O-GlcNAcylation of GSDMD promotes mitochondrial dysfunction and hepatocyte injury in MASLD

Background/Aims: Cell-membrane gasdermin D (GSDMD) plays a key role as a pyroptosis executor of metabolic dysfunction-associated steatohepatitis (MASH). However, the mitochondrial translocation of GSDMD occurs even during the metabolic dysfunction-associated steatotic liver (MASL) phase, which involves mainly hepatic steatosis without tremendous inflammatory responses or sustainable hepatic cell death; the underlying mechanism is unknown. We investigated the role of GSDMD in the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD). Methods: Liver tissues from patients with MASLD and control subjects were obtained to evaluate GSDMD expression and distribution. GSDMD△hep mice and their floxed littermate controls, GSDMDfl/fl mice, were fed a high-fat, high-cholesterol diet (HFHCD) for 4 and 12 weeks to develop MASL or MASH, respectively. Additionally, GSDMD△hep mice were administered an adeno-associated virus 8 (AAV8) vector expressing wild-type GSDMD (GSDMD-WT) or GSDMD O-GlcNAcylation site mutants (GSDMD-T174/S178A) and were provided with an HFHCD or a methionine-choline-deficient diet. Results: GSDMD expression and mitochondrial translocation were increased in patients and experimental mice with MASLD. The translocation of GSDMD-NT to mitochondria impaired the mitochondrial membrane potential and promoted the leakage of cytochrome c and mitochondrial DNA, thus inducing early hepatocyte apoptosis, and was required for pyroptosis in MASLD. O-GlcNAcylation at T173/T175 (in humans) or T174/S178 (in mice) of GSDMD was responsible for GSDMD mitochondrial translocation and mitochondrial dysfunction. Targeting O-GlcNAcylation with Celecoxib suppressed GSDMD mitochondrial translocation and the development of MASLD. Conclusions: Targeting mitochondrial GSDMD-mediated programed cell death (PCD) pathways may provide new insights into the potential theoretical basis for MASLD interventions.