Protection of insulin-secreting INS-1 cells against oxidative stress through adenoviral-mediated glutathione peroxidase overexpression.
OBJECTIVE
A large fraction of an islet graft can be lost early following allotransplantation from various non specific mechanisms including oxidative stress. Overexpression of antioxidant enzymes could confer a beneficial effect on islets exposed to reactive oxygen and nitrogen species. We examined the viability of beta cells driven to overexpress glutathione peroxidase (GPx) and exposed to a superoxide donor (hypoxanthine/xanthine oxidase HX/XO) and a nitric oxide donor (3-morpholinosydnonimine SIN-1).


METHODS
Cultured INS-1 rat-derived insulin-secreting cells were transfected by an E1-deleted adenovirus carrying GPx cDNA (AdGPx). Additional experiments were performed with an adenovector carrying Cu/Zn superoxide dismutase cDNA (AdSOD). Cellular viability was tested by the WST-1 colorimetric assay and functionality by static incubation.


RESULTS
AdGPx increased GPx activity within 48 hours from 0 (untransfected cells) to 60 +/- 11 U/g (cells transfected at an MOI of 25: 1). GPx overexpression significantly reduced cytotoxicity induced by HX/XO from 10.81 +/- 1.41 to 5.42 +/- 2.62% at 10 mU/ml and from 61.19 +/- 4.17 to 52.9 +/- 4.39% at 20 mU/ml (p=0.0002, transfected cells vs control cells). Doses of SIN-1 from 600 to 1000 micromol/l resulted in cytotoxicity ranging from 17.66 +/- 3.48 to 45.97 +/- 6.48% in control cells and from 5.65 +/- 1.37 to 35.80 +/- 5.59% in AdGPx transfected cells (p=0.015). The combination of AdGPx and AdSOD did not exhibit any synergistic cytoprotective effect. Control cells exposed to a HX/XO stress exhibited a reduction in glucose-theophylline stimulated insulin secretion by half, while stressed GPx overexpressing-cells maintained the same insulin secretion level than non-stressed cells.


CONCLUSIONS
Adenoviral-induced overexpression of GPx enhances the resistance of a rat beta cell line to both reactive oxygen (ROS) and reactive nitrogen species (RNS) cytotoxicity. Transposition of these findings to human islet transplantation with a clinically-relevant procedure deserves further investigations.
Department of Cell Therapy, University Hospital, Grenoble, France.
C
C
Moriscot
M J
MJ
Richard
M C
MC
Favrot
P Y
PY
Benhamou
D000256:Adenoviridae; D000818:Animals; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects*; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D005979:Glutathione Peroxidase / Q000235:genetics* / Q000378:metabolism; D019271:Hypoxanthine / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007340:Insulinoma; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000494:pharmacology; D018384:Oxidative Stress / Q000502:physiology*; D010190:Pancreatic Neoplasms; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D014969:Xanthine Oxidase / Q000494:pharmacology
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D007328:Insulin; D011994:Recombinant Proteins; D019271:Hypoxanthine; C002385:linsidomine; D008981:Molsidomine; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D005947:Glucose