivo experiments, db/db mice fed a high-fat diet were utilized as the MASLD model. Following BHB intervention, changes in glycolipid metabolism and lipid accumulation in liver were assessed. Hepatic expression of lipid oxidation-related genes (e.g., PPARα) was quantified via qPCR; hepatic Pan-Kbhb and H3K9bhb levels were detected using immunohistochemistry and immunofluorescence. For in vitro experiments, a palmitic acid (PA)-induced AML12 hepatocyte model was established. After BHB treatment, intracellular lipid accumulation was visualized via Oil Red O and BODIPY staining; PPARα and downstream lipid oxidation gene expression was measured by qPCR and Western blotting. Total protein and histone Kbhb were evaluated using immunofluorescence and Western blotting. Results BHB effectively mitigated lipid accumulation in the livers of db/db mice and PA-induced AML12 cells, while upregulating PPARα and its downstream lipid oxidation-related target genes. Simultaneously, BHB elevated total protein Kbhb and histone H3K9bhb modifications in hepatic cells. Critically, blocking Kbhb (via A485, an inhibitor of the acyltransferase P300, or an acyl-CoA synthetase 2 inhibitor) led to significant downregulation of PPARα and its target gene expression. Conclusion BHB alleviates lipid accumulation in the liver of MASLD by promoting the expression of PPARα and its downstream lipid oxidation-related genes in the hepatocytes, which is associated with histone Kbhb modification.