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Abnormal exocrine–endocrine cell cross-talk promotes β-cell dysfunction and loss in MODY8

Nature Metabolism volume 4, pages 76–89 (2022)Cite this article

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Abstract

MODY8 (maturity-onset diabetes of the young, type 8) is a dominantly inherited monogenic form of diabetes associated with mutations in the carboxyl ester lipase (CEL) gene expressed by pancreatic acinar cells. MODY8 patients develop childhood-onset exocrine pancreas dysfunction followed by diabetes during adulthood. However, it is unclear how CEL mutations cause diabetes. In the present study, we report the transfer of CEL proteins from acinar cells to β-cells as a form of cross-talk between exocrine and endocrine cells. Human β-cells show a relatively higher propensity for internalizing the mutant versus the wild-type CEL protein. After internalization, the mutant protein forms stable intracellular aggregates leading to β-cell secretory dysfunction. Analysis of pancreas sections from a MODY8 patient reveals the presence of CEL protein in the few extant β-cells. The present study provides compelling evidence for the mechanism by which a mutant gene expressed specifically in acinar cells promotes dysfunction and loss of β-cells to cause diabetes.

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Fig. 1: MUT CEL protein is transferred from acinar cells to β-cells.
Fig. 2: Intracellular localization of MUT CEL protein after uptake by β-cells.
Fig. 3: MUT CEL protein forms stable insoluble aggregates after internalization by pancreatic β-cells.
Fig. 4: Accumulation of MUT CEL protein aggregates reduces proliferation and impairs function of β-cells.
Fig. 5: Function of β-like cells derived from MUT hPSC lines is impaired.
Fig. 6: MUT CEL protein is taken up by primary human pancreatic islet cells.
Fig. 7: Analysis of the islet and acinar grafts.
Fig. 8: Histological analysis of MODY8 donor pancreas.

Data availability

RNA-seq data in EndoC-βH1 cells have been deposited into the National Center for Biotechnology Information’s Gene Expression Omnibus under accession no. GSE185430. Other data that support the findings of the present study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank E. Tjora (University of Bergen) for providing skin biopsies of a MODY8 family, A. B. Goldfine (Joslin) for providing skin biopsies from a non-family control, D. Hoem (Haukeland University Hospital) for providing pancreatic tissue from a MODY8 patient, A. K. K. Teo (Joslin) for establishing control skin fibroblast cultures and expanding fibroblasts, B. Slipp (Joslin) for technical assistance, O. Ijaduola (Joslin) for maintaining NSG mice, H. Pan and J. Dreyfuss (Joslin Bioinformatics & Biostatistics Core) for analysing RNA-seq data, and C. Cahill (Joslin Advanced Microscopy Core) for assistance with confocal microscopy and processing samples for electron microscopy. We thank P. R. Njølstad (University of Bergen) for support and discussions throughout the study and S. Bonner-Weir (Joslin) and M. Solimena (Dresden) for discussions. We thank the IIDP for providing human pancreatic islets (NIH grant no. 2UC4DK098085) and Prodo Labs for providing human pancreatic acinar and islet tissues. Flow cytometry experiments were performed in the Joslin Flow Cytometry Core, supported by the Diabetes Research Center (DRC) (nos. P30DK036836 and S10 OD021740-01). R.N.K. acknowledges support from the NIH (grant nos. R01 DK067536 and R01 DK103215). A.M. acknowledges support from the Western Norway Regional Health Authority (Helse Vest, no. 912057) and the Research Council of Norway (FRIMEDBIO, no. 289534). H.R. acknowledges support from Bergen Forskningsstiftelse (no. BFS2014REK02), Diabetesforbundet, Novo Nordisk Foundation (no. NNF17OC0027258), Johan Selmer Kvanes legat and the Western Norway Regional Health Authority (grant no. 911985), and D.H. acknowledges support from NIH/National Institute of Diabetes and Digestive and Kidney Diseases (grant no. R01DK096239).

