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 beta cells.
OBJECTIVE
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.
Kwan
Edwin P
EP
Department of Medicine, University of Toronto, Toronto, Ontario, Canada.
Gao
Xiaodong
X
Leung
Yuk M
YM
Gaisano
Herbert Y
HY
D000818:Animals; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D004789:Enzyme Activation; D005089:Exocytosis / Q000502:physiology*; D052216:Glucagon-Like Peptide 1 / Q000502:physiology; D020662:Guanine Nucleotide Exchange Factors / Q000502:physiology*; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism*; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D008561:Membrane Fusion / Q000502:physiology; D008856:Microscopy, Fluorescence; D051381:Rats; D017207:Rats, Sprague-Dawley; D015290:Second Messenger Systems / Q000187:drug effects / Q000502:physiology; D022142:Secretory Vesicles / Q000502:physiology; D013449:Sulfonamides / Q000494:pharmacology
C473217:8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate; C482601:Epac protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D007328:Insulin; D007546:Isoquinolines; D013449:Sulfonamides; D005576:Colforsin; C050086:N(6)-benzoyl-cyclic AMP; D052216:Glucagon-Like Peptide 1; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide