Uncoupling protein-2 participates in cellular defense against oxidative stress in clonal beta-cells

The role of uncoupling protein-2 (UCP-2) in beta -cells is presently unclea r. We have tested the notion that UCP-2 participates in beta -cell defense against oxidants. Expression of the UCP-2 gene in clonal beta -cells (INS-1 ) was decreased by 45% after 48 h of culture with vitamin E and selenite. W hen INS-1 cells were exposed to 200 muM H2O2 for 5 min, the cell viability (MTT assay) decreased to 85 +/- 1, 61 +/- 1, 40 +/- 2, and 39 +/- 2% of con trol when measured respectively 30 min, 2 h, 6 h, and 16 h after H2O2 expos ure. At corresponding time points UCP-2 mRNA levels were 1.01 +/- 0.09, 1.5 3 +/- 0.15 (P < 0.05), 1.44 +/- 0.18 (P = 0.06), and 1.12 +/- 0,09 fold of control, i.e., transiently increased. We next tested whether overexpression of UCP-2 could enhance resistance of <beta>-cells toward H2O2 toxicity. A cotransfection method using EGFP as a suitable marker and a human cDNA UCP- 2 construct was used for transient overexpression of UCP-2, Transfected cel ls expressed the gene about 30-fold more than normal cells. After exposure to H2O2 (200 mum, 5 min), the survival of UCP-2 overexpressing cells was me asured 30-45 min later by flow cytometry. Survival was 13 +/- 0.05% higher than control (EGFP only) cells, P < 0.004 for difference. The results indic ate that oxidative stress induces UCP-2 expression in <beta>-cells, and tha t UCP-2 serves a role in beta -cell defense against oxidative stress. (C) 2 001 Academic Press.