Author information

Author notes

  1. These authors contributed equally: Helge Raeder, Anders Molven.

Authors and Affiliations

  1. Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, MA, USA

    Sevim Kahraman, Ercument Dirice, Giorgio Basile, Danielle Diegisser, Manoj K. Gupta, Jiang Hu & Rohit N. Kulkarni

  2. Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    Sevim Kahraman, Giorgio Basile & Rohit N. Kulkarni

  3. Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, USA

    Sevim Kahraman, Giorgio Basile & Rohit N. Kulkarni

  4. Department of Pharmacology, New York Medical College of Medicine, Valhalla, NY, USA

    Ercument Dirice & Danielle Diegisser

  5. Gade Laboratory for Pathology, Department of Clinical Medicine, University of Bergen, Bergen, Norway

    Jahedul Alam & Anders Molven

  6. Center for Diabetes Research, Department of Clinical Science, University of Bergen, Bergen, Norway

    Bente B. Johansson, Helge Raeder & Anders Molven

  7. Cancer Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

    Ling Huang & Senthil K. Muthuswamy

  8. Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

    Ling Huang & Senthil K. Muthuswamy

  9. Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Chew-Li Soh & Danwei Huangfu

  10. Department of Pediatrics, Haukeland University Hospital, Bergen, Norway

    Helge Raeder

  11. Department of Pathology, Haukeland University Hospital, Bergen, Norway

    Anders Molven

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  1. Sevim Kahraman
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Contributions

S.K. conceived the idea, designed and performed the experiments, analysed the data and wrote the manuscript. E.D. and G.B. performed transplantation experiments. E.D. contributed to confocal imaging. D.D. contributed to cell culture experiments, western blotting, immunohistochemistry and confocal imaging. M.K.G. performed a Seahorse assay. J.H. contributed to immunohistochemistry. L.H. and S.K.M. contributed to acinar differentiation of hiPSC and hESC lines. C.L.S. and D.H. contributed to generation of isogenic hESC lines. H.R. provided MODY8 patient-derived skin biopsies and contributed to conceptual discussions. B.B.J. established fibroblast cultures from skin biopsies. B.B.J. and A.M. provided CEL plasmids and stable OE HEK293 cell lines, and contributed to conceptual discussions. A.M. and J.A. contributed to immunohistochemistry on MODY8 patient pancreas sections. R.N.K. conceived the idea, contributed to discussions, designed the experiments, supervised the project and wrote the manuscript. All the authors reviewed, commented on and edited the manuscript.

Corresponding author

Correspondence to Rohit N. Kulkarni.

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Nature Metabolism thanks Guy Rutter and the other, anonymous, reviewers for their contribution to the peer review of this work. The primary handling editor was Isabella Samuelson

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Extended data

Extended Data Fig. 1 Reduced secretion of mutant CEL protein from 266-6 acinar cells and endocytosis-mediated uptake by β-cells.

a, Mouse acinar cells (266-6) transfected with empty vector (EV), wild-type CEL (WT), or mutant CEL (MUT) plasmids. Cell lysates (L) and medium (M) were collected and labeled with anti-V5 antibody to measure V5-tagged CEL levels. b, Western blots, representative of three independent experiments, show low molecular mass forms of wild-type and mutant human CEL proteins in lysates and the fully glycosylated, high molecular mass forms secreted into media. c-e, The quantification of three biologically independent samples. Fold change relative to EV. Lysate CEL levels were normalized to β-actin. f, Determination of the concentration of CEL protein in conditioned medium by quantitative immunoblotting (n = 2 biologically independent samples) using recombinant mouse CEL proteins (rmCEL). g, A standard curve for rmCEL. h, The concentrations of wild-type and mutant CEL proteins were estimated according to the standard curve. i, Recipient β-cells were treated with conditioned medium obtained from HEK293 donor cells for 30 min at 37 °C or at 4 °C (n = 3 biologically independent samples). j, The quantification of three biologically independent samples. Fold change relative to WT 37 °C. V5-tagged CEL levels were normalized to α-tubulin levels. k, Percentage of CEL + β-cells detected by immunostaining after treatment with Wortmannin to block endocytosis (n = 3 biologically independent samples). l, Western blot quantification of β-cells treated with conditioned medium obtained from HEK293 donor cells treated with DMSO or exosome inhibitor GW4869. Fold change relative to WT DMSO (n = 3 biologically independent samples). m, Conditioned media collected from HEK293 donor cells stably transfected with EV, WT, or MUT plasmids was subjected to protein aggregation assay (n = 9 biologically independent samples). Fluorescence signal generated by Proteostat detection dye was measured. Aggregated lysozyme (20 μg) and monomeric lysozyme (20 μg) were used as positive and negative controls, respectively (n = 3 independent samples). Data are expressed as fold change relative to WT. Data are presented as mean values ± SEM. One-way ANOVA followed by Tukey’s multiple comparison test (c,d,j,k-m), two-tailed t-test used for (e). Dashed line is added for easy comprehension (b,f,i).

