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Cited by 33Pancreatic function and diabetesCellular transport and secretionCalcium signaling and nucleotide metabolism

Activation of Exchange Protein Directly Activated by Cyclic Adenosine Monophosphate and Protein Kinase A Regulate Common and Distinct Steps in Promoting Plasma Membrane Exocytic and Granule-to-Granule Fusions in Rat Islet β Cells

Kwan, Edwin P., Gao, Xiaodong, Leung, Yuk M., Gaisano, Herbert Y.·2007·Pancreas
DOI 10.1097/mpa.0b013e318073d1c9
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Abstract

Objectives: Using FM1-43 epifluorescence imaging and electron microscopy, we recently reported that glucagon-like peptide (GLP-1)-mediated cyclic adenosine monophosphate (cAMP) potentiation of insulin secretion markedly promotes the number of plasma membrane (PM) exocytic sites and insulin secretory granule (SG)-to-granule fusions underlying compound and sequential exocytosis. Methods: Here, we used FM1-43 imaging to dissect the distinct contributions of putative GLP-1/cAMP activated substrates-exchange protein directly activated by cAMP (EPAC) and protein kinase A (PKA)-in mediating these exocytic events. Results: Like GLP-1, cAMP activation by forskolin increased the number of PM exocytic sites (2.3-fold), which were mainly of the robust-sustained (55.8%) and stepwise-multiphasic (37.7%) patterns corresponding to compound and sequential SG-SG exocytosis, respectively, with few monophasic hotspots (6.5%) corresponding to single-granule exocytosis. Direct activation of EPAC by 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cAMP also increased the number of exocytic sites, but which were mainly multiphasic (60%) and monophasic (40%) hotspots. Protein kinase A inhibition by H89 blocked forskolin-evoked robust-sustained hotspots, while retaining multiphasic (47%) and monophasic (53%) hotspots. Consistently, PKA activation (N6-benzoyladenosine-3′,5′-cAMP) evoked only multiphasic (60%) and monophasic (40%) hotspots. These results suggested that PKA activation is required but alone is insufficient to promote compound SG-SG fusions. 8-(4-Chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cAMP plus N6- benzoyladenosine-3′,5′-cAMP stimulation completely reconstituted the effects of forskolin, including increasing the number of exocytic sites, with a similar pattern of robust-sustained (42.6%) and stepwise (39.6%) hotspots and few monophasic (17.8%) hotspots. Conclusions: The EPAC and PKA modulate both distinct and common exocytic steps to potentiate insulin exocytosis where (a) EPAC activation mobilizes SGs to fuse at the PM, thereby increasing number of PM exocytic sites; and (b) PKA and EPAC activation synergistically modulate SG-SG fusions underlying compound and sequential exocytoses. Abbreviations: CICR - calcium-induced calcium release, 8-CPT-2′-O-Me-cAMP - 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate, EPAC - exchange protein directly activated by cAMP, IBMX - 3-isobutyl-1-methylxanthine, GLP-1 - glucagon-like peptide 1, N6-Bnz-cAMP - N6-benzoyladenosine-3′,5′-cyclic monophosphate, PKA - protein kinase A, PM - plasma membrane, RRP - readily releasable pool, SG - secretory granule, SUR-1 - sulfonylurea receptor 1

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Crossref · 10.1097/mpa.0b013e318073d1c9

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Authors
Kwan, E. P., Gao, X., Leung, Y. M., & Gaisano, H. Y.
Affiliations
University of Toronto (CA)
ORCID
Venue
Pancreas
Publisher
Ovid Technologies (Wolters Kluwer Health)
Published
2007-10
Volume
35
Issue
3
Pages
e45-e54
ISSN
0885-3177 (ISSN-L)
DOI
10.1097/mpa.0b013e318073d1c9
OpenAlex
W2022674028
Cited by
33
References
37
Citations by year
2023: 2 · 2019: 1 · 2016: 1 · 2015: 2 · 2014: 5 · 2013: 3 · 2012: 7
FWCI
3.65
Type
journal article
Language
en
Open access
closed
Locations
  • Pancreas (publishedVersion)
  • PubMed (publishedVersion)
Topics
  • Health Sciences › Medicine › Surgery › Pancreatic function and diabetes
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Physiology › Calcium signaling and nucleotide metabolism
Keywords
Epac, protein kinase A, exocytosis, insulin secretory granules, compound exocytosis, glucagon-like peptide-1, cyclic AMP, pancreatic beta cells, forskolin, H89, Epac1, Epac2, FM1-43
MeSH terms
Insulin Secretion, Cyclic AMP, Animals, Enzyme Activation, Exocytosis, Colforsin, Insulin, Islets of Langerhans, Isoquinolines, Male, Membrane Fusion, Microscopy, Fluorescence, Sulfonamides, Second Messenger Systems, Rats, Sprague-Dawley, Cyclic AMP-Dependent Protein Kinases, Guanine Nucleotide Exchange Factors, Secretory Vesicles, Rats, Glucagon-Like Peptide 1

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