Metabolic control of resistance of human epithelial cells to H2O2 and NO stresses

Abstract

The carbon flux through the oxidative branch of the pentose phosphate pathway (PPP) can be viewed as an integrator of the antioxidant mechanisms via the generation of NADPH. It could therefore be used as a control point of the cellular response to an oxidative stress. Replacement of glucose by galactose sensitized the human epithelial cell line HGT-1 to H2O2 stress. Here we demonstrate that, due to the restricted galactose flux into the PPP, the H2O2 stress led to early cellular blebbing followed by cell necrosis, these changes being associated with a fall in the NADPH/NADP+ ratio and GSH depletion. H2O2 cytotoxicity was prevented by adding 2-deoxyglucose (2dGlc). This protection was associated with an increased flow of 2-deoxyglucose 6-phosphate into the oxidative branch of the PPP together with the prevention of the NADPH/NADP+ fall and the maintenance of intracellular GSH redox homoeostasis. Inhibitors of enzyme pathways connecting the PPP to GSH recycling abolished the 2dGlc protection. In carbohydrate-free culture conditions, 2dGlc dose-dependent protective effect was paralleled by a dose-dependent influx of 2dGlc into the PPP leading to the maintenance of the intracellular redox status. By contrast, in Glc-fed cells, the PPP was not a control point of the cellular resistance to H2O2 stress as they maintained a high NADPH/NADP+ ratio. Both 2dGlc and Glc inhibited, through the maintenance of GSH redox status, NO cytotoxicity on galactose-containing Dulbecco's modified Eagle's medium (Gal-DMEM)-fed cells. 2dGlc did not prevent the fall of ATP content in NO-treated Gal-DMEM-fed cells, indicating that NO cytotoxicity was essentially due to the disruption of GSH redox homoeostasis and not to the alteration of ATP production by the mitochondrial respiratory chain. The maintenance of ATP content in NO-treated glucose-fed cells was due to their ability to derive their energy from anaerobic glycolysis. In conclusion, Gal-DMEM and 2dGlc-supplemented Gal-DMEM provide a useful system to decipher and organize into a hierarchy the targets of several stresses at the level of intact barrier epithelial cells.