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.