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

Abstract

Oxidative stress and TNFα 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 TNFα-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 TNFα 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 TNFα killing by reducing the TNFα-triggered NF-κB activity. GSSG prevented TNFα-induced activation of IKK-β, an upstream kinase in the NF-κB signaling pathway, by inducing IKK-β glutathionylation (IKK-β-SSG formation). In animal studies, in comparison to a control diet, HFD consumption resulted in increased hepatic IKK-β-SSG formation, leading to suppressed IKK-β activation and subsequent NF-κB 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 TNFα-mediated cytotoxicity. In conclusion, our data suggest that GSSG is a potent and clinically relevant sensitizer for TNFα-induced hepatotoxicity in NAFLD, which represents a potential therapeutic target for NAFLD.

Activation of the AMPK-SIRT1 pathway contributes to protective effects of Salvianolic acid A against lipotoxicity in hepatocytes and NAFLD in mice

“…Western-blot was performed as described previously (Dou et al, 2018) and the following antibodies were used: phosphorylated-SAPK/JNK (Thr183/Tyr185) rabbit antibody, SAPK/JNK rabbit antibody, phosphorylated-p44/42 MAPK (Erk1/2) (Thr202/ Tyr204) rabbit antibody, p44/42 MAPK (Erk1/2) rabbit antibody, phosphorylated-p38 MAPK (Thr180/Tyr182) rabbit antibody, p38 MAPK rabbit antibody, phosphorylated-PKA (Thr197) rabbit antibody, PKA rabbit antibody, phosphorylated-AMPKα (Thr172) rabbit antibody, AMPKα rabbit antibody, SIRT1 rabbit antibody, and GAPDH rabbit antibody (Cell Signaling Technology, Danvers, MA). The antibodies were diluted according to the manufacturer's instructions at 1: 5,000 for GAPDH and 1:1,000 for the others.…”

The Role of the Transsulfuration Pathway in Non-Alcoholic Fatty Liver Disease

“…In silico and in vitro analyses indicate that the initial cellular response to increased hepatic FFAs and hepatic steatosis is an increase in cellular GSH concentration [ 99 , 100 ]. Studies evaluating diet-induced NAFLD animal models have found increased as well as decreased GSH levels in plasma and the liver [ 7 , 77 , 101 , 102 ]. The ratio between GSH and glutathione disulfide (GSSG) (GSH/GSSG—an early marker of oxidative stress) was decreased in NAFLD models [ 7 , 102 ].…”

“…Studies evaluating diet-induced NAFLD animal models have found increased as well as decreased GSH levels in plasma and the liver [ 7 , 77 , 101 , 102 ]. The ratio between GSH and glutathione disulfide (GSSG) (GSH/GSSG—an early marker of oxidative stress) was decreased in NAFLD models [ 7 , 102 ]. In addition, an increased influx of FFA for a prolonged period causes depletion of GSH in the livers of rats [ 77 ].…”

Fat and Sugar—A Dangerous Duet. A Comparative Review on Metabolic Remodeling in Rodent Models of Nonalcoholic Fatty Liver Disease

“…Furthermore, a high-fat and high-cholesterol diet potentially contributes to hepatic steatosis and oxidative stress [174,265,283]. Generally, a HF diet is associated with an altered cellular antioxidant defense that contributes to TNF-α-induced hepatotoxicity in NAFLD [284,285]. To a considerable extent, the importance of fatty acids lies in the ability of cell membranes to adapt their composition based on dietary FAs, influencing their vulnerability to cellular oxidative damage and the antioxidant response [262,[286][287][288].…”