Plecita-Hlavata 2020 Antioxid Redox Signal

From Bioblast
Publications in the MiPMap
Plecitá-Hlavatá L, Engstová H, Holendová B, Tauber J, Špaček T, Petrásková L, Křen V, Špačková J, Gotvaldová K, Ježek J, Dlasková A, Smolková K, Ježek P (2020) Mitochondrial superoxide production decreases on glucose-stimulated insulin secretion in pancreatic β cells due to decreasing mitochondrial matrix NADH/NAD+ ratio. Antioxid Redox Signal 33:789-815.

» PMID: 32517485 Open Access

Plecita-Hlavata Lydie, Engstova Hana, Holendova Blanka, Tauber Jan, Spacek Tomas, Petraskova Lucie, Kren Vladimir, Spackova Jitka, Gotvaldova Klara, Jezek Jan, Dlaskova Andrea, Smolkova Katarina, Jezek Petr (2020) Antioxid Redox Signal

Abstract: 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 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 NADHF, 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 NADPHF (iNAP1 fluorescence monitoring) at the expense of matrix NADHF, 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 NADPHF (iNAP3 monitored) decreased. With decreasing glucose, the suppressor of Complex III site Q electron leak (S3QEL) suppressor caused a higher Complex I IF site contribution, but a lower superoxide fraction ascribed to the Complex III site IIIQo. Thus, the diminished matrix NADHF/NAD+F decreased Complex I flavin site IF 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 NADHF/NAD+F (NADPHF/NAD+F) at increasing cytosolic NADPHF 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. Keywords: Complex I, NADH/NAD+ ratio, Fluorescence lifetime imaging, Glucose-stimulated insulin secretion, Mitochondrial superoxide generation, Pancreatic β cells Bioblast editor: Plangger M O2k-Network Lab: CZ Prague Jezek P

Labels: MiParea: Respiration 

Organism: Rat  Tissue;cell: Islet cell;pancreas;thymus  Preparation: Intact cells 

Coupling state: LEAK, ROUTINE, ET  Pathway: ROX  HRR: Oxygraph-2k 


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