Statistical analysis

For all experiments, two-sided t-tests were used to evaluate differences between two groups and analysis of variance (ANOVA) was used for multiple groups. To perform statistical analysis, at least three independent experiments from three different organ donors were performed for human islets; at least three independent experiments were performed for rat islets and pancreatic β-cell lines, as reported in figure legends. Data are presented as mean±s.d. For all in vivo animal studies, eight mice per group is required to reliably detect changes in glycemic control and β-cell mass based on historical data to achieve statistical significance level of α=0.05.

Animal experiments

All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Genomics Institute of the Novartis Research Foundation and strictly followed the NIH guidelines for humane treatment of animals. The double transgenic RIP-DTA male mice (Tg(Ins2-rtTA)2Efr Tg(teto-DTA)1Gfi/J; Jackson Laboratory, Stock # 008755) were bred in-house and were maintained in a 12-h light/12-dark cycle environment with controlled temperature, humidity and ventilation. Animals were housed 3–5 mice per cage and had free access to water and standard laboratory chow. To induce pancreatic β-cell ablation and hyperglycaemia, doxycycline (200 μg ml−1, Sigma-Aldrich) was provided in the drinking water for 5 days. Blood glucose levels were measured from tail-vein blood with a glucometer (AlphaTrak2 Monitoring System; Abbott). Experimental groups of 14 male mice each were randomized based on similar body weights (28.8±2.4 g), age (82±2 days), and blood glucose levels (428±96 mg dl−1). Group sizes of eight animals each are sufficient to reliably detect changes in glycaemic control and β-cell mass based on historical data with reference compounds; an additional six mice were added to each group to assess total pancreatic insulin levels. Doxycycline-induced diabetic mice were treated with either vehicle (0.5% MC/0.5% Tween-80), GNF7156 (50 mg kg−1 formulated in 0.5% MC/0.5% Tween-80, once a day) or GNF4877 (50 mg kg−1 formulated in 0.5% MC/0.5% Tween-80, twice a day). A sub-set of animals were not given doxycycline treatment and were used as a non-diabetic vehicle–control group. All compounds were administered daily via oral gavage (4 ml kg−1). Body weight and blood glucose were monitored every third day. An oral glucose tolerance test was performed after an overnight fast on day 12. For the insulin tolerance test, mice were fasted for 4 h before the administration of insulin (Novolin, Novo Nordisk, 0.7 U kg−1 body weight, i.p.). Pancreatic insulin content was determined by MSD (meso-scale discovery). Insulin ELISA (enzyme-linked immunosorbent assay) kit from whole pancreas lysate from mice (n=6) treated for 15 days with vehicle or GNF4877 (50 mg kg−1, twice daily). To determine β-cell proliferation and β-cell mass by morphological analyses, pancreata from the rest of animals (n=8) were rapidly dissected, fixed in formalin, sectioned and immunostained for insulin, Ki67 and BrdU on day 16. Male NSG mice, aged 8 weeks, were obtained from the Jackson Laboratory (Strain NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ; Stock number 005557) and housed according to IACUC guidelines. Baseline body weight and tail-vein blood glucose measurements were recorded before diabetic induction. To obtain a diabetic state, animals were given an intraperitoneal dose of STZ (135 mg kg−1) and blood glucose was monitored before transplant. Mice were grouped according to body weight and blood glucose level on the day of transplants (72 h after STZ treatment).

Transplantation experiments

Human islets (obtained commercially from Prodo Laboratories Inc.) were recovered for 48 h before transplantation. On the day of transplantation, mice were anaesthetized with a single i.p. injection of ketamine/xylazine (ketamine 75 mg kg−1; xylazine 10 mg kg−1). While mice were prepared, islets were counted according to IEQs and prepared into islet suspension of 800 IEQ/mouse in cold PBS. Islets were inserted into the renal pocket and the renal capsule was closed and the kidney was placed back into the abdominal cavity. The incision was closed and mice were given buprenorphrine and flunixin analgesics via subcutaneous injection. Sham kidneys received an equivalent volume of saline injected into the renal capsule. Mice recovered from surgery for 4 days following the transplant and body weight was monitored regularly. After 4 days, mice were given an oral dose of 0.5% MC/Tween vehicle or GNF4877 (50 mg kg−1; twice a day). In addition, mice were given an i.p. dose of BrdU (Life Technologies Catalogue number 00-0103; aqueous solution at 0.01 ml g−1 according to manufacturer instructions) once daily.

