NADPH oxidase-2 does not contribute to β-cell glucotoxicity in cultured pancreatic islets from C57BL/6J mice

- NOX2 was confirmed as a negative modulator of glucose-stimulated insulin secretion.
- 1 day-culture under glucotoxic condition increased β-cell cytosolic thiol oxidation.
- 3 weeks-culture under glucotoxic condition altered β-cell function and survival.
- NOX2 did not contribute to in vitro β-cell glucotoxicity.

High glucose-induced oxidative stress and increased NADPH oxidase-2 (NOX2) activity may contribute to the progressive decline of the functional β-cell mass in type 2 diabetes. To test that hypothesis, we characterized, in islets from male NOX2 knockout (NOX2-KO) and wild-type (WT) C57BL/6J mice cultured for up to 3 weeks at 10 or 30 mmol/l glucose (G10 or G30), the in vitro effects of glucose on cytosolic oxidative stress using probes sensing glutathione oxidation (GRX1-roGFP2), thiol oxidation (roGFP1) or H2O2 (roGFP2-Orp1), on β-cell stimulus-secretion coupling events and on β-cell apoptosis.After 1-2 days of culture in G10, the glucose stimulation of insulin secretion (GSIS) was ∼1.7-fold higher in NOX2-KO vs. WT islets at 20-30 mmol/l glucose despite similar rises in NAD(P)H and intracellular calcium concentration ([Ca2+]i) and no differences in cytosolic GRX1-roGFP2 oxidation.After long-term culture at G10, roGFP1 and roGFP2-Orp1 oxidation and β-cell apoptosis remained low, and the glucose-induced rises in NAD(P)H, [Ca2+]i and GSIS were similarly preserved in both islet types. After prolonged culture at G30, roGFP1 and roGFP2-Orp1 oxidation increased in parallel with β-cell apoptosis, the glucose sensitivity of the NADPH, [Ca2+]i and insulin secretion responses increased, the maximal [Ca2+]i response decreased, but maximal GSIS was preserved. These responses were almost identical in both islet types.In conclusion, NOX2 is a negative regulator of maximal GSIS in C57BL/6J mouse islets, but it does not detectably contribute to the in vitro glucotoxic induction of cytosolic oxidative stress and alterations of β-cell survival and function.