Glutathione disulfide sensitizes hepatocytes to TNFα-mediated cytotoxicity via IKK-β S-glutathionylation: a potential mechanism underlying non-alcoholic fatty liver disease - PubMed (original) (raw)
Glutathione disulfide sensitizes hepatocytes to TNFα-mediated cytotoxicity via IKK-β S-glutathionylation: a potential mechanism underlying non-alcoholic fatty liver disease
Xiaobing Dou et al. Exp Mol Med. 2018.
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.
Conflict of interest statement
The authors declare that they have no conflict of interest.
Figures
Fig. 1. NAFLD development is associated with increased TNFα production and dysregulated hepatic glutathione homeostasis.
Male C57BL/6 mice were fed with control and high-fat diets (HFD) for 12 weeks. a Liver TG levels. b Plasma ALT levels. c Histological examination (H&E and Oil Red O staining of liver tissue) and NAFLD histological activity scores. d Plasma TNFα and IL-6 levels. e TNFα levels in hepatic tissues. f GSH and GSSG levels in the liver. g Hepatic GSSG/GSH ratio. h Western blot analysis of long-term HFD feeding on hepatic. 4-HNE-protein adduct formation. Data are expressed as the mean ± SD (n = 6). *p < 0.05 versus control
Fig. 2. Intracellular glutathione imbalance sensitizes hepatocytes to TNFα cytotoxicity.
a, b Both HepG2 (a) and NCTC1469 cells (b) were exposed to complete DMEM containing H2O2 (0.2 mM) for the indicated time periods. Intracellular GSH and GSSG levels were measured, and the GSSG/GSH ratios were calculated. All values are denoted as the mean ± SD from three or more independent studies. Bars with different characters differ significantly (p < 0.05). c, d H2O2 sensitizes hepatocytes to TNFα-induced cell death. HepG2 (c) and NCTC cells (d) were pretreated with H2O2 (0.1 and 0.2 mM) for 2 h before the addition of TNFα (40 ng/mL). Cell death was measured 16 h later by LDH release assay. Bars with different characters differ significantly (p < 0.05). e Hoechst 33342 staining. Arrows denote apoptotic bodies. f Western blot analysis of PARP cleavage. HepG2 cells were pretreated with H2O2 (0.2 mM) for 2 h, followed by TNFα (40 ng/mL) stimulation. Whole-cell lysates were collected and subjected to western blot for the detection of PARP cleavage. g DNA fragmentation ELISA assay. h Caspase-3 activities. Bars with different characters differ significantly (p < 0.05)
Fig. 3. Cellular GSSG accumulation renders hepatocytes susceptible to TNFα hepatotoxicity.
a Schematic illustration of the glutathione synthesis pathway. HepG2 cells were pretreated with BSO (0.5 mM), a potent inhibitor of gamma-glutamylcysteine synthetase, for 2 h before H2O2 (0.2 mM) addition. b Intracellular GSH and GSSG levels. All values are expressed as the mean ± SD from three or more independent studies. Bars with different characters differ significantly (p < 0.05). c BSO pretreatment had no effect on TNFα-induced cell death. HepG2 cells were pretreated with either BSO (0.5 mM) or H2O2 (0.2 mM) for 2 h before TNFα (40 ng/mL) addition. Cell death was determined by the LDH release assay 16 h later. All values are expressed as the mean ± SD from three or more independent studies. Bars with different characters differ significantly (p < 0.05). d, e Inhibition of glutathione reductase (GR) significantly increased the intracellular GSSG levels without affecting GSH levels. HepG2 cells were treated with BCNU (0.1 mM), a chemical inhibitor of GR, and the intracellular GSH and GSSG concentrations were measured at the indicated time points. All values are expressed as the mean ± SD from three or more independent studies. Bars with different characters differ significantly (p < 0.05). f, g GS inhibition sensitizes hepatocytes to TNFα-induced cell death. HepG2 cells were pretreated with BCNU (0.1 and 0.2 mM) for 2 h (f) or transfected with GS siRNA overnight (g) before TNFα (40 ng/mL) addition. Cell death was measured by LDH assay 16 h later. All values are expressed as the mean ± SD from three or more independent studies. Bars with different characters differ significantly (p < 0.05). *p < 0.05 versus untreated cells
Fig. 4. GSSG accumulation induces cellular protein _S_-glutathionylation.