Histology

Pancreas specimens were harvested, weighed, fixed in 10% neutral buffered formalin and processed using standard paraffin processing techniques. Five micrometre serial-step sections were mounted onto Superfrost Plus slides and air-dried overnight. Samples were coded before histologic analyses resulting in blinded processing and staining. Routine hematoxylin and eosin (H&E) stains were performed on all specimens. Immunostaining was performed using the Ventana Discovery XT system. All epitope retrieval was done on the instrument using CC1 reagent. Chromogenic insulin was detected using guinea pig anti-insulin (Dako Corp A0564) at 1:3,200 and the Blue Map kit, after serum blocking and endogenous biotin blocking was performed, slides were then counterstained with Nuclear Fast Red. Fluorescent insulin and Ki67 double stains used a cocktail of the Dako anti-insulin at 1:1,600 and rabbit monoclonal Ki67 (Thermo RM9106) at 1:100, and were detected using a cocktail of Alexa Fluor 488 and 594-labelled secondary antibodies. Auto-fluorescence was quenched in red blood cells and reduced in connective tissue by treatment with 10 mM copper sulfate. Slides were counterstained with DAPI. 21 slides were collected per block. All slides were imaged on the Hamamatsu Nanozoomer and evaluated for β-cell mass and proliferation.

Automated image analysis

Automated β-cell mass calculation using MATLAB software: tissue masks were created using chromogenic insulin slides. Co-localization of islets in chromogenic and fluorescent insulin slides was identified by overlaying these two stains to verify the detection of islet regions of interest. Islet regions of interests were then mapped to chromogenic insulin slides to search for β-cell area by chromagen staining. Total tissue area was calculated from tissue mask area, whereas β-cell area is calculated from chromogenic insulin stained area for each section. β-Cell mass was calculated by multiplying the islet/pancreas area ratio by pancreas weight.

Automated evaluation of intra-islet versus extra-islet proliferation

Insulin/Ki67 stained slides were generated as described above. Rulesets were written to detect and exclude stain artifacts, tissue folds and lymph nodes from pancreas sections. Approximately 500,000 nuclei were counted per pancreas section and binned into either ‘intra-islet’ region (defined by the fluorescent insulin stain) or ‘extra-islet’ region (remaining tissue). Nuclei with Ki67 stain intensity higher than a pre-defined threshold were classified as Ki67 positive. In each region, intra- or extra-islet, numbers of Ki67-positive nuclei was then divided by total number of nuclei per region. After blinded, automated analysis was complete, images were decoded.

Cell culture and islet isolation

R7T1 β-cells were obtained from Dr S. Efrat (Tel Aviv University)49 and expanded in growth medium (DMEM with 15% horse serum and 2.5% fetal bovine serum (FBS)) in the presence of 10 μg ml−1 doxycycline. HEK293 and TM3 cells were grown according to instructions from American Type Culture Collection (www.atcc.org/). INS1E cells were cultured in RPMI containing 10 mM HEPES, 1 mM sodium pyruvate, 50 μg ml−1 β mercaptoethanol and 10% FBS. Rat islets were isolated by the standard collagenase digestion method from the pancreata of adult Sprague–Dawley rats (200–250 g) and cultured in RPMI medium (Invitrogen) with 10% FBS (Thermo Scientific). In brief, 9 ml of ice-cold Collagenase V (Sigma) solution was injected into the pancreas via the common bile duct. After dissection, the pancreas was incubated for 35 min at 37 °C and then further dissociated by repeated pipetting by using a 10-ml pipette. Islets were purified by Histopaque 1.077 (Sigma) density gradient centrifugation. Islets were allowed to recover from the isolation procedure for overnight in RPMI medium containing 10% FBS in non-tissue culture-treated petri dishes to prevent attachment. Human islets were obtained from Prodo Laboratories, Inc (Irvine, CA, USA), University of Minnesota and University of Miami, in accordance with internal review board (IRB) ethical guidelines for the use of human tissue. Typical viability was 80–90% and purity was>85%. Human islets were cultured in PIM-S medium (Prodo Laboratories, Inc.) containing 5% human AB serum. All cells were maintained at 37 °C, with 5% CO 2 in a humidified atmosphere.

Glucose stimulated insulin secretion from intact islets

Human islets were evaluated for glucose stimulated insulin secretion using standard methods. Secreted insulin was monitored using an ELISA (Meso-scale Devices) and normalized to total DNA (PicoGreen, Invitrogen).

Rat and human islet proliferation assays

Rat or human β-cell proliferation assays were performed using dissociated primary rat or human islets. Islets were treated with the indicated compounds in the presence of EdU for 4 days before fixing and staining. EdU incorporation was measured by click reaction with AlexaFluor-647-azide (Invitrogen). For intact islet assays, primary human islets were treated for 7 or 8 days (as indicated in the legend). Rat dissociated islets were cultured in RPMI medium containing 10% FBS and human dissociated islets were cultured in PIM-S media (Prodo laboratories) with 10% human antibody serum.

R7T1 proliferation assay

Reversibly immortalized mouse β-cells (R7T1)49 express SV40 T antigen under a tetracycline inducible promoter. R7T1 cells were expanded in the presence of doxycycline. For experiments on proliferation, R7T1 cells were growth arrested by removal of doxycycline for 2 days and plated into 384-well plates at a density of 3,000 cells per well in growth medium. β-Cell proliferation was assessed using CellTiter Glo (Promega) after 4 days. Fold increase in cell number was calculated by normalizing compound-treated wells to the median of DMSO-treated wells.

