ABSTRACT: Objective: Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by hepatic insulin resistance (IR) and impaired lipid–glucose metabolism. Sodium butyrate (NaB), a short-chain fatty acid (SCFA), may improve metabolic homeostasis, but its actions in MASLD-related IR remain unclear. This study investigated the therapeutic effects and mechanisms of NaB in palmitate (PA)-induced insulin-resistant hepatocytes and high-fat diet (HFD)-induced MASLD mice. Methods: PA-treated HepG2 cells received NaB (0.5–1.5 mM). Lipid accumulation, glucose uptake, glycogen content, and metabolic gene and protein expression were analyzed using biochemical assays, staining, qPCR, Western blotting, and RNA-seq. In vivo, C57BL/6 mice were fed an HFD for 16 weeks and then treated orally with NaB (200 or 400 mg/kg/day) for 8 weeks. Metabolic phenotypes, liver histology, and major signaling pathways were analyzed. Results: In PA-induced HepG2 cells, NaB dose-dependently decreased triglyceride, total cholesterol, low-density lipoprotein cholesterol, and non-esterified fatty acid levels, while increasing high-density lipoprotein cholesterol, glucose uptake, and glycogen storage. NaB suppressed lipogenesis (↓SREBP1c, ↓FASN), enhanced fatty acid oxidation (↑PGC-1α, ↑PPARα, ↑CPT1A), restored GPR43–CaMKK2–adenosine monophosphate-activated protein kinase (AMPK)α–SIRT1 signaling, and activated PI3K/AKT signaling (↑p-IRS1, ↑p-PI3K, ↑p-AKT, ↑GLUT2), thereby reducing gluconeogenic markers (↓FOXO1, ↓PCK1, ↓G6PC1) and increasing glycogen synthase (↑GS). Transcriptomics confirmed enrichment of PPARα and metabolic pathways. In HFD-fed mice, NaB reduced body weight, improved lipid profiles, alleviated steatosis, improved glucose tolerance and insulin sensitivity, and restored liver glycogen, with greater effects at the higher dose. Conclusion: NaB alleviates hepatic steatosis and IR in MASLD by activating AMPK and PI3K/AKT pathways, promoting fatty acid oxidation, and enhancing glycogen synthesis. NaB may serve as a promising multi-target metabolic modulator for MASLD.
