Fig. 6.

Overnutrition-induced β-cell differentiation requires existing β-cells. A: schematic representation of the transgene Tg(−1.2ins:htBid^TE-ON; LR) used to inducibly ablate insulin-expressing cells in the presence of doxycycline and tebufenozide. In the transgene, the proapoptotic gene truncated BID (tBID) is under the control of a TRE-based promoter that is activated by a TE-ON driven by the zebrafish insulin promoter. An α-crystalline-driven tagRFP was used to mark transgene carriers. B: Tg(−1.2ins:H2BmCherry) (Control) or double transgenic Tg(−1.2ins:htBid^TE-ON; LR); Tg(−1.2ins:H2BmCherry) (Ablation) larvae were incubated for 48 h with doxycycline and tebufenozide or drug-free medium and allowed to recover for 40 h in drug-free medium. Islets were then imaged with equivalent imaging parameters for β-cells. Note the mCherry+ acellular debris (arrows) that remained 48 h after drug washout. The images are confocal projections, and scale bars indicate 10 μm. C: total free glucose level of 6 dpf Tg(−1.2ins:H2BmCherry) (tBID−) and Tg(−1.2ins:htBid^TE-ON; LR); Tg(−1.2ins:H2BmCherry) (tBID+) larvae treated with or without doxycycline and tebufenozide. Significantly increased free glucose levels were observed in Dox + Tbf-induced Tg(−1.2ins:htBid^TE-ON; LR); Tg(−1.2ins:H2BmCherry) larvae. D: Dox- and Tbf-treated 6 dpf Tg(−1.2ins:H2BmCherry) (−) and double-transgenic Tg(−1.2ins:htBid^TE-ON; LR); Tg(−1.2ins:H2BmCherry) (+), and control double-transgenic Tg(−1.2ins:htBid^TE-ON; LR); Tg(−1.2ins:H2BmCherry) (+) larvae were cultured in 5% egg yolk or nutrient-free medium for 8 h. Overnutrition did not significantly affect β-cell number following β-cell ablation in unfed and yolk-fed double-transgenic larvae, but control animals responded normally. All values are means ± SE; n are shown inside of the bars. Groups labeled with different letters are significantly different from each other (P < 0.05).