Artemisinins Target GABAA Receptor Signaling and Impair α Cell Identity

Abstract

Type 1 diabetes is characterized by the destruction of pancreatic β cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional β-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABA_A receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic β cell mass from α cells.

Keywords: ARX translocation; GABA-receptor signaling; artemisinins; chemical biology; diabetes; gephyrin; insulin secretion; pancreatic endocrine transdifferentiation; regenerative medicine; β cell.

Graphical abstract

Figure 1

A Cell-Line Model of Transcription-Factor-Mediated Transdifferentiation for Identifying Functional ARX Inhibitors (A) Representative images of Min6-ARX cells stained for insulin and the Myc-tag present on the ARX overexpression construct with and without induction with 1 μg/ml doxycycline for 72 hr. Scale bars, 5 μm. (B) Quantification of insulin reduction upon ARX overexpression, detected by immunofluorescence as in (A) (data indicate means and SD from 50 wells). ^∗p < 0.001. (C) High-content screen of Min6-ARX cells concomitantly treated with 1 μg/mL doxycycline and 10 μM compound for 72 hr, analyzed for cell number and insulin protein levels by immunofluorescence. Means and SD for DMSO-treated wells are indicated in red. (D) Validation of active compounds from the primary screen in αTC1 cells, analyzed for cell number and insulin protein levels by immunofluorescence following compound treatment for 72 hr. (E) Insulin immunofluorescence in αTC1 cells treated with 10 μM artemether or control DMSO for 72 hr. Scale bars, 5 μm. (F) Quantitative proteome analysis of αTC1 cells treated with 10 μM artemether for 72 hr compared to control DMSO-treated cells. (G) Western blot for glucagon in αTC1 cells treated with increasing concentrations of artemether for 72 hr. (H) Immunofluorescence staining for the Myc-tag on overexpressed ARX in Min6-ARX cells. Cells were induced with 1 μg/mL doxycycline for 24 hr and concomitantly treated with either 10 μM artemether or control DMSO. Scale bars, 5 μm. (I) Quantification of the cells with higher nuclear/cytoplasmic ARX staining ratio from immunofluorescence images as in (H). (J) Western blot for nuclear ARX in cells treated as in (H). See also Figures S1 and S2.

Figure 2

Artemether Induces Insulin Expression in α Cells by Targeting Gephyrin (A) Chemical proteomics to identify proteins that specifically bind to artemisinins. (B) Volcano plot of interacting proteins identified by chemical proteomics. Both p values and fold change were calculated based on two biological replicates. (C) Western blot for gephyrin in αTC1 cells treated with increasing doses of artemether for 72 hr. (D) Immunofluorescence for insulin and gephyrin in αTC1 cells treated with artemether and/or shGephyrin. Scale bars, 25 μm. shRNA treatment was for 16 hr, followed by selection with 4 μg/mL puromycin for 3 days and artemether treatment at a concentration of 10 μM for 3 days. (E) Quantification of insulin and gephyrin intensity in single cells from (D). See also Figure S3.

Figure 3

Artemether Increases GABA-Receptor Signaling in α Cells (A) Western blot for GABA-receptor subunits in αTC1 cells treated with 1 μM artemether for 72 hr. (B) Representative immunofluorescence images of αTC1 cells treated with 10 μM artemether or control DMSO for 72 hr. Scale bars, 5 μm. (C) GO term enrichment analysis of RNA-seq data from αTC1 cells treated with 10 μM artemether versus control DMSO for 72 hr. (D) Representative calcium responses to depolarizing solutions containing 15 and 50 mM KCl of αTC1 cells pre-treated with 10 μM artemether or control DMSO for 72 hr. (E) Basal Fura-2 ratio levels (F340/F380) in αTC1 cells treated with 10 μM artemether versus control DMSO for 72 hr (boxplots show 10^th, 25^th, 50^th, 75^th, and 90^th percentiles). ^∗∗∗p < 0.001, Mann-Whitney rank-sum test. (F) Percentage of αTC1 cells generating detectable calcium responses upon 15 mM KCl containing solution (mean ± SEM). ^∗p < 0.05 (n = 6 + 6). (G) Average calcium responses to the solution containing 50 mM KCl (mean ± 99% confidence interval). (H) Dot-density plots of mean Δratio level after stimulation by 15 mM KCl application without or with 100 μM muscimol in αTC1 cells pre-treated with 10 μM artemether alone, 6.25 μM picrotoxin alone, or the combination of the two for 72 hr. ^∗∗∗p < 0.001, Wilcoxon signed-rank test. (I) Intracellular chloride intensity in αTC1 cells treated with 10 μM artemether for 72 hr by live-cell fluorescence imaging (n = 3). ^∗p < 0.001. Error bars represent mean ± SD. (J) Quantification of insulin intensity in αTC1 cells treated with GABA-receptor antagonist gabazine (10 μM, n = 6), picrotoxin (6.25 μM, n = 4), or salicylidene salycylhydrazide (5 μM, n = 4), alone or in combination with 10 μM artemether for 72 hr (data indicate means and SD). ^∗p = 0.006; ^∗∗p < 0.0001. (K) Quantification of insulin intensity in αTC1 cells treated with 10 μM artemether or GABA-receptor agonist etifoxine (12.5 μM), muscimol (50 μM), vigabatrin (100 μM), halothane (20 μM), NS11394 (10 μM), zolpidem (6.25 μM), or gaboxadol (200 μM) for 72 hr (data indicate means and SD; n = 4). ^∗p < 0.05; ^∗∗p < 0.005. See also Figure S4.

