Fig. 4

Fig. 4

Different levels of H₂O₂ induce beta-cell proliferation and quiescence. (A) Quantification of the number of Tg(ins:FUCCI-S/G2/M)⁺ beta-cells after continuous treatment with increasing concentrations of H₂O₂ from 3 to 4 dpf. The treatment with 50 μM H₂O₂ (n = 89 larvae) increased the number of Tg(ins:FUCCI-S/G2/M)⁺ beta-cells compared to controls (n = 104 larvae) (p = 0.025) whereas the treatments with 500 and 1000 μM H₂O₂ decreased the number of Tg(ins:FUCCI-S/G2/M)⁺ beta-cells (n = 38 larvae for 500 H₂O₂ μM; n = 63 larvae for controls; p = 0.03) (n = 46 larvae for 1000 μM H₂O₂; n = 78 larvae for controls; p = 0.0004). (B) Quantification of the number of Tg(ins:FUCCI-S/G2/M)⁺ beta-cells in 3 dpf larvae treated with increasing concentrations of H₂O₂ for 1.5 hours followed by a wash-out. The number of Tg(ins:FUCCI-S/G2/M)⁺ beta-cells was examined 24 hours after the initiation of the treatment. The short treatment with 200 and 500 μM H₂O₂ increased the number of Tg(ins:FUCCI-S/G2/M)⁺ beta-cells compared to controls (n = 51 larvae for 200 μM H₂O₂; n = 86 larvae for controls; p = 0.02) (n = 68 larvae for 500 μM H₂O₂; n = 96 for larvae for controls; p = 0.02). (C) Confocal projections of the principal islets of 4 dpf Tg(ins:H2B-GFP; ins:dsRED) larvae treated from 3 to 4 dpf with 50 μM H₂O₂. (D) Quantification of the average number of beta-cells marked with H2B-GFP in controls and H₂O₂-treated larvae. H₂O₂-treatement increased beta-cell number (n = 30 larvae) compared to controls (n = 26 larvae) (p = 0,046). Error bars = SEM. (E) Bar chart showing the increase in the number of INS-1 cells after H₂O₂ or NAC-treatments. Data from four independent experiments and > 3.5 × 10⁵ cells per condition were normalized to the mean of control treated cells ( = 100%) and analyzed for significance using a one-way ANOVA with Dunnett’s multiple comparison test. Error bars indicate S.E.M. Cartoons with permission by Luis Delgadillo.