Difference between revisions of "Plecita-Hlavata 2020 Antioxid Redox Signal"
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|abstract=Glucose-stimulated insulin secretion (GSIS) in pancreatic β cells was expected to enhance mitochondrial superoxide formation. Hence, we elucidated relevant redox equilibria. | |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 | 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<sub>2</sub>O<sub>2</sub> (mitoHyPer, subtracting mitoSypHer emission). Novel double-channel fluorescence lifetime imaging, approximating free mitochondrial matrix NADHF, indicated its ∼20% decrease. Matrix NAD<sup>+</sup><sub>F</sub> increased on GSIS, indicated by the FAD-emission lifetime decrease, reflecting higher quenching of FAD by NAD<sup>+</sup><sub>F</sub>. The participation of pyruvate/malate and pyruvate/citrate redox shuttles, elevating cytosolic NADPH<sub>F</sub> (iNAP1 fluorescence monitoring) at the expense of matrix NADH<sub>F</sub>, was indicated, using citrate (2-oxoglutarate) carrier inhibitors and cytosolic malic enzyme silencing: All changes vanished on these manipulations. <sup>13</sup>C-incorporation from <sup>13</sup>C-L-glutamine into <sup>13</sup>C-citrate reflected the pyruvate/isocitrate shuttle. Matrix NADPH<sub>F</sub> (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 III<sub>Qo</sub>. Thus, the diminished matrix NADH<sub>F</sub>/NAD<sup>+</sup><sub>F</sub> 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 | Mutually validated methods showed decreasing superoxide release into the mitochondrial matrix in pancreatic β cells on GSIS, due to the decreasing matrix NADH<sub>F</sub>/NAD<sup>+</sup><sub>F</sub> (NADPH<sub>F</sub>/NAD<sup>+</sup><sub>F</sub>) at increasing cytosolic NADPH<sub>F</sub> levels. The developed innovative methods enable real-time NADH/NAD<sup>+</sup> and NADPH/NADP<sup>+</sup> 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. | 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. |
Revision as of 21:43, 8 January 2021
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. |
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) 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.
• Bioblast editor: Plangger M
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2021-01