Figure 4

Hormone Secretion Controls Pancreatic Cell-type Stability (A) ELISA for secreted glucagon in αTC1 cells treated with 10 μM artemether for 72 hr (n = 4). ^∗p < 0.05. Error bars represent mean ± SD. (B) Immunofluorescence quantification of insulin intensity in αTC1 cells with treated with 10 μM artemether and 10 ng/ml glucagon for 72 hr (n = 4). ^∗p < 0.001. Error bars represent mean ± SD. (C) Representative images of insulin and glucagon immunofluorescence in αTC1 cells treated with 10 μM prohormone convertase inhibitor (PCi) for 72 hr. Scale bars, 5 μm. (D) qRT-PCR of islet genes in αTC1 cells following treatment with 10 μM PCi for 72 hr. All data were normalized to DMSO control (n = 3). ^∗p = 0.010; ^∗∗p = 0.019; ^∗∗∗p = 0.002; ^∗∗∗∗p < 0.001. Error bars represent mean ± SD.

Figure 5

Artemether Increases β Cell Mass In Vivo (A) Representative images of insulin reporter cells in zebrafish larvae. β cells in ins:caspase8;ins:E2Crimson embryos were ablated by treatment with 2 μM dimerizer AP20187 from 3 dpf to 5 dpf. Then, larvae were treated with 5 μM artemether or control DMSO for 4 days (8–12 dpf) and immunostained for dsRed and DAPI. Scale bar, 15 μm. (B) Quantification of numbers of insulin reporter cells in zebrafish larvae described in (A). Shown are cell numbers from individual animals, and the means and SD are indicated for the three groups. ^∗p = 0.0005. (C) Glucose measurement in pooled larvae extracts, treated as in (A) and normalized to non-ablated animals (means and SD are indicated for at least four independent larvae pools for each condition). (D) Representative staining for insulin and glucagon in mouse pancreas following a 3-month treatment with 1 mg/mL artesunate in drinking water versus control DMSO. Scale bar, 30 μm. (E) Quantification of islets size in sections from mouse pancreata following a 3-month treatment with artesunate in drinking water (120–180 islets per animal). ^∗p < 0.001. Shown are medians, 25%–75% (boxes) and 10%–90% confidence intervals. (F) Co-staining of RFP and insulin in RFP-labeled lineage-tracing mouse islets after treatment with 10 μM artemether or control DMSO for 24 hr. Scale bar, 10 μm. (G) Quantification of RFP/insulin double-positive cells in mouse islet from (F) (means and SD are indicated from two replicates). ^∗p = 0.019. (H) Blood glucose levels after an overnight fast in a rat β cell ablation model. β cells were ablated with 60 mg/kg streptozotocin; on day 9 post-ablation, animals were assigned to treatment and control groups based on matching fasting glucose levels and were treated with 20 mg/kg artemether p.o. for 7 days followed by 200 mg/kg artemether p.o. for 16 days. (Measured values in individual animals, means and SD, n = 10 per group, p = 0.005). (I) Oral glucose tolerance test of animals described in (H). Means and SE are indicated for ten animals from each group. (J) Area under the curve from the oral glucose tolerance test described in (I). Shown are measurements in individual animals, and the means and SD are indicated for the groups; p = 0.004. See also Figure S5.

