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

AIMS: Whether H2 O2 contributes to the glucose-dependent stimulation of insulin secretion (GSIS) by pancreatic β cells is highly controversial. We used two H2 O2 -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 H2 O2 concentrations. We then tested the effects of low H2 O2 and menadione concentrations on insulin secretion.

RESULTS: RoGFP2-Orp1 was more sensitive than HyPer to H2 O2 (response at 2-5 vs. 10 μM) 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 μM exogenous H2 O2 . 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 mM glucose in the cytosol and 10 mM glucose in the mitochondria, and were partly mimicked by the other nutrients. Exogenous H2 O2 (1-15 μM) did not affect insulin secretion. By contrast, menadione (1-5 μM) did not increase basal insulin secretion but reduced the stimulation of insulin secretion by 20 mM glucose.

INNOVATION: Subcellular changes in β cell H2 O2 levels are better monitored with roGFP2-Orp1 than HyPer/SypHer. Nutrients acutely lower mitochondrial H2 O2 levels in β cells and promote degradation of exogenously supplied H2 O2 in both cytosolic and mitochondrial compartments.

CONCLUSION: The GSIS occurs independently of a detectable increase in β cell cytosolic or mitochondrial H2 O2 levels. Antioxid. Redox Signal. 00, 000-000.