Nutrient stimulation of rat pancreatic beta cells acutely improves their cytosolic and mitochondrial defenses against H2O2
(2016) 52nd EASD annual meeting — Location: Munich (13.September.2016)
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- Background and aims: NADPH and H2O2 have been proposed as metabolic coupling factors in the glucose stimulation insulin secretion (GSIS) in pancreatic β cells. However, as NADPH favors enzymatic H2O2 degradation, their common operation is possible only if their production are spatially or temporally disconnected. Here, we used (mt-)roGFP2-Orp1 to monitor dynamic changes in cytosolic and mitochondrial H2O2 in β cells acutely stimulated by glucose and other nutrients. This ratiometric fluorescent probe is specifically oxidized byH2O2 owing to its Orp1 moiety, and is reduced by thioredoxin- and glutathione-dependent antioxidant systems. Materials and methods: Male Wistar rat pancreatic islets were isolated and dispersed in clusters of ≥5 cells. (mt-)roGFP2-Orp1 was expressed (adenovirus infection, CMV promotor) in the cytosol or mitochondrial matrix of islet cells. The fluorescence ratio of the probe was measured every 30 s (λexc 405/485 nm; λem 535 nm) during perifusion with Krebs solution containing various glucose concentrations (Gn = n mMglucose), other nutrients and low concentrations of freshly added exogenous H2O2. The ratio was expressed relative to the ratios measured after sequential addition of 10 mM dithiothreitol (minimum ratio set to 0%) and 100 μM aldrithiol (maximum ratio set to 100%) at the end of each experiment. Results: Both probes were mainly expressed in β cells: after coinfection with an adenovirus coding DsRed under the control of the rat insulin promoter, (mt-)roGFP2-Orp1 were expressed in 74-85% of DsRed-positive cells vs. 14-21% of DsRed-negative cells. RoGFP2-Orp1 was diffusely expressed in the cytosol and nucleus, and mt-roGFP2-Orp1 colocalized with MitoTracker Red (confocal microscopy, obj 100X). During perifusion with Krebs containing G10, roGFP2-Orp1 fluorescence ratio was low (~5% of min/max ratio) while mt-roGFP2-Orp1 was more oxidized (~15%), suggesting H2O2 is more concentrated in the mitochondrial matrix than cytosol of β cells. roGFP2-Orp1 was significantly oxidized by ≥2 μM exogenous H2O2 and mt-roGFP2-Orp1 by ≥15 μM H2O2, the effect of 15 μM on both probes being slowly reversible upon removal of H2O2. Under control conditions, roGFP2-Orp1 fluorescence ratio was unaffected by sequential changes from G10 to G0.5 then to G30. In contrast, roGFP2-Orp1 oxidation by 15 μM H2O2 was significantly higher at G0.5 than at G5, with no difference between G5 and G30. In the mitochondrial matrix, glucose also reduced mt-roGFP2-Orp1 oxidation, but this effect was maximal only at G10 (vs. G5 in the cytosol) and was observed both in the absence and presence of exogenous H2O2. Interestingly, the effects of glucose on (mt-)roGFP2-Orp1 oxidation were reproduced by addition of 20 mM monomethylsuccinate, 10 mM Leu + 10 mM Gln, or 10 mM alpha-ketoisocaproate (KIC) to G0.5 (cytosolic and mitochondrial probes) or G5 (mitochondrial probe only), either in the presence of 15 μM exogenous H2O2 (both probes) or in its absence (mitochondrial probe only). However, only KIC, a keto-acid that degrades H2O2, more effectively protected β cells against H2O2 than G10. Conclusion: Glucose and other nutrients acutely improve the ability of rat pancreatic β cells to defend their cytosol and mitochondrial matrix against low concentrations of endogenous and exogenous H2O2. These results do not support a role for H2O2 as a metabolic coupling factor in GSIS.
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Deglasse, J.-P., El-Hawat, V., & Jonas, J.-C. (2016). Nutrient stimulation of rat pancreatic beta cells acutely improves their cytosolic and mitochondrial defenses against H2O2. Diabetologia : clinical and experimental diabetes and metabolism, 59 Suppl. 1, S190. https://doi.org/10.1007/s00125-016-4046-9 (Original work published 2016)