Glucose acutely reduces cytosolic and mitochondrial H₂O₂ in rat pancreatic beta-cells.

Aims: Whether H2O2 contributes to the glucose-dependent stimulation of insulin secretion (GSIS) by pancreatic beta cells is highly controversial. We used two H2O2-sensitive probes, roGFP2-Orp1 (reduction/oxidation-sensitive enhanced green fluorescent protein fused to oxidant receptor peroxidase 1) and HyPer (hydrogen peroxide sensor) with its pH-control SypHer, to test the acute effects of glucose, monomethyl succinate, leucine with glutamine, and alpha-ketoisocaproate on beta cell cytosolic and mitochondrial H2O2 concentrations. We then tested the effects of low H2O2 and menadione concentrations on insulin secretion. Results: RoGFP2-Orp1 was more sensitive than HyPer to H2O2 (response at 2-5 vs. 10 mu M) and less pH-sensitive. Under control conditions, stimulation with glucose reduced mitochondrial roGFP2-Orp1 oxidation without affecting cytosolic roGFP2-Orp1 and HyPer fluorescence ratios, except for the pH-dependent effects on HyPer. However, stimulation with glucose decreased the oxidation of both cytosolic probes by 15 mu M exogenous H2O2. The glucose effects were not affected by overexpression of catalase, mitochondrial catalase, or superoxide dismutase 1 and 2. They followed the increase in NAD(P)H autofluorescence, were maximal at 5mM glucose in the cytosol and 10mM glucose in the mitochondria, and were partly mimicked by the other nutrients. Exogenous H2O2 (1-15 mu M) did not affect insulin secretion. By contrast, menadione (1-5 mu M) did not increase basal insulin secretion but reduced the stimulation of insulin secretion by 20mM glucose. Innovation: Subcellular changes in beta cell H2O2 levels are better monitored with roGFP2-Orp1 than HyPer/SypHer. Nutrients acutely lower mitochondrial H2O2 levels in beta cells and promote degradation of exogenously supplied H2O2 in both cytosolic and mitochondrial compartments. Conclusion: The GSIS occurs independently of a detectable increase in beta cell cytosolic or mitochondrial H2O2 levels.