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

Aims: Glucose-stimulated insulin secretion (GSIS) in pancreatic β cells was expected to enhance mitochondrial superoxide formation. Hence, we elucidated relevant redox equilibria. Results: Unexpectedly, INS-1E cells at transitions from 3 (11 mM; pancreatic islets from 5 mM) to 25 mM glucose decreased matrix superoxide release rates (MitoSOX Red monitoring validated by MitoB) and H(2)O(2) (mitoHyPer, subtracting mitoSypHer emission). Novel double-channel fluorescence lifetime imaging, approximating free mitochondrial matrix NADH(F,) indicated its ∼20% decrease. Matrix NAD(+)(F) increased on GSIS, indicated by the FAD-emission lifetime decrease, reflecting higher quenching of FAD by NAD(+)(F). The participation of pyruvate/malate and pyruvate/citrate redox shuttles, elevating cytosolic NADPH(F) (iNAP1 fluorescence monitoring) at the expense of matrix NADH(F), was indicated, using citrate (2-oxoglutarate) carrier inhibitors and cytosolic malic enzyme silencing: All changes vanished on these manipulations. (13)C-incorporation from (13)C-L-glutamine into (13)C-citrate reflected the pyruvate/isocitrate shuttle. Matrix NADPH(F) (iNAP3 monitored) decreased. With decreasing glucose, the suppressor of Complex III site Q electron leak (S3QEL) suppressor caused a higher Complex I I(F) site contribution, but a lower superoxide fraction ascribed to the Complex III site III(Qo). Thus, the diminished matrix NADH(F)/NAD(+)(F) decreased Complex I flavin site I(F) superoxide formation on GSIS. Innovation: Mutually validated methods showed decreasing superoxide release into the mitochondrial matrix in pancreatic β cells on GSIS, due to the decreasing matrix NADH(F)/NAD(+)(F) (NADPH(F)/NADP(+)(F)) at increasing cytosolic NADPH(F) levels. The developed innovative methods enable real-time NADH/NAD(+) and NADPH/NADP(+) monitoring in any distinct cell compartment. Conclusion: 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.