Mitochondrial Superoxide Production Decreases on Glucose-Stimulated Insulin Secretion in Pancreatic β Cells Due to Decreasing Mitochondrial Matrix NADH/NAD + Ratio

Glucose-stimulated insulin secretion (GSIS) in pancreatic β cells was expected to enhance mitochondrial superoxide formation. Hence, we elucidated relevant redox equilibria. Unexpectedly, INS-1E cells at transitions from 3 (11 m ; pancreatic islets from 5 m ) to 25 m glucose decreased matrix superoxide release rates (MitoSOX Red monitoring validated by MitoB) and H O (mitoHyPer, subtracting mitoSypHer emission). Novel double-channel fluorescence lifetime imaging, approximating free mitochondrial matrix NADH indicated its ∼20% decrease. Matrix NAD increased on GSIS, indicated by the FAD-emission lifetime decrease, reflecting higher quenching of FAD by NAD . The participation of pyruvate/malate and pyruvate/citrate redox shuttles, elevating cytosolic NADPH (iNAP1 fluorescence monitoring) at the expense of matrix NADH , was indicated, using citrate (2-oxoglutarate) carrier inhibitors and cytosolic malic enzyme silencing: All changes vanished on these manipulations. C-incorporation from C-L-glutamine into C-citrate reflected the pyruvate/isocitrate shuttle. Matrix NADPH (iNAP3 monitored) decreased. With decreasing glucose, the suppressor of Complex III site Q electron leak (S3QEL) suppressor caused a higher Complex I I site contribution, but a lower superoxide fraction ascribed to the Complex III site III . Thus, the diminished matrix NADH /NAD decreased Complex I flavin site I superoxide formation on GSIS. Mutually validated methods showed decreasing superoxide release into the mitochondrial matrix in pancreatic β cells on GSIS, due to the decreasing matrix NADH /NAD (NADPH /NADP ) at increasing cytosolic NADPH levels. The developed innovative methods enable real-time NADH/NAD and NADPH/NADP monitoring in any distinct cell compartment. The export of reducing equivalents from mitochondria adjusts lower mitochondrial superoxide production on GSIS, but it does not prevent oxidative stress in pancreatic β cells.