Source data

Extended Data Fig. 2 Accumulation of mutant CEL protein aggregates reduces proliferation and impairs function of β-cells.

a, Representative FACS plots showing percentage of AnnexinV and Zombie Near Infrared (NIR) stained β-cells treated with conditioned medium for 10 days to assess apoptosis levels (n = 3 biologically independent samples). b-d, Heatmap showing differentially expressed genes involved in cellular senescence (b), Insulin secretion (c), glycolysis/gluconeogenesis (top panel), oxidative phosphorylation (lower panel) (d) in β-cells treated with conditioned medium for 10 days (EV n = 9, WT n = 10, MUT n = 10 biologically independent samples). e, Mitochondrial respiration profile of EndoC-βH1 cells exposed to conditioned media for 10 days. Cells were challenged with oligomycin (15 μM), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (10 μM), and Rotenone (1.1 µM) and actinomycin (25 μM) (left panel). Quantification of basal (middle panel) and maximal respiration (right panel) capacity (EV n = 5, WT n = 4, MUT n = 5 biologically independent samples). Data are presented as mean values ± SEM. One-way ANOVA followed by Tukey’s multiple comparison (a,e).

Extended Data Fig. 3 Generation of hESC lines expressing mutant CEL.

a, Schematic of CRISPR-Cas9 strategy for generation of CEL mutant hESC lines. Exons and introns are represented by boxes and lines, respectively. A 100-nt single strand DNA (ssDNA) carrying the patient specific mutation (c.1686delT) and two gRNAs (gRNA1, gRNA2) were used to target the first repeat of VNTR in the CEL gene exon 11. PAM sequences in the gRNAs are indicated in purple. Heterozygous and compound heterozygous mutant cell lines were generated. Deletions are indicated in red boxes. b, Alignment of the C-terminal end of WT and MUT CEL proteins. WT and MUT amino acid sequences are indicated in blue and red, respectively. Small deletions cause frameshift in the first repeat of VNTR (Rep1) and create a shorter protein. Each asterisk denotes deletion of an amino acid. c, DNA sequencing of isogenic hESC lines carrying deletion mutations in the CEL gene. Asterisks show the mutation sites. d, Normal karyotype of WT and MUT hESC lines generated by CRISPR-Cas9 technology.

Extended Data Fig. 4 Generation and characterization of MODY8 disease-specific hiPSCs.

a, MODY8 family pedigree. Solid symbols denote diabetes. NN, no mutation; NM, mutation. b, Outline of the episomal reprogramming approach. Details are given in the Methods section. c, Representative bright-field images of hiPSC colonies derived from family and non-family controls (Fam Ctr n = 4, and Non-Fam Ctr n = 4 independent clones) and from mutation carriers with or without diabetes (Mut + Dia+ n = 8, Mut+ Dia- n = 6 independent clones). Scale bar is 1 mm. d, Normal karyotype of hiPSCs derived from controls and MODY8 patients (Non-Fam Ctr n = 2, Fam Ctr n = 2, Mut+ Dia- n = 4, Mut+ Dia+ n = 4 biologically independent samples). e, DNA sequencing confirmed presence of c.1686delT deletion (asterisk) in hiPSCs derived from MODY8 patients (Non-Fam Ctr n = 2, Fam Ctr n = 2, Mut+ Dia- n = 4, Mut+ Dia+ n = 4 biologically independent samples). f, Immunostaining for pluripotency markers OCT4 (red), SOX2 (green), SSEA4 (red) in hiPSCs derived from controls and mutation carriers (Non-Fam Ctr n = 2, Fam Ctr n = 2, Mut+ Dia- n = 4, Mut+ Dia+ n = 4 biologically independent samples). Nuclei stained with DAPI (blue). Scale bar is 500 μm. g, Control and MODY8 hiPSCs formed teratoma approximately 13 weeks after implantation into immunodeficient mice. Non-Fam Ctr n = 3, Fam Ctr n = 3, Mut+ Dia- n = 4, Mut+ Dia+ n = 4 biologically independent samples. Data are presented as mean values ± SEM. The difference between control vs mutant lines is not significant. Two-tailed t-test. h, Control and mutant hiPSCs formed teratoma (approximately 2 cm diameter) after injection into right leg muscle of immunodeficient mice (Non-Fam Ctr n = 3, Fam Ctr n = 3, Mut+ Dia- n = 4, Mut+ Dia+ n = 4 biologically independent samples). i, Representative images of teratomas stained with hematoxylin and eosin (HE) (Non-Fam Ctr n = 3, Fam Ctr n = 3, Mut+ Dia- n = 4, Mut+ Dia+ n = 4 biologically independent samples). Arrows show differentiated tissues including neural rosettes (ectoderm), cartilage (mesoderm), epithelial tissue (endoderm). Scale bar 50 μm.

