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Leader β-cells coordinate Ca2+ dynamics across pancreatic islets in vivo

Nature Metabolism volume 1, pages 615–629 (2019)Cite this article

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Abstract

Pancreatic β-cells form highly connected networks within isolated islets. Whether this behaviour pertains to the situation in vivo, after innervation and during continuous perfusion with blood, is unclear. In the present study, we used the recombinant Ca2+ sensor GCaMP6 to assess glucose-regulated connectivity in living zebrafish Danio rerio, and in murine or human islets transplanted into the anterior eye chamber. In each setting, Ca2+ waves emanated from temporally defined leader β-cells, and three-dimensional connectivity across the islet increased with glucose stimulation. Photoablation of zebrafish leader cells disrupted pan-islet signalling, identifying these as likely pacemakers. Correspondingly, in engrafted mouse islets, connectivity was sustained during prolonged glucose exposure, and super-connected ‘hub’ cells were identified. Granger causality analysis revealed a controlling role for temporally defined leaders, and transcriptomic analyses revealed a discrete hub cell fingerprint. We thus define a population of regulatory β-cells within coordinated islet networks in vivo. This population may drive Ca2+ dynamics and pulsatile insulin secretion.

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Fig. 1: Glucose-stimulated Ca2+ influx imaged in vivo in living zebrafish.
Fig. 2: Ca2+ dynamics and connectivity in zebrafish: slow imaging acquisition (frame rate 0.1 Hz).
Fig. 3: Ca2+ dynamics and connectivity examined in zebrafish during rapid image acquisition.
Fig. 4: Ablation of temporally defined ‘leader’ cells (but not ‘follower’ cells) alters islet responsivity to glucose in vivo in zebrafish.
Fig. 5: Ca2+ waves and connectivity revealed using islets expressing GCaMP6f throughout the cell population under insulin promoter control.
Fig. 6: Binarized and Granger causality analysis corroborate the existence of super-connected leader cells in mouse islets in vivo.

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Data availability

The data that support the findings of this study and the MATLAB codes for the various connectivity analyses described above are available from the corresponding authors upon request. Zebrafish islet RNA-seq data are deposited at the Gene Expression Omnibus repository with accession no. GSE123662.

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Acknowledgements

V.S. was supported by a Diabetes UK Harry Keen Clinician Scientist 15/0005317. G.A.R. was supported by a Wellcome Trust Senior Investigator Award (no. WT098424AIA), Wellcome Trust Investigator Award (212625/Z/18/Z), MRC Programme grants (nos. MR/R022259/1, MR/J0003042/1 and MR/L020149/1) and Experimental Challenge Grant (DIVA, no. MR/L02036X/1), MRC (no. MR/N00275X/1), Diabetes UK (nos. BDA/11/0004210, BDA/15/0005275 and BDA 16/0005485) and Imperial Confidence in Concept grants, and a Royal Society Wolfson Research Merit Award. I.L. was supported by Diabetes UK Project Grant no. 16/0005485 and D.J.H. by a Diabetes UK R.D. Lawrence Fellowship (no. 12/0004431), a Wellcome Trust Institutional Support Award, and Medical Research Council (no. MR/N00275X/1) and Diabetes UK (no. 17/0005681) Project Grants. N.N. received funding from the DFG–Center for Regenerative Therapies Dresden, Cluster of Excellence at TU Dresden and the German Center for Diabetes Research (DZD), as well as research grants from the German Research Foundation (DFG), the European Foundation for the Study of Diabetes, The International Research Training Group (IRTG 2251), and the DZD. L.J.B.B. was supported by a Sir Henry Wellcome Postdoctoral Fellowship (Wellcome Trust, no. 201325/Z/16/Z) and a Junior Research Fellowship from Trinity College, Oxford. This project has received funding from the European Research Council (under the European Union’s Horizon 2020 research and innovation programme (Starting Grant no. 715884 to D.J.H.) and from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement no. 115881 (RHAPSODY) to G.A.R. and P.M. This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation programme and European Federation of Pharmaceutical Industries and Associations. We would like to thank P.-O. Berggren (Karolinska Institute, Sweden and Imperial College London), A. Caicedo and R. Rodriguez (University of Miami), and P. Chabosseau, M.-S. Nguyen-Tu and B. Owen (Imperial College London) for valuable advice and support with surgery and imaging. We thank R. Callingham (Imperial College London) for assistance with human islet culture.

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Author notes

  1. These authors contributed equally: Victoria Salem, Luis Delgadillo Silva.

Authors and Affiliations

  1. Department of Medicine, Imperial College London, London, UK

    Victoria Salem, Kinga Suba, Eleni Georgiadou, S. Neda Mousavy Gharavy, Aldara Martin-Alonso, Gaelle Carrat, Isabelle Leclerc & Guy A. Rutter

  2. Center for Regenerative Therapies Dresden, Technische Universität Dresden, Dresden, Germany

    Luis Delgadillo Silva, Nadeem Akhtar, Sumeet Pal Singh & Nikolay Ninov

  3. Facility for Imaging by Light Microscopy, Imperial College London, London, UK

    David C. A. Gaboriau & Stephen M. Rothery

  4. Loughborough University, Loughborough, UK

    Theodoros Stylianides

  5. Department of Diabetes, Faculty of Life Science and Medicine, King’s College London, London, UK

    Timothy J. Pullen

  6. Institute of Metabolism and Systems Research, University of Birmingham, Edgbaston, UK

    David J. Hodson

  7. Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners, Birmingham, UK

    David J. Hodson

  8. Centre of Membrane Proteins and Receptors, University of Birmingham and University of Nottingham, Nottingham, UK

    David J. Hodson

  9. Clinical Islet Laboratory and Clinical Islet Transplant Program, University of Alberta, Edmonton, Alberta, Canada

    A. M. James Shapiro

  10. University of Pisa, Pisa, Italy

    Piero Marchetti

  11. Radcliffe Department of Medicine, University of Oxford, Oxford, UK

    Linford J. B. Briant

  12. Imperial College London, London, UK

    Walter Distaso

  13. Paul Langerhans Institute Dresden of the Helmholtz Center Munich at the University Hospital Carl Gustav Carus of TU Dresden, German Center for Diabetes Research, Dresden, Germany

    Nikolay Ninov

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  1. Victoria Salem
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Contributions

V.S., N.N. and G.A.R. designed and supervised the study. L.D.S. and N.A. performed the zebrafish experiments. V.S., K.S., A.M.A. and I.L. undertook the mouse studies. G.C. and K.S. performed virus preparations. D.C.A.G., S.M.R., K.S., L.D.S. and V.S. developed movement correction macros. E.G., S.N.M.G., N.A., T.S., D.J.H. and L.B. contributed to connectivity analysis. D.J.H. and L.B. provided the code for connectivity analysis. V.S. and W.D. developed connectivity and Granger scripts and undertook all connectivity analyses. P.M. and A.M.J.S. provided human islets. K.S. and V.S. undertook studies on these preparations. T.J.P., N.A. and S.P.S. performed transcriptomic and bioinformatics analyses. G.A.R., V.S., L.D.S. and N.N. wrote the manuscript with contributions from all authors.

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Correspondence to Victoria Salem, Nikolay Ninov or Guy A. Rutter.

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G.A.R. has received grant funding from Servier and is a consultant for Sun Pharma. All others authors declare no competing interests.

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Salem, V., Silva, L.D., Suba, K. et al. Leader β-cells coordinate Ca2+ dynamics across pancreatic islets in vivo. Nat Metab 1, 615–629 (2019). https://doi.org/10.1038/s42255-019-0075-2

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