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

Abstract

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 H₂O₂ (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. ¹³C-incorporation from ¹³C-L-glutamine into ¹³C-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.

Keywords: Complex I; NADH/NAD+ ratio; fluorescence lifetime imaging; glucose-stimulated insulin secretion; mitochondrial superoxide generation; pancreatic β cells.