Screening for insulin-independent pathways that modulate glucose homeostasis identifies androgen receptor antagonists
- Max Planck Institute for Heart and Lung Research, Germany
- CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Austria
- German Centre for Cardiovascular Research, Germany
Figures
Figure 1 with 1 supplement
insulin is crucial for zebrafish metabolic homeostasis.
(A) CRISPR target site in the insulin gene, with PAM sequence highlighted, and the resulting 16 bp deletion allele (below). (A’) Schematic of wild-type Insulin protein and the predicted mutant …
Figure 1—figure supplement 1
ins, and not insb, is the predominant paralog expressed in zebrafish pancreatic islets.
(A) Brightfield images of 72 hpf wild-type, ins mutant and insb mutant larvae. (B–C) Confocal plane images of the pancreatic islet in 72 hpf wild-type and insb mutant larvae stained for Insulin …
Figure 2 with 1 supplement
Highly efficient endoderm transplant technique rescues ins mutants to adulthood.
(A) Schematic depicting the endoderm transplantation protocol; sox32 mRNA-injected ins +/+ donor cells (orange) were transplanted into host embryos (blue) to form chimeric animals. (B–B’’) Confocal …
Figure 2—figure supplement 1
sox32 mRNA-injected cells contribute to host endoderm upon transplantation.
(A–A”) Confocal images of a 48 hpf host embryo injected with H2B-mCherry mRNA (magenta) and transplanted with Tg(sox17:GFP) expressing donor endoderm showing the chimeric islet (yellow arrowhead). (B…
Figure 3 with 1 supplement
Small molecule screen in ins mutants reveals insulin-independent modulators of glucose metabolism.
(A) Relative glucose levels in 120 hpf ins mutant larvae after 36 hr of treatment with anti-diabetic drugs (dapagliflozin, pioglitazone, metformin) or reported insulin mimetics (MLR1023, Fraxidin) …
Figure 3—figure supplement 1
A 96-well plate-adapted protocol to measure glucose levels is suitable for small molecule screening.
(A) Plotting the measured glucose levels interpolated from the regular standard curve (blue) shows a shifted curve in the presence of lysis buffer (orange) with a dynamic range still suitable for …
Figure 4 with 1 supplement
Androgen receptor (AR) antagonism reduces glucose levels in hyperglycemic larvae and adults.
(A) Relative glucose levels in ins mutants at 120 hpf upon treatment with various AR antagonists starting at 84 hpf; mean ± SEM, n = 3–7 replicates. (B) Glucose levels in 72 hpf ins mutants after …
Figure 4—figure supplement 1
Flutamide reduces glucose levels in a dose-dependent manner, possibly exerting its effects through liver gluconeogenic enzymes.
(A) Dose response curve of the glucose lowering effect of flutamide in ins mutant larvae, treated from 84 to 120 hpf; mean ± SEM, n = 3 replicates. (B) Wholemount in situ hybridization for ar …
Tables
Key resources table
| Reagent type or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Danio rerio) | insbns102 | This paper | 16 bp deletion allele of ins (Gene ID: 30262) | |
| Antibody | α-Insulin (Guinea Pig Polyclonal) | Dako | A0564 | (1:300) |
| Sequence-based reagent | sox32 | Kikuchi et al., 2001 | (RNA) | |
| Commercial assay or kit | mMessage mMachine SP6 Transcription Kit | ThermoFisher | AM1340 | |
| Commercial assay or kit | Glucose assay kit | Merck | CBA086 | |
| Software, algorithm | ZEN Blue 2012 | Zeiss, Germany | ||
| Software, algorithm | GraphPad Prism 7 | GraphPad Software, California |
Additional files
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Supplementary file 1
List of proteins with Log2FC > 1 or Log2FC < −1 from proteomic analyses comparing 120 hpf ins mutant and wild-type animals.
- https://doi.org/10.7554/eLife.42209.011
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Transparent reporting form
- https://doi.org/10.7554/eLife.42209.012