Extended Data Fig. 5 Differentiation of control hiPSCs to generate S6 β-like cells.

a, Differentiation protocol used to differentiate hPSCs towards insulin expressing β-like cells. Details are given in Supplementary Table 4. b, Control hiPSCs (family and non-family control) were differentiated to S6 and expression levels of genes specific to β-cells were measured. Two differentiation batches of control hiPSCs (Batch1 n = 4, Batch2 n = 2 biologically independent samples) and human islets collected from five donors were used. Donor information is provided in Supplementary Table 1. β-Actin was used as a housekeeping control. Fold change relative to S0. Data are presented as mean values ± SEM. c, Control hiPSCs (N65-51 family control, n = 2 biologically independent samples) were differentiated to S6 and co-stained for several pancreatic markers; INS/GCG, NKX6.1/PDX1, CHGA/GCG, PDX1/ISL1, NeuroD1/PDX1, NKX2.2/PDX1. Nuclei stained with DAPI (blue). Scale bar is 500 μm.

Extended Data Fig. 6 Differentiation of gene-edited hESCs and patient-derived hiPSCs to definitive endoderm stage.

a, FACS analysis of wild-type and mutant hESC lines differentiated to S1. + / + n = 12, +/1686delT n = 3, +/1698delA n = 3, +/1690_1702del n = 3, +/1683_1704del n = 3, 1686delT/1702delG n = 3 biologically independent samples. Data are presented as mean values ± SEM. No difference was detected in wild-type vs. each mutant line by two-tailed multiple t-test corrected using Holm-Sidak method. b, FACS analysis of control and mutant hiPSC lines differentiated to S1. Control n = 8, Mut+ Dia- n = 6, Mut+ Dia+ n = 7 biologically independent samples. Data are presented as mean values ± SEM. No difference was detected in control vs Mut+ Dia- and vs. Mut+ Dia+ by two-tailed multiple t-test corrected using Holm-Sidak method. c, Immunostaining images, representative of three biologically independent samples, showing DE cells stained for SOX17 (green) and OCT4 (red). Nuclei stained with DAPI (blue). Scale bar is 500 μm.

Extended Data Fig. 7 Differentiation of gene-edited hESCs and patient-derived hiPSCs to pancreatic progenitor stage.

a, FACS analysis of wild-type and mutant hESC lines differentiated to S4. + / + n = 16, +/1686delT n = 4, +/1698delA n = 4, +/1690_1702del n = 4, +/1683_1704del n = 4, 1686delT/1702delG n = 4 biologically independent samples. Data are presented as mean values ± SEM. No difference was detected in wild-type vs. each mutant line by two-tailed multiple t-test corrected using Holm-Sidak method. c, Representative FACS plots of control and mutant (with or without diabetes) hiPSC lines differentiated to S4. b, FACS analysis of control and mutant hiPSC lines differentiated to S4. Control n = 6, Mut+ Dia- n = 5, Mut+ Dia+ n = 4 biologically independent samples. Data are presented as mean values ± SEM. No difference was detected in control vs Mut+ Dia- and vs Mut+ Dia+ by two-tailed multiple t-test corrected using Holm-Sidak method. c, Immunostaining images, representative of three biologically independent samples, showing PP cells for PDX1 (green) and NKX6.1 (red). Nuclei stained with DAPI (blue). Scale bar is 500 μm.

