First quantitative high-throughput screen in zebrafish identifies novel pathways for increasing pancreatic β-cell mass
- Johns Hopkins University, United States
- Georgia Regents University, United States
- University of Macau, China
Figures
Figure 1 with 1 supplement
Screening resources, design, and controls.
(A) Transgenic line used for the primary screen, Tg(ins:PhiYFP-2a-nsfB, sst2:tagRFP)lmc01 (β/δ reporter; Walker et al., 2012), the insulin promoter drives YFP-expression in β cells (yellow), the soma…
Figure 1—figure supplement 1
ARQiv-HTS system.
(A) Robotics-integrated ARQiv-HTS system (all units Hudson Robotics unless otherwise indicated). (1) Micro10× liquid handlers, (2) SOLO automated pipettor, (3) COPAS-XL (Complex Object Parametric Ana…
Figure 2 with 1 supplement
ARQiv data and screen flow chart.
(A–C) Example of MATLAB/R-generated real-time data plots provided for each drug plate; note, data for YFP are shown; however, plots were provided for both fluorophores. (A) Boxplots of Drug to DMSO …
Figure 2—figure supplement 1
Observation of 2o islet formation in live β/δ-reporter larvae after drug treatment.
(A, B) Representative in vivo confocal images—brightfield and fluorescence images merged—of pancreata in β/δ-reporter larvae following treatment with 0.1% DMSO (A) or a representative Hit I drug (B, …
Figure 3 with 2 supplements
Validation of endocrine differentiation induction: precocious 2° islet assay.
(A, B) Representative confocal images—brightfield and fluorescence images merged—of dissected pancreata (dashed lines) from neurod:EGFP transgenic larvae treated from 3 to 5 dpf with 0.1% DMSO (A) …
Figure 3—figure supplement 1
Validation of endocrine differentiation induction: precocious 2° islet assay (neurod reporter).
Precocious 2° islet assays were performed as per Figure 3. (A–J) Representative confocal images—brightfield and fluorescence images merged—of dissected pancreata (dashed lines) from neurod:EGFP …
Figure 3—figure supplement 2
Validation of endocrine differentiation induction: precocious 2° islet assay (pax6b reporter).
Precocious 2° islet assays were performed as per Figure 3. (A–L) Representative confocal images—brightfield and fluorescence images merged—of dissected pancreata (dashed lines) from pax6b:EGFP …
Figure 4
Validation of increased β-cell proliferation: cell counts.
(A) Quantification of β-cell numbers following incubation of ins:hmgb1-EGFP transgenic larvae from 3 to 5 dpf in one of 30 Hit compounds, 0.1% DMSO, or the Notch inhibitor RO4929097 (5 µM). 15 …
Figure 5 with 1 supplement
NF-κB pathway inhibition induces endocrine differentiation.
(A, B) Representative confocal images—brightfield and fluorescence images merged—of dissected pancreata (dashed lines) from neurod:EGFP transgenic larvae treated from 3 to 5 dpf with NF-κB signaling …
Figure 5—figure supplement 1
Thioctic acid and parthenolide inhibit NF-κB signaling.
(A–E) Confocal images of 5 dpf pancreata (dashed lines) from 6xNFκB:EGFP/Tp1:hmgb1-mCherry larvae treated with indicated compounds or DMSO control from 3dpf to 5dpf. The NF-κB reporter showed …
Figure 6 with 2 supplements
Serotonin signaling stimulates β-cell proliferation in a cell type-specific manner.
(A) β-cell quantification following 25 μM serotonin or 25 μM fluoxetine treatment of ins:hmgb1-eGFP transgenic larvae from 3 to 5 dpf indicates enhanced serotonin signaling increases β-cell numbers …
Figure 6—figure supplement 1
Amcinonide increases β-cell mass by inducing hyperglycemia.
Absolute glucose values in zebrafish larvae treated with selected lead compounds. For each drug, 3 treatments (20 embryos each treatment) were measured. The absolute glucose levels per embryo were …
Figure 6—figure supplement 2
Paroxetine injection stimulates β-cell proliferation in mice.
Paroxetine effects on β-cell proliferation in postnatal mice assayed at postnatal day 14 (P14; A–C): EdU and DMSO (A), or EdU and 1 mM paroxetine (B), were injected into mice daily from P7 to P14. (A…
Author response image 1
Tables
Table 1
Lead I drugs: inducers of endocrine differentiation
| Drug name | >2° islet # | ARQiv (µM)* | >2° islet (µM)* | |
|---|---|---|---|---|
| 1 | N-Acetylmuramic acid | +++ | 0.5 | 12.5 |
| 2 | Methylthiouracil | ++ | 0.25 | 12.5 |
| 3 | Dimethindene (S, +) maleate | ++ | 1 | 1 |
| 4 | Thioctic acid | + | 0.5 | 5 |
| 5 | Biperiden HCl | + | 1 | 12.5 |
| 6 | Parthenolide | + | 4 | 5 |
| 7 | Maprotiline | + | 0.5 | 5 |
| 8 | Estradiol diacetate → Beta-estradiol | + | 1 | 12.5 |
| 9 | Oxacillin | + | 1 | 5 |
| 10 | Benzamidine | + | 0.125 | 20 |
| 11 | Dexetimide | + | 1 | 1 |
| 12 | Decitabine | − | 0.5 | n/a |
| 13 | N-Acetylaspartic acid | − | 4 | n/a |
| 14 | Nevirapine | − | 0.25 | n/a |
| 15 | Ethynodiol diacetate | − | 0.25 | n/a |
| 16 | Vitamin K3 | − | 0.25 | n/a |
| 17 | Fosphenytoin sodium | − | 0.25 | n/a |
| 18 | Thiram | − | 0.5 | n/a |
| 19 | BOC-S-acetaminomethyl-L-cysteine→N-Acetyl-L-cysteine | − | 0.5 | n/a |
| 20 | Tretinoin | − | 0.25 | n/a |
| 21 | Iodine | nd | 0.25 | nd |
| 22 | Bayberry wax | nd | 0.25 | nd |
| 23 | 1,5-Bis (succinimidooxycarbonyloxy) pentane | nd | 0.5 | nd |
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The 23 Hit I drugs are listed. 20 were tested for induction of endocrine differentiation, that is, precocious 2° islet formation. Compounds are ordered according to the results of the validation screen, 11 drugs were confirmed as leads (++ = p < 0.01′; + = p < 0.05), 9 failed (−). *optimal response concentration for the ARQiv and validation screens. n/a: not applicable; nd: not determined, n = 5–10 larvae per condition, experiment repeated 3 times.
