Glycogen Synthase Kinase 3 β Hepatocyte Deletion Attenuates Ferroptosis and Metabolic Dysfunction-associated Steatohepatitis in Mice.

BACKGROUND & AIMS: Metabolic dysfunction-associated steatohepatitis (MASH) is characterized by toxic lipid-induced cellular stress (lipotoxicity), which culminates in lethal and sublethal hepatocyte injury and a sterile fibroinflammatory response. We previously reported that pharmacological inhibition of glycogen synthase kinase 3 (GSK3) ameliorates murine MASH. However, the hepatocyte-specific role of GSK3β in lipotoxic injury and the fibroinflammatory response in MASH remains unclear. METHODS: We generated hepatocyte-specific Gsk3β knockout (Gsk3β(ΔHep)) mice by crossing Gsk3β(fl/fl) mice with albumin-Cre mice. Mice were fed either a choline-deficient high-fat diet (CDHFD) or a high-fat, fructose, and cholesterol (FFC) diet to induce MASH. RESULTS: Metabolic parameters and hepatic lipidomic were similar between FFC-fed Gsk3β(ΔHep) and Gsk3β(fl/fl) mice. The NanoString Metabolic Pathways Panel on liver tissues showed upregulation of NAD, mitochondrial function, and oxidative phosphorylation signaling pathways in FFC-fed Gsk3β(ΔHep) mice compared with Gsk3β(fl/fl) mice. In vitro studies in palmitate-treated hepatocytes showed that mitochondrial morphology, biogenesis, contact with lipid droplets, and respiration were improved, whereas mitochondrial DNA release and Ferroptosis Suppressor Protein 1 (FSP1) phase separation were reduced with pharmacological GSK3 inhibition or in hepatocytes isolated from Gsk3β(ΔHep) mice. Similarly, liver injury, lipid peroxidation, ferroptosis markers, and circulating mitochondrial DNA levels were reduced in Gsk3β(ΔHep) mice with MASH. Furthermore, Gsk3β(ΔHep) mice with MASH had reduced hepatic expression of proinflammatory genes, myeloid cell infiltration, NETosis, and showed significant downregulation of fibrosis signaling pathways. CONCLUSIONS: Gsk3β(ΔHep) reduced liver injury, mitochondrial DNA release, inflammation, and fibrosis in mice with MASH, secondary to improved mitochondrial bioenergetics and reduced ferroptosis. Therefore, GSK3β may be a potential therapeutic target for human MASH.