Figure 6

Artemether Affects α Cell Identity in Human α Cells (A) Representative immunofluorescence images of sectioned human islets treated with 10 μM artemether and control DMSO for 3 hr, co-stained for nuclei (DAPI), ARX, glucagon, or C-peptide. Scale bars, 100 μm. (B) Representative images of dissociated human islet cells stained for ARX after treatment with 10 μM artemether and/or 6.25 μM picrotoxin for 3 hr. Scale bars, 5 μm. (C) Quantification of ARX localization from human islets cells treated as in (B) (data indicate means and SD from six imaged wells). Boxes indicate 25^th and 75^th percentiles, horizontal lines medians, and whiskers minima and maxima. (D) Multidimensional scaling plot representing transcriptomes of single human islet cells treated as intact islets with either control DMSO (gray circles) or 10 μM artemether (black circles) for 24 hr or 72 hr. (E) Expression pattern of GABA-receptor subunits in human α cells from (D). (F) Immunofluorescence staining of dissociated human pancreatic islet cells for nuclei (DAPI), gephyrin, and GABRG2 receptor treated as intact islets with either control DMSO or 10 μM artemether for 72 hr. Scale bar, 10 μm. (G) Gene set enrichment analysis using a gene set representing all genes significantly higher expressed in human α cells compared to β cells, comparing cumulative expression levels from single α cell transcriptomes obtained from islets treated with control DMSO or 10 μM artemether for 72 hr. (H) Cumulative expression levels from single α or β cell transcriptomes obtained from islets treated with control DMSO or 10 μM artemether for 72 hr. See also Figure S6 and Table S1.

Figure 7

Artemether Enhances Insulin Secretion in Human Islets (A) RT-qPCR assay for ARX expression in human islets treated with control DMSO or 10 μM artemether for 72 hr. Error bars represent mean ± SD. (B) Measurement of intracellular insulin content in human islets treated with control DMSO or 10 μM artemether for 72 hr. Error bars represent mean ± SD. (C) Measurement of glucose-stimulated insulin secretion in human islets treated with control DMSO or 10 μM artemether for 72 hr (two replicates each donor; ^∗∗p < 0.005; ^∗p ≤ 0.05). Error bars represent mean ± SD. (D) Proposed mechanism of action of artemether. By stabilizing gephyrin, artemether increases GABA-receptor signaling in α cells, thereby preventing glucagon secretion. Decreased extracellular glucagon concentration induces loss of α cell identity and increase of insulin expression. See also Figure S6.

Figure S1

A Min6 Cell Line for Inducible ARX Overexpression, Related to Figure 1 (A) Western blot for the Myc-tag in three clonal Min6 cell lines allowing inducible overexpression of Myc-ARX. Myc-ARX expression was induced with 1 μM doxycycline for 16 hr. Histone H2B or tubulin was used as a loading control. (B) Western blot for the ARX as in (A). (C) Gene expression after 24 hr ARX induction. (D) Time course of gene expression after ARX induction for 3 and 6 days. (E) Relative gene expression changes in ARX inducible clones after 24 hr ARX induction.

Figure S2

Structure Activity Relationships of Artemisinins, Related to Figure 1 (A) Artemether dose-dependence of insulin protein maintenance quantified by immunofluorescence in induced Min6-ARX cells treated for 72 hr (Mean and SD). (B) Artemether dose-dependence of insulin protein induction quantified by immunofluorescence in αTC1 cells treated for 72 hr. (C) Insulin intensity in αTC1 cells following 72 hr treatment with artemisinin analogs at concentrations achieving maximum insulin induction: DMSO (0), 0.27 μM Artesunate (1), 4.5 μM Deoxyarteether (2), 2.5 μM Arteether (3), 0.17 μM N-hydroxy-11-azaartemisinin (4), 7.5 μM artemistene (5), 0.1 μM artemisone (6), 2.5 μM artemisiten (7) and 67.5 μM anhydro dihydro artemisinin (8) (Mean and SD from 3 wells). (D) Quantification of insulin intensity in αTC1 cells following treatment with the ROS inducer elesclomol for 72 hr. (Mean and SD from 4 wells). (E) Quantification of insulin intensity in αTC1 cells following treatment with artemether in combination with N-Acetyl-Cysteine for 72 hr (^∗p = 0.034). All panels show mean and SD from three biological replicates.

Figure S3

Artemether Increases Gephyrin Protein Levels and GABA-Receptor Signaling, Related to Figure 2 (A) Quantification of insulin intensity in αTC1 cells following treatment with SERCA inhibitor for 72 hr (n.s.,p > 0.05). Error bars represent mean ± SD. (B) Western blot for p-Akt and Akt of lysates from αTC1 cells treated with multiple doses of artemether for 72 hr. (C) Measurement of PI3P in αTC1 cells treated with artemether for 72 hr (^∗p = 0.007; ^∗∗p = 0.002). Error bars represent mean ± SD. (D) List of all significantly enriched proteins identified by chemical proteomics in artesunate pull-downs. (E) Immunofluorescence staining for gephyrin in α cells treated with 10 μM artemether for 72 hr (scale bar 25 μm). (F) Expression of GABA-receptor subunits in αTC1 cells treated with artemether for 72 hr detected by RNA-seq, measured as RPKM (Reads Per Kilobase of transcript per Million mapped reads). Error bars represent mean ± SD. (G) Costaining of αTC1 cells treated with 10 μM fluorescently labeled artelinic acid for 2 hr for gephyrin (scale bar 10 μm). (H) Western blot for validating gephyrin knockdown by shRNA. αTC1 cells were infected with shGephyrin, selected with 4 μg/ml puromycin and lysed after 7 days. Ponceau S staining is used as a loading control.

