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Mitochondrial Superoxide Production Decreases Upon Glucose-Stimulated Insulin Secretion in Pancreatic β-cells Due to Decreasing Mitochondrial Matrix NADH/NAD

Mitochondrial Superoxide Production Decreases Upon Glucose-Stimulated Insulin Secretion in Pancreatic β-cells Due to Decreasing Mitochondrial Matrix NADH/NAD

Lydie Plecitá-Hlavatá, Hana Engstová, Blanka Holendova, Jan Tauber, Tomas Spacek, Lucie Petrásková, Vladimir Kren, Jitka Špačková, Klára Gotvaldová, Jan Jezek, Andrea Dlasková, Katarína Smolková, Petr Jezek

Antioxid Redox Signal. 2020 Jun 10.

PMID: 32517485

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 upon 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 upon these manipulations. ¹³C-incorporation from ¹³C-1-glutamine into ¹³C-citrate reflected the pyruvate/isocitrate shuttle. Matrix NADPH_F, (iNAP3 monitored) decreased. With decreasing glucose, the 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 upon GSIS.
Innovation:
Mutually validated methods showed decreasing superoxide release into the mitochondrial matrix in pancreatic β-cells upon 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 upon GSIS, but does not prevent oxidative stress in pancreatic β-cells.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP890888127	S3QEL 2		890888-12-7	Price

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