194 - Mitochondrial matrix H2O2 release in INS-1E cells monitored by the H2O2-selective fluorescent probe Mito-HyPer

Abstract

Mitochondria of pancreatic β-cells act as the perfect glucose sensor substantiating glucose-stimulated insulin secretion (GSIS). Despite controversies, whether this leads or not to the increased superoxide release into the matrix, it has been recently indicated that H2O2 degradation is favored in the matrix upon GSIS [1]. We attempted to study this in model β-cells, INS-1E cells [2], using confocal microscopy time-resolved monitoring with Mito-HyPer. We found a substantial negative drift in fluorescence F485/F405 ratio, indicating degradation and possible slow H2O2 efflux in INS-1E cells pre-incubated with 3 mM or 11 mM glucose. After glucose addition to 25 mM, the H2O2 decrease was enhanced by several folds, which was prevented by inhibition of mitochondrial redox shuttles exporting NADPH to the cell cytosol on the expense of matrix NADH. We conclude that pancreatic β-cells do not create mitochondrial oxidative stress upon GSIS.