Keratin 8 Destabilization via O-GlcNAcylation Promotes Insulin Resistance in metabolic dysfunction associated steatotic liver disease.

BACKGROUND: Keratin 8 (K8) serves as a core component of intermediate filaments within the hepatocellular cytoskeleton, with its functionality critically regulated by post-translational modifications. O-GlcNAcylation critically participates in liver disease progression. This study aimed to identify the O-GlcNAcylation sites on K8 and elucidate their roles and molecular mechanisms in MASLD. METHODS: Using genetically modified (K8-deficient) and high-fat diet-induced MASLD mice, along with palmitic acid-treated hepatocytes, we assessed K8 O-GlcNAcylation dynamics and filament stability via immunoblotting, affinity assays, co-immunoprecipitation, and O-linked HexNAc proteomic analysis. Metabolic parameters and mTOR/PI3K/AKT signaling were evaluated. Viral vectors mediating K8 knockdown and S9A-mutant or wild-type K8 replacement were employed for site-directed mutagenesis studies. RESULTS: MASLD models showed elevated K8 O-GlcNAcylation and increased O-GlcNAc transferase expression. Proteomics identified Ser9 as the key modification site. Ser9 O-GlcNAcylation destabilized K8 filaments, increased soluble K8, and suppressed mTOR/PI3K/AKT phosphorylation. S9A-K8 mutation reduced O-GlcNAcylation, restored cytoskeletal stability and insulin signaling, and improved glucose tolerance and hepatic steatosis. CONCLUSIONS: Ser9-dependent K8 O-GlcNAcylation drives MASLD-associated insulin resistance by disrupting cytoskeletal integrity and impairing insulin signaling. Targeting this modification site restores metabolic disorders, providing a potential novel therapeutic target for MASLD.