NADPH oxidase-2 does not contribute to β-cell glucotoxicity in cultured pancreatic islets from C57BL/6J mice.
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+]iand 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+]iand insulin secretion responses increased, the maximal [Ca2+]iresponse 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.
Université catholique de Louvain, Institute of Experimental and Clinical Research, Pole of Endocrinology, Diabetes and Nutrition, Brussels, Belgium; Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
Arnaldo H
AH
de Souza
Université catholique de Louvain, Institute of Experimental and Clinical Research, Pole of Endocrinology, Diabetes and Nutrition, Brussels, Belgium.
Laila R B
LRB
Santos
Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
Leticia P
LP
Roma
Université catholique de Louvain, Institute of Experimental and Clinical Research, Pole of Endocrinology, Diabetes and Nutrition, Brussels, Belgium.
Mohammed
M
Bensellam
Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
Angelo R
AR
Carpinelli
Université catholique de Louvain, Institute of Experimental and Clinical Research, Pole of Endocrinology, Diabetes and Nutrition, Brussels, Belgium. Electronic address: jean-christophe.jonas@uclouvain.be.
Jean-Christophe
JC
Jonas
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003600:Cytosol / Q000378:metabolism; D005947:Glucose / Q000633:toxicity*; D051273:Glucose Transporter Type 2 / Q000235:genetics / Q000378:metabolism; D054477:Glutaredoxins / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051547:Heme Oxygenase-1 / Q000235:genetics / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology*; D008297:Male; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D000074662:NADPH Oxidase 2 / Q000172:deficiency / Q000378:metabolism*; D010084:Oxidation-Reduction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism; D046509:Tissue Culture Techniques
C516006:Glrx protein, mouse; D051273:Glucose Transporter Type 2; D054477:Glutaredoxins; D007328:Insulin; D012333:RNA, Messenger; D013438:Sulfhydryl Compounds; D049452:Green Fluorescent Proteins; D051547:Heme Oxygenase-1; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D005978:Glutathione; D005947:Glucose
Apoptosis; Insulin secretion; NOX2; Pancreatic β-cell; roGFP sensors