Mitochondrial Superoxide Production Decreases on Glucose-Stimulated Insulin Secretion in Pancreatic β Cells Due to Decreasing Mitochondrial Matrix NADH/NAD <sup>+</sup> Ratio
Lydie Plecitá‐Hlavatá, Hana Engstová, Blanka Holendová, Jan Tauber, Tomáš Špaček, Lucie Petrásková, Vladimı́r Křen, Jitka Špačková, Klára Gotvaldová, Jan Ježek, Andrea Dlasková, Katarína Smolková, Petr Ježek
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 m M ; pancreatic islets from 5 m M ) to 25 m M 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.