The activation of pentose phosphate pathway flux by hydrogen peroxide is not regulated by NADPH-mediated feedback inhibition

Abstract

Oxidative stress induces a rerouting of metabolic flux from glycolysis to the pentose phosphate pathway. One proposed mechanism involves negative feedback via tonic inhibition of glucose-6-phosphate dehydrogenase by NADPH. However, recent evidence shows that NADPH levels do not decrease five seconds after hydrogen peroxide (H2O2) treatment. This finding is inconsistent with the canonical model wherein feedback inhibition loop is modulated by NADPH-depletion. This inconsistency prompts us to test the involvement of feedback inhibition at high temporal resolution. We employed genetically encoded fluorescent indicators for H2O2 (HyPerRed) and NADPH (iNap1) expressed in epithelial HEK293 cells. These tools enable simultaneous real-time, single-cell monitoring of NADPH and H2O2. Glucose sustains NADPH levels under acute oxidative stress in the first seconds following H2O2 exposure. This result contradicts the reported feedback inhibition, which is considered one of the fundamental mechanisms to explain the acute rerouting of glycolysis to PPP. Furthermore, pharmacological inhibition of G6PDH suggests that the PPP is the primary source of cytosolic NADPH under oxidative stress. Monitoring NADPH levels following G6PDH inhibition allows for the assessment of the NADPH consumption flux. This parameter is low under baseline conditions, but rises dramatically under oxidative stress. Our results support an anticipatory phenomenon that maintains NADPH levels under acute H2O2 exposure, thereby discarding the proposed feedback inhibition loop. This work offers a new perspective on the regulatory nuances of a metabolic pathway implicated in aging, cancer and a plethora of pathological conditions associated with the deleterious consequences of oxidative stress.