Oxidative pentose phosphate pathway and glucose anaplerosis support maintenance of mitochondrial NADPH pool under mitochondrial oxidative stress

Mitochondrial NADPH protects cells against mitochondrial oxidative stress by serving as an electron donor to antioxidant defense systems. However, due to technical challenges, it still remains unknown as to the pool size of mitochondrial NADPH, its dynamics, and NADPH/NADP⁺ ratio. Here, we have systemically modulated production rates of H₂O₂ in mitochondria and assessed mitochondrial NADPH metabolism using iNap sensors, ¹³C glucose isotopic tracers, and a mathematical model. Using sensors, we observed decreases in mitochondrial NADPH caused by excessive generation of mitochondrial H₂O₂, whereas the cytosolic NADPH was maintained upon perturbation. We further quantified the extent of mitochondrial NADPH/NADP⁺ based on the mathematical analysis. Utilizing ¹³C glucose isotopic tracers, we found increased activity in the pentose phosphate pathway (PPP) accompanied small decreases in the mitochondrial NADPH pool, whereas larger decreases induced both PPP activity and glucose anaplerosis. Thus, our integrative and quantitative approach provides insight into mitochondrial NADPH metabolism during mitochondrial oxidative stress.