The oxidative
pentose phosphate cycle (OPPC) is necessary to maintain cellular reducing capacity during periods of increased oxidative stress. Metabolic flux through the OPPC increases stoichiometrically in response to a broad range of chemical
oxidants, including those that generate
reactive oxygen species (ROS). Here we show that OPPC sensitivity is sufficient to detect low levels of ROS produced metabolically as a function of the percentage of O2. We observe a significant decrease in OPPC activity in cells incubated under severe and moderate
hypoxia (ranging from <0.01 to 4% O2), whereas
hyperoxia (95% O2) results in a significant increase in OPPC activity. These data indicate that metabolic ROS production is directly dependent on
oxygen concentration. Moreover, we have found no evidence to suggest that ROS, produced by mitochondria, are needed to stabilize
hypoxia-inducible factor 1alpha (HIF-1alpha) under moderate
hypoxia.
Myxothiazol, an inhibitor of mitochondrial electron transfer, did not prevent HIF-1alpha stabilization under moderate
hypoxia. Moreover, the levels of HIF-1alpha that we observed after exposure to moderate
hypoxia were comparable between rho0 cells, which lack functional mitochondria, and the wild-type cells. Finally, we find no evidence for stabilization of HIF-1alpha in response to the non-toxic levels of H2O2 generated by the
enzyme glucose oxidase. Therefore, we conclude that the
oxygen dependence of the
prolyl hydroxylase reaction is sufficient to mediate HIF-1alpha stability under moderate as well as severe
hypoxia.