Glucose Acutely Reduces Cytosolic and Mitochondrial H 2 O 2 in Rat Pancreatic Beta Cells

摘要

Aims: Whether H_(2)O_(2) contributes to the glucose-dependent stimulation of insulin secretion (GSIS) by pancreatic β cells is highly controversial. We used two H_(2)O_(2)-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 α-ketoisocaproate on β cell cytosolic and mitochondrial H_(2)O_(2) concentrations. We then tested the effects of low H_(2)O_(2) and menadione concentrations on insulin secretion. Results: RoGFP2-Orp1 was more sensitive than HyPer to H_(2)O_(2) (response at 2–5 vs. 10 μ 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 μ M exogenous H_(2)O_(2). 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 5 m M glucose in the cytosol and 10 m M glucose in the mitochondria, and were partly mimicked by the other nutrients. Exogenous H_(2)O_(2) (1–15 μ M ) did not affect insulin secretion. By contrast, menadione (1–5 μ M ) did not increase basal insulin secretion but reduced the stimulation of insulin secretion by 20 m M glucose. Innovation: Subcellular changes in β cell H_(2)O_(2) levels are better monitored with roGFP2-Orp1 than HyPer/SypHer. Nutrients acutely lower mitochondrial H_(2)O_(2) levels in β cells and promote degradation of exogenously supplied H_(2)O_(2) in both cytosolic and mitochondrial compartments. Conclusion: The GSIS occurs independently of a detectable increase in β cell cytosolic or mitochondrial H_(2)O_(2) levels.