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.

A necrotic cell death model in a protist

“…31 As an example, some mammalian cells could be protected from H 2 O 2 -induced necrotic cell death by 2-DOG flowing into the pentose phosphate pathway and producing NADPH; exogenous glucose led only or mostly to glycolysis, and the pentose phosphate pathway was favored only when 2-DOG was added instead of glucose. 32 In starved Dictyostelium cells, for unknown reasons exogenous glucose cannot undergo glycolysis. In both cases, (Le Goffe et al 32 , this report), ATP was not produced.…”

“…32 In starved Dictyostelium cells, for unknown reasons exogenous glucose cannot undergo glycolysis. In both cases, (Le Goffe et al 32 , this report), ATP was not produced.…”

Role of glycolysis inhibition and poly(ADP‐ribose) polymerase activation in necrotic‐like cell death caused by ascorbate/menadione‐induced oxidative stress in K562 human chronic myelogenous leukemic cells

“…The NADPH is regenerated then by the hexose monophosphate shunt that utilizes glucose-6-phosphate. 24 This metabolic pathway should be enhanced in case of NAD 1 depletion because cells will displace the NAD(P)/NAD(P)H equilibrium to restore the NAD 1 levels.…”

Mitochondrial hexokinases, novel mediators of the antiapoptotic effects of growth factors and Akt

“…However, this needs to be directly verified using a catalytically inactive mutant of hexokinase. Other investigators have demonstrated that 2-deoxyglucose-6-phosphate, generated via the action of hexokinase, can be metabolized via the pentose phosphate pathway (PPP) in quantities sufficient to support redox coupling critical for cell survival (Le Goffe et al, 1999Goffe et al, , 2002 and redox coupling between this pathway and caspase-2 activation has also recently been described (Nutt et al, 2005), so potential metabolic contributions distal to glucose phosphorylation cannot be excluded (see below).…”