Activation of the pentose phosphate pathway flux by hydrogen peroxide is not regulated by NADPH‐mediated feedback inhibition

Background and Purpose 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 5‐s after hydrogen peroxide treatment. This finding is inconsistent with the canonical model wherein a feedback inhibition loop is modulated by NADPH depletion. This inconsistency prompted us to test the involvement of feedback inhibition at high temporal resolution. Experimental Approach We employed genetically encoded fluorescent indicators for hydrogen peroxide (HyPerRed) and NADPH (iNap1) expressed in human embryonic kidney HEK293 cells. These tools enabled simultaneous real‐time, single‐cell monitoring of NADPH and hydrogen peroxide. Key Results Glucose sustained NADPH levels under acute oxidative stress in the first seconds following hydrogen peroxide exposure. This result contradicts the reported feedback inhibition, which is considered one of the fundamental mechanisms to explain the acute rerouting of glycolysis to the pentose phosphate pathway. Furthermore, pharmacological inhibition of G6PDH suggests that the pentose phosphate pathway is the primary source of cytosolic NADPH under oxidative stress. Monitoring NADPH levels following G6PDH inhibition allowed assessment of the NADPH consumption flux, a parameter that rises markedly under oxidative stress. Conclusion and Implications Our results support an anticipatory phenomenon that maintains NADPH levels under acute hydrogen peroxide exposure, thereby discarding the proposed feedback inhibition loop. This work offers a new perspective on the regulatory nuances of a metabolic pathway involved in ageing, cancer and many other pathological conditions.