(158v) A Systemic Approach to Understanding Compartmentalized NADPH Metabolism Under Mitochondrial Oxidative Stress

NADPH, a reducing power that prevents oxidative stress and promotes reductive biosynthesis, is considered highly compartmentalized in eukaryotes. Dynamic measurements of NADPH within different compartments of living cells have been challenging using available methods, limiting our understanding of the roles of NADPH in subcellular level. Here, we have systemically modulated production rates of hydrogen peroxide (H2O2) in mitochondria and assessed dynamics of both mitochondrial and cytosolic NADPH. iNap sensors revealed mitochondrial NADPH decreases during excessive generation of mitochondrial H2O2 with its ratio to NADP⁺ decreasing 67-fold. The mitochondrial NADPH pool was observed to decrease when cytosolic H2O2 increased, whereas the cytosolic NADPH pool was maintained and did not change during increased mitochondrial H2O2. Glucose isotopic tracer analysis further demonstrated that increased activity in the pentose phosphate pathway (PPP) accompanies small decreases in the mitochondrial NADPH pool, whereas larger reductions induce both PPP activity and glucose anaplerosis. In summary, our study demonstrates that mitochondrial oxidative stress elevates a cytosolic PPP activity and glucose anaplerosis upon reduction of mitochondrial NADPH, indicating a communication between mitochondria and cytosol that supports maintenance of mitochondrial NADPH homeostasis.