Glucose Acutely Reduces Cytosolic and Mitochondrial HO in Rat Pancreatic Beta Cells

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