Table 2
Lead II drugs: increased β-cell number
| Drug name | > β-cell # | ARQiv (µM)* | > β-cell (µM)* | |
|---|---|---|---|---|
| 1 | Promethazine HCl | ++++ | 0.125 | 1 |
| 2 | Paroxetine HCl | ++++ | 4 | 1 |
| 3 | Biperiden HCl | ++++ | 1 | 12.5 |
| 4 | Decitabine | ++++ | 0.5 | 25 |
| 5 | Amitriptyline | ++++ | 2 | 1 |
| 6 | N-Acetylaspartic acid | ++++ | 4 | 1 |
| 7 | Nevirapine | ++++ | 0.25 | 25 |
| 8 | Tretinoin | ++++ | 0.25 | 1 |
| 9 | Ethynodiol diacetate | +++ | 0.25 | 1 |
| 10 | Pasiniazid | ++ | 2 | 1 |
| 11 | NCS-382 | ++ | 0.25 | 1 |
| 12 | Amcinonide | + | 0.25 | 5 |
| 13 | Thioctic acid | + | 0.5 | 5 |
| 14 | Promazine HCl | + | 4 | 10 |
| 15 | D-Gluconic acid calcium salt | + | 2 | 25 |
| 16 | Benzalkonium chloride | − | 2 | n/a |
| 17 | Chloroacetoxyquinoline | − | 0.125 | n/a |
| 18 | Acyclovir | − | 2 | n/a |
| 19 | Diphenhydramine | − | 0.5 | n/a |
| 20 | Reserpine | − | 2 | n/a |
| 21 | Resorcinol monoacetate | − | 2 | n/a |
| 22 | Khellin | − | 2 | n/a |
| 23 | Butenafine HCl | − | 0.5 | n/a |
| 24 | Phenothrin | − | 0.25 | n/a |
| 25 | Nomifensin maleate | − | 2 | n/a |
| 26 | Ethopropazine HCl | − | 1 | n/a |
| 27 | Fosphenytoin sodium | − | 0.25 | n/a |
| 28 | Thiram | − | 0.5 | n/a |
| 29 | Vitamin K3 | − | 0.25 | n/a |
| 30 | BOC-S-acetaminomethyl-L-cysteine → N-Acetyl-L-cysteine** | − | 0.5 | n/a |
| 31 | RIAA 94 | nd | 0.5 | nd |
| 32 | Trientine | nd | 1 | nd |
| 33 | Beta propiolactone | nd | 2 | nd |
| 34 | Emodic acid | nd | 0.5 | nd |
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All 23 Hit II drugs (non-shaded), as well as 2 Hit I validated compounds with high SSMD values (shaded light gray), and 9 Hit I ‘fails’ (shaded dark gray), are listed. The top 30 drugs were tested for increased β-cell numbers: 15 were validated as leads (++++ = p < 0.0001; +++ = p < 0.001; ++ = p < 0.01; + = p < 0.05), 15 failed (−). *optimal response concentration for the ARQiv and validation screens; **substituted compound due to availability issues; n/a: not applicable; nd: not determined. n = 5–10 larvae per condition, experiment repeated 2–3 times.
Additional files
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Supplementary file 1
ARQiv Hit calls. The 46 compounds implicated as Hit Calls following the ARQiv screen and initial visual assessments of enhanced 2° islet formation are listed. Compounds are ordered according to SSMD value. Hit I and Hit II subsets are indicated by a check mark in the corresponding column. In addition, Hit I compounds that were evaluated in β-cell proliferation assays alongside Hit II compounds are indicated as ‘tested’. Also listed are clinical indication, FDA approval status, or naming convention for compounds approved by FDA counterparts in other countries.
- https://doi.org/10.7554/eLife.08261.018
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Supplementary file 2
Outstanding Hit calls. The 131 compounds implicated in the primary screen but which remain to be further evaluated are listed here in subgroups according to their implicated pharmacological activity (shaded subsets). Subcategories included: neuromodulators, glucocorticoids, and retinoids—also implicated by Tsuji et al.,—as well as 11 other categories unique to our study. n/a: not applicable.
- https://doi.org/10.7554/eLife.08261.019
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Source code 1
R-based code developed for plotting sample size data.
- https://doi.org/10.7554/eLife.08261.020
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Source code 2
R-based code for processing a series of drug and control plates configured.
- https://doi.org/10.7554/eLife.08261.021