a–c Exogenous H2O2 (a) and BCNU (b) treatment increased the intracellular glutathionylated protein levels. HepG2 cells were incubated with exogenous H2O2 (0.1 and 0.2 mM) or BCNU (0.1 and 0.2 mM) for 2 h. Whole-cell lysates were collected and subjected to western blot for the detection of intracellular GSSG levels
Fig. 5. Increased cellular protein _S_-glutathionylation sensitizes hepatocytes to TNFα hepatotoxicity.
a Diamide, a protein _S_-glutathionylation induction agent, increased intracellular protein-GSSG adduct formation. HepG2 cells were incubated with exogenous diamide (0.1 and 0.2 mM) for 2 h. Whole-cell lysates were collected and used for the detection of the intracellular GSSG levels by western blot analysis. b Diamide pretreatment sensitized hepatocytes to TNFα-induced cell death. HepG2 cells were pretreated with diamide (0.1 and 0.2 mM) for 2 h before TNFα (40 ng/mL) addition. Cell death was measured by the LDH assay 16 h later. All values are expressed as the mean ± SD from three or more independent studies. Bars with different characters differ significantly (p < 0.05)
Fig. 6. GSSG suppresses TNFα-stimulated NF-κB transactivation via induction of IKK-β glutathionylation.
a H2O2, BCNU, or diamide pretreatment suppressed NF-κB-mediated gene expression after TNFα stimulation. HepG2 cells were pretreated with H2O2, BCNU or diamide for 2 h before TNFα (40 ng/mL) addition. Total RNA was isolated, and NF-κB-targeted gene expression was determined by real-time RT-PCR. All values are expressed as the mean ± SD from three or more independent studies. *p < 0.05, **p < 0.01 versus TNFα-treated cells. b H2O2 pretreatment suppresses TNFα-induced nuclear p65/DNA-binding activity. HepG2 cells were pretreated with H2O2 (0.2 mM) for 2 h before TNFα (100 ng/mL) addition. Four hours later, nuclear fractions were isolated and subjected to ELISA for the measurement of p65/DNA-binding activities. All values are expressed as the mean ± SD from three or more independent studies. *p < 0.05 versus TNFα-treated cells. c H2O2 pretreatment abolished the TNFα-stimulated increase in IκB-α phosphorylation. HepG2 cells were pretreated with H2O2 (0.2 mM) for 2 h before TNFα (100 ng/mL) stimulation, and cell lysates were collected 15 min after TNFα exposure for western blot analysis of phosphorylated IκB-α. d H2O2 pretreatment inhibited TNFα-stimulated IKK-β activation/phosphorylation. HepG2 cells were pretreated with H2O2 for 2 h before TNFα (100 ng/mL) stimulation, and cell lysates were collected at the indicated time points for western blot analysis of the phosphorylated IKK-β levels. e H2O2, BCNU, or diamide pretreatment increased the IKK-β-SSG levels. HepG2 cells were treated with H2O2, BCNU, or diamide for 1 h, and cell lysates were collected and subjected to immunoprecipitation and western blot analysis for glutathionylated IKK- β. IB immunoblotting, IP immunoprecipitation
Fig. 7. Long-term HFD consumption increases IKK-β glutathionylation and suppresses NF-κB activation in the liver.
Male C57BL/6 mice were fed with either a control diet or high-fat diet (HFD) for 12 weeks. a Hepatic protein-SSG adduct levels. b Hepatic glutathionylated IKK-β levels. c Hepatic IKK-β phosphorylation. d Hepatic nuclear p65/DNA-binding activity. e NF-κB-targeted gene expression in the liver. Data are expressed as the mean ± SD (n = 6). *p < 0.05 versus control. f Hepatic JNK and caspase-3 activation
Fig. 8. Long-term HFD consumption reduces hepatic expression of glutaredoxins (Grxs).
Male C57BL/6 mice were fed with either a control diet or high-fat diet (HFD) for 12 weeks. a Hepatic expression of Grx1 and Grx2. b Cdcl2, a chemical inhibitor of Grx, increased protein-SSG formation in hepatocytes. HepG2 cells were incubated with Cdcl2 (1 μM) for 16 h. Whole-cell lysates were collected and subjected to western blot analysis for the detection of the intracellular protein-SSG levels. c Cdcl2 pretreatment sensitized hepatocytes to TNFα-induced cell death. HepG2 cells were pretreated with Cdcl2 (1 μM) for 2 h before TNFα (40 ng/mL) addition. Cell death was measured by LDH assay 16 h later. All values are expressed as the mean ± SD from three or more independent studies. *p < 0.01 versus untreated cells
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