Fusion pore regulation by cAMP/Epac2 controls cargo release during insulin exocytosis

Abstract

Regulated exocytosis establishes a narrow fusion pore as initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of polypeptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in diabetes and neurodegenerative disease. Here we report that the cAMP-sensor Epac2 (Rap-GEF4) controls fusion pore behavior by acutely recruiting two pore-restricting proteins, amisyn and dynamin-1, to the exocytosis site in insulin-secreting beta-cells. cAMP elevation restricts and slows fusion pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2-/- (Rapgef4-/-) mice. Consistently, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 receptor agonists and sulfonylureas) activate this pathway and thereby paradoxically restrict hormone release. We conclude that Epac2/cAMP controls fusion pore expansion and thus the balance of hormone and transmitter release during insulin granule exocytosis.

Data availability

Source data file has been provided for Fig 7. All raw data are available on the Dryad Digital Repository (https://doi.org/10.5061/dryad.6b604g8).

The following data sets were generated

Article and author information

Author details

  1. Alenka Guček

    Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4453-1498
  2. Nikhil R Gandasi

    Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
    Competing interests
    The authors declare that no competing interests exist.
  3. Muhmmad Omar-Hmeadi

    Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8893-7348
  4. Marit Bakke

    Department of Biomedicine, University of Bergen, Bergen, Norway
    Competing interests
    The authors declare that no competing interests exist.
  5. Stein O Døskeland

    Department of Biomedicine, University of Bergen, Bergen, Norway
    Competing interests
    The authors declare that no competing interests exist.
  6. Anders Tengholm

    Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4508-0836
  7. Sebastian Barg

    Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
    For correspondence
    sebastian.barg@mcb.uu.se
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4661-5724

Funding

Swedish Research Council (2014-02575)

  • Anders Tengholm
  • Sebastian Barg

Norwegian Research Council

  • Marit Bakke

Helse-Bergen

  • Marit Bakke

Olga Jönssons stipend

  • Alenka Guček

P O Zetterlingsstiftelse

  • Alenka Guček

Swedish Research Council (2017-00956)

  • Anders Tengholm
  • Sebastian Barg

Swedich Research Council (2018-02871)

  • Anders Tengholm
  • Sebastian Barg

European Foundation for the Study of Diabetes

  • Anders Tengholm
  • Sebastian Barg

Diabetes Wellness Network Sweden

  • Anders Tengholm
  • Sebastian Barg

Swedish Diabetes Society

  • Anders Tengholm
  • Sebastian Barg

Swedish Society for Medical Research

  • Nikhil R Gandasi

Hjärnfonden

  • Sebastian Barg

NovoNordisk

  • Nikhil R Gandasi
  • Anders Tengholm
  • Sebastian Barg

Family Ernfors Foundation

  • Alenka Guček
  • Anders Tengholm
  • Sebastian Barg

European Foundation for the Study of Diabetes

  • Nikhil R Gandasi
  • Anders Tengholm
  • Sebastian Barg

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: This study was performed in strict accordance with European and Swedish legislation, fundamental ethical principles and approved by the Regional Ethics Board Uppsala (license number 31, 1-32).

Copyright

© 2019, Guček et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,302
    views
  • 413
    downloads
  • 42
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Alenka Guček
  2. Nikhil R Gandasi
  3. Muhmmad Omar-Hmeadi
  4. Marit Bakke
  5. Stein O Døskeland
  6. Anders Tengholm
  7. Sebastian Barg
(2019)
Fusion pore regulation by cAMP/Epac2 controls cargo release during insulin exocytosis
eLife 8:e41711.
https://doi.org/10.7554/eLife.41711

Share this article

https://doi.org/10.7554/eLife.41711

Further reading

    1. Cell Biology
    Kaili Du, Hongyu Chen ... Dan Li
    Research Article

    Niemann–Pick disease type C (NPC) is a devastating lysosomal storage disease characterized by abnormal cholesterol accumulation in lysosomes. Currently, there is no treatment for NPC. Transcription factor EB (TFEB), a member of the microphthalmia transcription factors (MiTF), has emerged as a master regulator of lysosomal function and promoted the clearance of substrates stored in cells. However, it is not known whether TFEB plays a role in cholesterol clearance in NPC disease. Here, we show that transgenic overexpression of TFEB, but not TFE3 (another member of MiTF family) facilitates cholesterol clearance in various NPC1 cell models. Pharmacological activation of TFEB by sulforaphane (SFN), a previously identified natural small-molecule TFEB agonist by us, can dramatically ameliorate cholesterol accumulation in human and mouse NPC1 cell models. In NPC1 cells, SFN induces TFEB nuclear translocation via a ROS-Ca2+-calcineurin-dependent but MTOR-independent pathway and upregulates the expression of TFEB-downstream genes, promoting lysosomal exocytosis and biogenesis. While genetic inhibition of TFEB abolishes the cholesterol clearance and exocytosis effect by SFN. In the NPC1 mouse model, SFN dephosphorylates/activates TFEB in the brain and exhibits potent efficacy of rescuing the loss of Purkinje cells and body weight. Hence, pharmacological upregulating lysosome machinery via targeting TFEB represents a promising approach to treat NPC and related lysosomal storage diseases, and provides the possibility of TFEB agonists, that is, SFN as potential NPC therapeutic candidates.

    1. Cell Biology
    Yan Song, Linda J Fothergill ... Gene W Yeo
    Research Article

    Dynamic interactions between gut mucosal cells and the external environment are essential to maintain gut homeostasis. Enterochromaffin (EC) cells transduce both chemical and mechanical signals and produce 5-hydroxytryptamine to mediate disparate physiological responses. However, the molecular and cellular basis for functional diversity of ECs remains to be adequately defined. Here, we integrated single-cell transcriptomics with spatial image analysis to identify 14 EC clusters that are topographically organized along the gut. Subtypes predicted to be sensitive to the chemical environment and mechanical forces were identified that express distinct transcription factors and hormones. A Piezo2+ population in the distal colon was endowed with a distinctive neuronal signature. Using a combination of genetic, chemogenetic, and pharmacological approaches, we demonstrated Piezo2+ ECs are required for normal colon motility. Our study constructs a molecular map for ECs and offers a framework for deconvoluting EC cells with pleiotropic functions.