Extended Data Fig. 8 Differentiation of gene-edited hESCs and patient-derived hiPSCs to β-like cells.

a, FACS analysis of wild-type and mutant hESC lines differentiated to S6. + / + n = 4, +/1686delT n = 4, +/1698delA n = 3, +/1690_1702del n = 4, +/1683_1704del n = 4, 1686delT/1702delG n = 4 biologically independent samples. Data are presented as mean values ± SEM. No difference was detected in wild-type vs. each mutant line by two-tailed multiple t-test corrected using Holm-Sidak method. b, FACS analysis of control and mutant hiPSC lines differentiated to S6. Control n = 8, Mut+ Dia- n = 6, Mut+ Dia+ n = 6 biologically independent samples. Data are presented as mean values ± SEM. No difference was detected in control vs. Mut+ Dia- and vs. Mut+ Dia+ by two-tailed multiple t-test corrected using Holm-Sidak method. c, Immunostaining images, representative of three biologically independent samples, showing β-like cells for CPEP (green) and GCG (red). Nuclei stained with DAPI (blue). Scale bar is 500 μm. d, GSIS was performed by stimulating wild-type or mutant S6 cells with 1 mM low glucose (LG) or 20 mM high glucose (HG) for an hour. The stimulation index was calculated as the fold increase in human C-peptide release measured in 20 mM over 1 mM glucose. +/+ n = 4, +/1686delT n = 4, 1686delT/1702delG n = 4 biologically independent samples. Data are represented as median with 25% to 75% percentile box and min/max whisker plots. Two-tailed multiple t-tests followed by Holm Sidak’s multiple comparison test. e, Stimulation index of iPSC-derived β-like cells. Control n = 4, Mut+ Dia- n = 4, Mut+ Dia+ n = 4 biologically independent samples. Data are represented as median with 25% to 75% percentile box and min/max whisker plots. Two-tailed multiple t-tests followed by Holm Sidak’s multiple comparison test.

Extended Data Fig. 9 Differentiation of gene-edited hESCs and patient-derived hiPSCs to acinar-like organoids.

a, Differentiation protocol used to differentiate hPSCs towards exocrine organoids. Details are given in the Methods section. b, Bright field images, representative of three biologically independent samples, show organoids that were derived from gene-edited hESCs. Scale bar is 100 μm. c, Expression levels of genes specific to exocrine pancreas were measured by RT-PCR (WT n = 5, MUT n = 5 biologically independent samples). Data are presented as mean values ± SEM. No difference was detected in wild-type vs. mutant by two-tailed t-test. Similar results were observed using three differentiation batches of hESC or hiPSC lines. β-Actin was used as a housekeeping control. Fold change relative to S0.

Extended Data Fig. 10 Analysis of WT and MUT S4 graft sections.

Representative immunostaining images of grafts derived from mutant (n = 4) or wild-type (n = 4) S4 cells stained for β-cell markers such as INS in green, PDX1, NKX2.2, NeuroD1, and NKX6.1 in red. Human foetal pancreas (34 weeks, n = 1 donor) was used as control. Donor information is given in Supplementary Table 1. Nuclei stained with DAPI (blue). Scale bar is 20 μm.

Supplementary information

Supplementary Information

Supplementary Fig. 1, Protocols and References.

Reporting Summary

Supplementary Tables

Supplementary Table 1 Donor information. Supplementary Table 2 Antibody information. Supplementary Table 3 Primer information. Supplementary Table 4 In vitro differentiation protocol.

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Unprocessed western blots for Fig. 1.

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Source Data Extended Data Figure 1

Unprocessed western blots for Extended Data Fig. 1.

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Kahraman, S., Dirice, E., Basile, G. et al. Abnormal exocrine–endocrine cell cross-talk promotes β-cell dysfunction and loss in MODY8. Nat Metab 4, 76–89 (2022). https://doi.org/10.1038/s42255-021-00516-2

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