Mitochondrial Targeting of Apollo-NADP+ Reveals that Palmitate-Induced Toxicity in Beta-Cells Involves a Drop in Mitochondrial NADPH/NADP+ Redox State

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We recently developed a genetically encoded homoFRET sensor to measure NADPH/NADP+ redox state based on changes in anisotropy/polarization due to oligomerization of glucose-6-phosphate dehydrogenase (Apollo-NADP+). We have demonstrated that Apollo-NADP+ is reversible, spectrally tuneable, pH independent at physiological ranges, and responds to physiological levels of NADP+. In cells, the NADPH/NADP+ redox state supports the scavenging of the reactive oxygen species H2O2 by the glutathione/thioredoxin antioxidant pathway. A major source of H2O2 production is the mitochondrial electron transport chain (ETC), with overproduction a characteristic of many metabolic disorders. Here, we develop mitochondrially-targeted Apollo-NADP+ and use it in conjunction with the hydrogen peroxide sensor HyPer to investigated the effect of glucolipotoxicity on the mitochondria of the beta-cell. Following long-chain fatty acid (LC-FFA) treatment, we found a significant depletion of NADPH in the mitochondria but not the cytoplasm. We are now investigating the effect of mitochondrial morphology on this response, and have evidence that preserving mitochondrial networks may provide a protective effect against LC-FFA toxicity. We are also investigating the mechanism behind this response, particularly in the context of the effect of LC-FFAs on mitochondrial NADPH-generating pathways. Overall, this project demonstrates the utility of intermolecular sensor design using homoFRET while revealing novel information on the mitochondrial NADPH/NADP+ redox state dynamics involved in beta-cell stress.