Adenoviral overexpression of Gsk3b and Dyrk1a and NFATC1

Adenoviruses (Welgen) were diluted in the culture media, with a final concentrations (Dyrk1a WT 1:20 K; Gsk3b 1:20 K; NFATc1: 1:4,000–8,000; β-catenin adeno, 1:50 K) and added to dispersed rat islet cells in 384-well plates. Cells were incubated for 4 days with or without GNF7156 or GNF4877 treatment, fixed with 4% paraformaldehyde and EdU incorporation was measured by click reaction with AlexaFluor-647-azide (Invitrogen) and quantified similarly to the dispersed islet proliferation assay.

β-Catenin and NFATc1 nuclear translocation

Dispersed rat islets were treated with GNF7156, GNF4877, GNF6324 or DMSO in 384-well plates according to the dispersed rat islet assay. At the end of the treatment, cells were fixed with 4% paraformaldehyde, blocked and permeabilized, and stained by standard immunofluorescence techniques for β-catenin (Cell signaling, #9562L; dilution 1:500), NFATc1 (Santa Cruz, rabbit polyclonal, SC-7294; dilution 1:200) or NFAT3c (Santa Cruz, SC-8321; dilution 1:200) and nuclear DNA was stained with DAPI (Molecular Probes; dilution 1:1,000). EdU incorporation was measured by click reaction with AlexaFluor-647-azide (Invitrogen). Plates were imaged with confocal microscope and analysed by Metamorph (Molecular Devices).

Binding model of aminopyrazine compounds to Dyrk1a

Flexible docking was performed using Glide 5.8 (Schrodinger, Inc, Portland, OR, 2012). The protein coordinates were taken from protein databank (2vx3.pdb). The grid box was centred on the co-crystallized ligand and extended 10 Å from the centre, with outer box extending an additional 20 Å. The ligand was docked using the standard precision algorithm and scored using GlideScore.

Single cell RNA sequencing

Whole islets from Sprague–Dawley rats were isolated followed by immediate hand picking to ensure removal of contaminating extra-islet cell populations. After isolation, whole islets were cultured in RPMI1640 (Gibco) containing 10% FBS and 5.5 mM glucose with either 0.1% DMSO or 3 μM GNF4877. Following 48 h of culture, islets were dissociated with 0.05% trypsin for 5 min at 37 °C into a single cell suspension and neutralized with media. Dispersed islets were passed through a 40 μm filter to remove un-dissociated cells and quantified by ViCell (Beckman Coulter).

Dispersed and quantified cells from islets were loaded into Fluidigm C1 IFC microfluidic chips according to manufacturer specifications (Fluidigm). Briefly, 60 μl of a 200 cell per μl mixture was combined with 40 μl of C1 suspension reagent. Five microlitre was loaded into the 10–17 μm Fluidigm IFC using the mRNA Seq: Cell Load (1,772x) protocol. After loading, the IFC was manually inspected by phase contrast microscopy to ensure single cell capture. Following inspection, lysis, reverse transcription, and PCR amplification were all performed on the IFC using the SMARTer Ultra Low RNA kit (Clontech) with the mRNA Seq: RT & Amp (1,772x) protocol. cDNA was quantified by PicoGreen assay (Life Technologies), normalized with C1 DNA dilution buffer to 0.1–0.3 ng μl−1, and sequencing libraries were prepared with the Nextera XT DNA Sample Preparation Index Kit (Illumina, PN FC-131-1002). Sequencing libraries were run on an Illumina HiSeq 1,000 sequencer using single 50 bp reads and one 96-well plate per lane. Of the 96 samples per IFC, 84 GNF4877-treated and 86 DMSO-treated samples containing greater than 100,000 mapped sequencing reads per sample and having a single verified cell per port were used for analysis. The average number of mapped sequencing reads was 1.26 million per cell. Reads were aligned to the mouse transcriptome using BWA (Li H. and Durbin R. (2009) Fast and accurate short read alignment with Burrows-Wheeler Transform, Bioinformatics, 25:1754-60. [PMID: 19451168]). Expression values are represented in reads per million.

To identify proliferating β-cells in GNF4877-treated islets, cells were assessed for Cyclin B1 (Ccnb1) and Insulin 1 (Ins1) gene co-expression. To uncover significant transcriptional changes between cell populations, the average of each gene’s expression in proliferating β-cells of GNF4877-treated islets was divided by the average expression in β-cells of DMSO-treated islets (Ccnb1/DMSO). To ensure these changes were statistically significant, an unpaired Student’s t-test was performed.

Gene ontology analysis was performed using DAVID (ref. 50) on those genes that had a Log2 fold change in Ccnb1 expressing β-cells of GNF4877 islets >1 with a P value of at least 0.05.

Volcano plot was generated using Spotfire (Tibco) and bivariate plots were generated in Prism (GraphPad).