Glutathione disulfide sensitizes hepatocytes to TNFα-mediated cytotoxicity via IKK-β S-glutathionylation: a potential mechanism underlying non-alcoholic fatty liver disease

Oxidative stress and TNF alpha are critically involved in the initiation and progression of non-alcoholic fatty liver disease (NAFLD). In this study, we investigated the effects of dysregulated glutathione homeostasis, a principal feature of oxidative stress, on TNF alpha-induced hepatotoxicity and its mechanistic implications in NAFLD progression. We showed that mice fed a high-fat diet (HFD) for 12 weeks developed hepatic steatosis and liver injuries, which were associated with not only TNF alpha overproduction but also hepatic glutathione dysregulation, characterized by GSH reduction and GSSG elevation. Moreover, consuming a HFD increased protein S-glutathionylation (protein-SSG formation) in the liver. Subsequent cell culture studies revealed that GSSG accumulation, as opposed to GSH reduction, sensitized hepatocytes to TNF alpha killing by reducing the TNF alpha-triggered NF-kappa B activity. GSSG prevented TNF alpha-induced activation of IKK-beta, an upstream kinase in the NF-kappa B signaling pathway, by inducing IKK-beta glutathionylation (IKK-beta-SSG formation). In animal studies, in comparison to a control diet, HFD consumption resulted in increased hepatic IKK-beta-SSG formation, leading to suppressed IKK-beta activation and subsequent NF-kappa B suppression. Furthermore, we found that HFD consumption also led to decreased hepatic expression of glutaredoxin, a key enzyme for de-glutathionylation. Similarly, CdCl2, a chemical inhibitor of glutaredoxin, sensitized hepatocytes to TNF alpha-mediated cytotoxicity. In conclusion, our data suggest that GSSG is a potent and clinically relevant sensitizer for TNF alpha-induced hepatotoxicity in NAFLD, which represents a potential therapeutic target for NAFLD.