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. 2018 Oct 22;8:15587. doi: 10.1038/s41598-018-33875-0

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Suppression of the TBK1/IKKε-PDE3 signaling axis promotes β-cell proliferation by increasing cAMP levels and mTOR activity. (A) Schematic of the TBK1/IKKε-PDE3 signaling that modulates cAMP-PKA-mTOR pathway. The sites of inhibition by PIAA and cilostamide are shown in red. (B) Quantification of cAMP levels (mean ± SD) at 48 hpa (0.4 ± 0.1 pmol/larva (DMSO) and 0.9 ± 0.0 pmol/larva (PIAA)). (C) Representative Western blot showing increased pS6K1 levels in PIAA-treated recovering larvae. (D-I’) Confocal images of [Tg(ins:CFP-NTR)^s892; Tg(ins:Kaede)^jh6] larvae at 48 hpa, concurrently treated with EdU and DMSO (D-D’), PIAA (E-E’), cilostamide (F-F’), a combination of PIAA and cilostamide (G-G’), a combination of PIAA and rapamycin (H-H’), or a combination of cilostamide and rapamycin (I-I’), respectively, from 0–48 hpa, stained for pRPS6 (blue). The number of EdU-incorporated (white arrows) and pRPS6-positive (white arrowheads) β-cells was increased in recovering larvae treated with both PIAA and cilostamide (G,G’) compared to individual compound-treated larvae (E-E’ and F-F’). Rapamycin substantially suppressed the PIAA- and cilostamide-dependent increases in the number of EdU-incorporated and pRPS6-positive β-cells (H-I’). (J) Quantification of the number (mean ± SD) of total regenerated β-cells (green bars) and regenerated β-cells that incorporated EdU with pRPS6 immunoreactivity (white bars) at 48 hpa (in D-I’; 5.0 ± 1.3 total regenerated β-cells, of which 0.3 ± 0.5 (DMSO), 17.8 ± 2.8, of which 8.4 ± 1.7 (PIAA), 12.7 ± 1.9, of which 5.0 ± 1.5 (cilostamide), 24.7 ± 1.2, of which 13.3 ± 0.6 (PIAA and cilostamide), 6.0 ± 2.0, of which 1.0 ± 1.0 (PIAA and rapamycin), and 5.2 ± 1.1, of which 0.8 ± 0.8 (cilostamide and rapamycin) incorporated EdU with pRPS6 immunoreactivity). (K) The percentage (mean ± SD) of regenerated β-cells that incorporated EdU with pRPS6 immunoreactivity at 48 hpa (in D-I’; 6.1 ± 9.5% (DMSO), 47.3 ± 7.5% (PIAA), 50.4 ± 8.8% (cilostamide), 54.2 ± 4.2% (PIAA and cilostamide), 13.9 ± 12.7% (PIAA and rapamycin), and 13.7 ± 13.0% (cilostamide and rapamycin)). Cells in 20 planes of confocal images from 10 individual larvae were counted per condition. ***P < 0.001.