Adjudin improves beta cell maturation, hepatic glucose uptake and glucose homeostasis

Aims/hypothesis Recovering functional beta cell mass is a promising approach for future diabetes therapies. The aim of the present study is to investigate the effects of adjudin, a small molecule identified in a beta cell screen using zebrafish, on pancreatic beta cells and diabetes conditions in mice and human spheroids. Methods In zebrafish, insulin expression was examined by bioluminescence and quantitative real-time PCR (qPCR), glucose levels were examined by direct measurements and distribution using a fluorescent glucose analogue, and calcium activity in beta cells was analysed by in vivo live imaging. Pancreatic islets of wild-type postnatal day 0 (P0) and 3-month-old (adult) mice, as well as adult db/db mice (i.e. BKS(D)-Leprdb/JOrlRj), were cultured in vitro and analysed by qPCR, glucose stimulated insulin secretion and whole mount staining. RNA-seq was performed for islets of P0 and db/db mice. For in vivo assessment, db/db mice were treated with adjudin and subjected to analysis of metabolic variables and islet cells. Glucose consumption was examined in primary human hepatocyte spheroids. Results Adjudin treatment increased insulin expression and calcium response to glucose in beta cells and decreased glucose levels after beta cell ablation in zebrafish. Adjudin led to improved beta cell function, decreased beta cell proliferation and glucose responsive insulin secretion by decreasing basal insulin secretion in in vitro cultured newborn mouse islets. RNA-seq of P0 islets indicated that adjudin treatment resulted in increased glucose metabolism and mitochondrial function, as well as downstream signalling pathways involved in insulin secretion. In islets from db/db mice cultured in vitro, adjudin treatment strengthened beta cell identity and insulin secretion. RNA-seq of db/db islets indicated adjudin-upregulated genes associated with insulin secretion, membrane ion channel activity and exocytosis. Moreover, adjudin promoted glucose uptake in the liver of zebrafish in an insulin-independent manner, and similarly promoted glucose consumption in primary human hepatocyte spheroids with insulin resistance. In vivo studies using db/db mice revealed reduced nonfasting blood glucose, improved glucose tolerance and strengthened beta cell identity after adjudin treatment. Conclusions/interpretation Adjudin promoted functional maturation of immature islets, improved function of dysfunctional islets, stimulated glucose uptake in liver and improved glucose homeostasis in db/db mice. Thus, the multifunctional drug adjudin, previously studied in various contexts and conditions, also shows promise in the management of diabetic states. Data availability Raw and processed RNA-seq data for this study have been deposited in the Gene Expression Omnibus under accession number GSE235398 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE235398). Graphical Abstract Supplementary Information The online version contains peer-reviewed but unedited supplementary material available at 10.1007/s00125-023-06020-4.

Calcium activity indicated by normalized fluorescence over time in beta cells.Each line represents one cell.Tg(ins:GCaMP6s);Tg(ins:H2BmCherry) zebrafish larvae were treated with 5% glucose from 3 to 4 dpf, then glucose was washed out and live calcium imaging was performed at 5 dpf.The addition of 10 µmol/l and 50 µmol/l Adjudin is indicated by the white dashed lines.Each line represents one cell.n=6 cells from 1 larva.(b) Insulin content in P0 islets.P0 islets were cultured in medium containing 11 mmol/l glucose and treated with DMSO or 10 µmol/l Adjudin for 1 day before GSIS, and islet insulin content was measured at the end of the GSIS.Student's t test.n=12 (DMSO), n=11 (Adjudin).Data are presented as mean ± SEM.
(c) Insulin content in adult islets.Adult islets were cultured in medium containing 11 mmol/l glucose and treated with DMSO or 10 µmol/l Adjudin for 1 day before GSIS, and islet insulin content was measured at the end of the GSIS.Student's t test.n=12 per treatment.Data are presented as mean ± SEM.
(e-f) Heatmap of beta cell maturation markers (e) and disallowed genes (f) in P0 islets.* indicates significantly regulated genes.
(g) Representative immunofluorescence images of whole mount staining of mouse islets to assess the expression of PDX1, NKX6.1 and MAFA in beta cells.Islets were cultured in medium containing 11 mmol/l glucose and treated with DMSO or 10 µmol/l Adjudin for 1 day before fixation and whole mount staining.Scale bar=10 µm.
(d) Representative immunofluorescence images from whole mount staining of mouse islets to assess proliferation in islets from P4, P7 and adult mice.Islets were cultured in medium containing 11 mmol/l glucose and treated with DMSO or 10 µmol/l Adjudin for 1 day, a final concentration of 20 µmol/l EdU was used for 2 h incubation at the end of the treatment.Scale bar=10 µm.
(c) Heatmap of the core signature for the gene set of "2-oxoglutarate metabolic process" (Fig. 2l), the range of colors (red, pink, light blue, and dark blue) shows the range of expression values (high, moderate, low, and lowest).
(e) Heatmap of the core signature for the gene set of "NADH metabolic process" (Fig. 2m), the range of colors (red, pink, light blue, and dark blue) shows the range of expression values (high, moderate, low, and lowest).(f) GSEA plot showing gene sets related to "type B pancreatic cell differentiation" in db/db islets.NES, normalized enrichment score.
(g) Heatmap of the core signature for the gene set of "type B pancreatic cell differentiation" in (f), the range of colors (red, pink, light blue, and dark blue) shows the range of expression values (high, moderate, low, and lowest).
(h) GSEA plot showing gene sets related to "positive regulation of insulin secretion involved in cellular response to glucose stimulus" in db/db islets.NES, normalized enrichment score.
(i) Heatmap of the core signature for the gene set of "positive regulation of insulin secretion involved in cellular response to glucose stimulus" in (h), the range of colors (red, pink, light blue, and dark blue) shows the range of expression values (high, moderate, low, and lowest).Heatmaps of expression changes in db/db islet with or without Adjudin treatment in genes related to cell cycle activator (a) and inhibitor (b), oxidative phosphorylation complexes I-V (c-g), inner (h) and outer (i) mitochondrial membrane transport, mitochondrial ribosomal proteins (j), mitochondrial genes (k), cell-cell contact (l) and chloride channels (m).* indicates significantly regulated genes.(a) Melt curve analysis of qPCR for genotyping larvae that were ins+/+ or ins-/-.
(b) Schematic of how human hepatocytes were conditioned in low physiological insulin levels (LI) and high physiological glucose levels (HG) for 7 days, followed by phasing out insulin, and treatment with DMSO, 3 µmol/l, 10 µmol/l or 30 µmol/l Adjudin for 24 hours.The glucose level in the medium was measured before and after compound treatment.
(c) Glucose production/consumption in the PHH spheroids treated as described in (b).Oneway ANOVA followed by Dunnett's multiple comparisons test.n=3 independent biological replicates per treatment.Data are presented as mean ± SEM.Tg(ins:H2BmCherry);Tg(ins:Flag-NTR) zebrafish larva at 5 dpf after 1 day of beta cell regeneration, with beta cells expressing H2BmCherry shown in red, calcium signal in green.The zebrafish larvae were treated with DMSO for 1 day after beta cell ablation from 3-4 dpf, and had calcium signal recorded at 5 dpf.An example video of live calcium imaging of beta cells in a Tg(ins:GCaMP6s);Tg(ins:H2BmCherry) fish at 5 dpf, with beta cells expressing H2BmCherry shown in red, calcium signal in green.The zebrafish larvae were treated with 5% glucose from 3-4 dpf, then glucose was washed out, and calcium signal was recorded at 5 dpf before and after acute treatment with Adjudin.

