Proteomic and Phosphoproteomic Profiling of High-Fat Diet-Induced Hepatic Steatosis in Mice

Background: Long-term high-fat diet (HFD) consumption induces hepatic lipid accumulation and metabolic dysregulation, but its integrated effects on the hepatic proteome and phosphoproteome remain incompletely understood. This study investigated HFD-induced hepatic molecular alterations using quantitative proteomics and phosphoproteomics. Methods: Eight-week-old male C57BL/6J mice were fed a control diet (CON) or HFD for 16 weeks, followed by phenotypic assessment, serum redox assays, and hepatic proteomic and phosphoproteomic analyses. Results: HFD-fed mice exhibited increased body weight, fat mass, liver weight, and marked hepatic lipid accumulation. Blood glucose levels and total pyruvate tolerance test area under the curve (AUC) were increased, whereas baseline-adjusted incremental AUC did not differ significantly. Serum malondialdehyde levels were elevated, whereas glutathione peroxidase activity was reduced, indicating impaired glucose homeostasis and redox balance. Proteomic analysis identified 15 differentially expressed proteins (4 upregulated and 11 downregulated), associated with lipid metabolism, biological oxidation, and xenobiotic metabolism. Gene set enrichment analysis identified significant positive enrichment of Class I peroxisomal membrane protein import and significant negative enrichment of cholesterol biosynthesis, gluconeogenesis, and xenobiotics. Phosphoproteomic analysis identified 8593 phosphoproteins and 28,180 phosphorylation sites, including 2045 differentially phosphorylated sites (1044 increased and 1001 decreased). Enrichment and exploratory network analyses implicated mitophagy, glucose metabolism, mTOR signaling, autophagy, oxidative phosphorylation, and cytoskeletal organization. Conclusions: Long-term HFD feeding induced obesity, glucose dysregulation, hepatic steatosis, and redox imbalance. Fatty acid oxidation and peroxisomal abundance patterns may reflect metabolic adaptation to lipid loading, while phosphosite abundance changes link nutrient sensing and autophagy regulation to the hepatic response.