Lack Of NADPH Oxidase-2 Does Not Improve Pancreatic β-cell Survival And Function In Male C57BL/6J Mouse Islets Cultured For 3 Weeks At High Glucose

Souza, Arnaldo;Santos, Laila RB;Roma, Leticia P;Carpinelli, Angelo R;Jonas, Jean-Christophe
(2016) American Diabetes Association 76th scientific sessions — Location: New-Orleans, LA, USA (10.June.2016)

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  • Souza, ArnaldoUCLouvain
    Author
  • Santos, Laila RBUCLouvain
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  • Roma, Leticia PGerman Cancer Research Center, Heidelberg, Germany
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  • Carpinelli, Angelo RUniversity of Sao Paulo, Brazil
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  • Jonas, Jean-Christopheorcid-logoUCLouvain
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Abstract
High glucose-induced oxidative stress may contribute to the progressive loss of functional β-cell mass in type 2 diabetes. To test whether superoxide production by NADPH oxidase-2 (NOX2) is involved in β-cell dysfunction and apoptosis under glucotoxic conditions, we characterized the acute and long-term effects of glucose on cytosolic glutathione/thiol oxidation (GRX1-roGFP2 and roGFP1 redox-sensitive probes), H2O2 concentration (roGFP2-Orp1 H2O2-sensitive probe), β-cell stimulus-secretion coupling events and apoptosis in islets from male NOX2 knockout (NOX2-KO) and wild-type (WT) C57BL/6J mice cultured for up to 3 weeks in RPMI medium containing 10 or 30 mmol/L glucose (G10 or G30). After 1-2 days of culture at G10, the acute glucose stimulation of insulin secretion (GSIS) was ~1.7-fold higher in NOX2-KO vs. WT islets despite similar rises in NAD(P)H autofluorescence and intracellular calcium ([Ca2+]i) and no differences in cytosolic glutathione oxidation. After long-term culture at G10, cytosolic thiol oxidation, H2O2 concentration and β-cell apoptosis remained low in both islet types. Also, the glucose-induced rises in NAD(P)H autofluorescence, [Ca2+]i and GSIS were similar to the results obtained after 1-2 days of culture, except for a progressive reduction of the difference in GSIS between NOX2-KO and WT islets. After prolonged culture at G30, cytosolic thiol oxidation and H2O2 concentration increased in parallel with β-cell apoptosis, the glucose sensitivity of the NAD(P)H, [Ca2+]i and insulin secretion responses increased, the maximal [Ca2+]i response decreased, but maximal GSIS (at G15 and G30) was preserved. These responses were almost identical in both islet types. In conclusion, NOX2 is confirmed as a negative regulator of GSIS in C57BL/6J mouse islets, but it does not detectably contribute to the long-term glucotoxic alterations of β-cell function and survival in vitro.
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Souza, A., Santos, L. R., Roma, L. P., Carpinelli, A. R., & Jonas, J.-C. (2016). Lack Of NADPH Oxidase-2 Does Not Improve Pancreatic β-cell Survival And Function In Male C57BL/6J Mouse Islets Cultured For 3 Weeks At High Glucose. Diabetes, 65 Suppl. 1, LB86. https://hdl.handle.net/2078.5/186355 (Original work published 2016)