ESM Video 2. Live calcium imaging of beta cells in a Adjudin
effects of Adjudin on beta cells in zebrafish.Related to Fig.1.
of Adjudin on P0 and adult islets.Related to Fig. 2. (a) Basal insulin secretion from GSIS shown in percentage of cellular insulin content.Student's t test.n=12 per treatment.**p < 0.01.Data are presented as mean ± SEM.
b) Heat map of cell cycle activators (a) and cell cycle inhibitors (b) in P0 islets.* indicates significantly regulated genes.
(f-n) Heatmap showing mitochondrial signatures in P0 islets.(f-j) oxidative phosphorylation complexes I-V.Genes encoding inner (k) and outer (l) mitochondrial membrane transport proteins.(m) genes encoding mitochondrial ribosomal proteins.(n) mitochondrial genes.* indicates significantly regulated genes.Representative immunofluorescence images from whole mount staining of mouse islets to assess expression of TOM20.Islets were cultured in medium containing 11 mmol/l glucose and treated with DMSO or 10 µmol/l Adjudin for 1 day before the fixation and whole mount staining.Scale bar=10 µm.(b) Representative image from western blot for TOM20, TFAM, E-caherin and beta-actin in mouse islets.Islets were cultured in medium containing 11 mmol/l glucose and treated with DMSO or 10 µmol/l Adjudin for 1 day before western blot.(c-e) Quantification of western blot bands in (b), each dot represent data from one biological sample.One-way ANOVA with Tukey's multiple-comparisons test.*p < 0.05, **p < 0.01, ***p < 0.001.n=3 per treatment.Data are presented as mean ± SEM.(f) Heat map of cell-cell contact genes in P0 islets.* indicates significantly regulated genes.(g) Representative immunofluorescence images from whole mount staining of mouse islets to assess expression of E-cadherin.Islets were cultured in medium containing 11 mmol/l glucose and treated with DMSO or 10 µmol/l Adjudin for 1 day before the fixation and whole mount staining.Scale bar=10 µm.(h) Heat map of genes associated with chloride channels.* indicates significantly regulated genes. of Adjudin on db/db islets.Related to Fig. 3. (a) qPCR analysis of the expression of beta cell maturation markers in db/db and db maps for db/db islets.Related to Fig.3.
results of insulin mutant fish and insulin-independent effects of Adjudin on human liver spheroids.Related to Fig. 4.
of Adjudin on beta cells in db/db mouse.Related to Fig. 5. (a) Quantification of relative intensity of NKX6.
:GCaMP6s);Tg(ins:H2BmCherry);Tg(ins:Flag-NTR) zebrafish larva at 5 dpf after 1 day of beta cell regeneration, with beta cells expressing H2BmCherry shown in red, calcium signal in green.The zebrafish larvae were treated with Adjudin for 1 day after beta cell ablation from 3-4 dpf, and had calcium signal recorded at 5 dpf.ESMVideo 3. Live calcium imaging of beta cells in a larva subjected to acute treatment with Adjudin.Related to ESM Fig. 1.
(d) Volcano plot for db/db islets.(e)Heatmap of beta cell maturation markers in db/db islets.* indicates significantly regulated genes.