Figure S4

Artemether Effect on Gephyrin Biology, Related to Figure 3 (A) Moco synthetase activity concentration in αTC1 cell treated with artemether for 72 hr (n = 3 ^∗p = 0.04). Error bars represent mean ± SD. (B) mTOR pull-down analyzed for interaction with gephyrin by western blot in αTC1 cells treated with control DMSO or 10 μM artemether for 72 hr. (C) Expression change of genes involved in synaptic transmission in αTC1 cells treated with 10 μM artemether for 72 hr. (D) Basal nuclear Fura-2 ratio levels (F340/F380) in αTC1 cells treated with 10 μM artemether versus control DMSO for 72 hr (boxplots showing 10^th, 25^th, 50^th, 75^th and 90^th percentiles; ^∗∗∗p < 0.001, Mann-Whitney rank sum test). (E) Basal cytoplasmic Fura-2 ratio levels (F340/F380) in αTC1 cells treated with 10 μM artemether versus control DMSO for 72 hr (boxplots showing 10^th, 25^th, 50^th, 75^th and 90^th percentiles; ^∗∗∗p < 0.001, Mann-Whitney rank sum test). (F) Analysis of chloride changes upon muscimol (100 μM) application in αTC1 cells treated with 10 μM artemether versus control DMSO for 72 hr (data presented as boxplots). (G) Voltage-clamp current recordings upon 100 ms polarization steps in control αTC1 cells in pseudophysiological conditions with K-gluconate in the patch pipette (left panel) and with CsCl-based internal solution (right panel); pink traces show the presence of voltage-gated slowly inactivated inward currents. (H) Current clamp recordings in αTC1 cells; the representative traces demonstrate similar “firing” properties upon 5 pA current injection (1 s). (I) Quantification of insulin intensity in αTC1 cells treated with GABA-receptor antagonists bicuculline (10 μM; ^∗∗∗p = 0.0001), furosemide (12.5 μM; ^∗∗p = 0.0014) or FG-7142 (50 μM; ^∗p = 0.043) alone or in combination with 10 μM artemether for 72 hr (Mean and SD from 4 wells).

Figure S5

Effect of Artemether on Pancreatic Islets In Vivo, Related to Figure 5 (A) Islet morphology and GFP expression as marker of α cells of Tg(Gcga:GFP)^ia1 zebrafish larvae treated from 26 hpf until 100 hpf with artemether. (B) Quantification of Glucagon-GFP positive cells and Insulin-mCherry positive cells per single zebrafish islets (8-10 fish per condition; ^∗p = 0.007, ^∗∗p = 0.088, ^∗∗∗p = 0.004, ^∗∗∗∗p = 0.003). Error bars represent mean ± SD. (C) Immunofluorescence for Sst, Pp, and BrdU co-stained with insulin or glucagon in mouse pancreas with or without artemether treatment.

Figure S6

Artemether-Induced Insulin/Glucagon Double-Positive Cells in Human Pancreatic Islets, Related to Figure 6 (A) Detection of insulin/glucagon double positive cells following treatment of intact human islets with 10 μM artemether for 72 hr (scale bar 10 μm). (B) Quantification of cell fractions in human islets treated with 10 μM artemether for 72 hr by individual donor. (C) Summary of data in B. (^∗p < 0.001, n = 5). Error bars represent mean ± SD.

Figure S7

Artemether Alters Human Pancreatic Islet Transcriptomes, Related to Figure 6 (A) GSEA in bulk human islet samples treated with 10 μM artemether or 10 mM GABA for 72 hr. (B) Overlap between the gene set upregulated by both GABA and artemether (core-upregulation) and exocrine or endocrine gene sets. Fisher’s exact test is used for the significance test. (C) GSEA for α cell specific genes in single human β cells from islets treated with artemether. (D) Repression of β cell genes in artmether treated beta cells. (E) Induction of expression of GNAS in human α cells after treatment of intact islets with 10 μM artemether for 72 hr. (F) Induction of expression of ABCC8 in human α cells after treatment of intact islets with 10 μM artemether for 72 hr.