ZFIN Figure: Andersson et al., 2012, Fig. S3

Fig. S3

NECA-enhanced β cell proliferation is not modified by elevated glucose levels and affects both young and older β cells, related to Figure 3.

(A-D) Because of the prominent effect of glucose on β cell proliferation, we tested how NECA interacts with hyperglycemia. To this end, we made use of Formoterol, a -2-adrenoceptor agonist that is known to generate drug-induced hyperglycemia in humans. Tg(ins:GFP) larvae were treated with DMSO/NECA and EdU in the absence or presence of Formoterol from 4-6 dpf to assess the effect of glucose on EdU incorporation in β cells during normal development. (A) Free glucose levels substantially increased after Formoterol treatment from 4-6 dpf, whereas NECA only had a marginal effect on free glucose levels. (B-C) Confocal images of Tg(ins:GFP) larvae treated with NECA and EdU in the absence (B) or presence (C) of Formoterol. β cells that incorporated EdU are indicated by arrows. (D) The number of β cells that incorporated EdU per larva following DMSO or NECA treatment did not change significantly in the presence of Formoterol. n = 10 larvae per group.

(E-H) To test if newly formed β cells were more responsive to NECA than slightly older ones, we examined proliferation and neogenesis of β cells simultaneously. To monitor neogenesis of β cells, we made use of Tg(ins:dsRed);Tg(ins:CFP-NTR) animals. In these larvae, β cells that are older than 24 hours are labeled with both Tg(ins:dsRed) and Tg(ins:CFP-NTR) (we visualized CFP by immunohistochemisty in green, and overlap of green and red is shown in yellow). In contrast, because the dsRed fluorescent protein is slow-folding, β cells younger than 24 hours are labeled only by Tg(ins:CFP-NTR) expression (green). By treating Tg(ins:dsRed);Tg(ins:CFP-NTR) larvae with MTZ from 3-4 dpf, DMSO or NECA from 4-6 dpf, and EdU from 5-6 dpf, we could determine whether the β cells that proliferated during the second day of regeneration were actually formed by β cell neogenesis during the first (4-5 dpf) or second (5-6 dpf) day of regeneration. (E) Schema for β cell ablation and treatment with DMSO or NECA and EdU. (F and G) Confocal images of Tg(ins:dsRed);Tg(ins:CFP-NTR) larvae showing EdU incorporation in β cells formed during 4-5 dpf (yellow cells with white nuclei; arrow) and 5-6 dpf (green cells with blue nuclei; arrowhead). (H) Percentage of β cells formed during 4-5 dpf (green bars) or 5-6 dpf (yellow bars) that incorporated EdU during 5-6 dpf in the presence of DMSO or NECA; these data show that NECA enhanced the proliferation of both new (green) and older (yellow) β cells. n = 14-18 larvae per group. Error bars represent SEM.

Expression Data

Expression Detail

Antibody Labeling

Phenotype Data

Phenotype Detail

Acknowledgments

This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image.

Reprinted from Cell Metabolism, 15(6), Andersson, O., Adams, B.A., Yoo, D., Ellis, G.C., Gut, P., Anderson, R.M., German, M.S., and Stainier, D.Y., Adenosine Signaling Promotes Regeneration of Pancreatic beta Cells In Vivo, 885-894, Copyright (2012) with permission from Elsevier. Full text @ Cell Metab.