Kwan Edwin P, Gao Xiaodong, Leung Yuk M, Gaisano Herbert Y
Department of Medicine, University of Toronto, Toronto, Ontario, Canada.
Pancreas. 2007 Oct;35(3):e45-54. doi: 10.1097/mpa.0b013e318073d1c9.
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.
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.
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.
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.
我们最近利用FM1-43荧光成像和电子显微镜技术报道，胰高血糖素样肽（GLP-1）介导的环磷酸腺苷（cAMP）增强胰岛素分泌作用显著促进了质膜（PM）胞吐位点的数量以及复合性和连续性胞吐作用中胰岛素分泌颗粒（SG）与颗粒之间的融合。
在此，我们使用FM1-43成像技术来剖析假定的GLP-1/cAMP激活底物——cAMP直接激活的交换蛋白（EPAC）和蛋白激酶A（PKA）——在介导这些胞吐事件中的不同作用。
与GLP-1一样，福斯高林激活cAMP增加了PM胞吐位点的数量（2.3倍），这些位点主要是持续强烈型（55.8%）和逐步多相型（37.7%）模式，分别对应复合性和连续性SG-SG胞吐作用，只有少数单相热点（6.5%）对应单个颗粒胞吐作用。8-（4-氯苯硫基）-2'-O-甲基腺苷-3',5'-cAMP直接激活EPAC也增加了胞吐位点的数量，但主要是多相型（60%）和单相型（40%）热点。H89抑制蛋白激酶A可阻断福斯高林诱发的持续强烈型热点，同时保留多相型（47%）和单相型（53%）热点。同样，PKA激活剂（N6-苯甲酰腺苷-3',5'-cAMP）仅诱发多相型（60%）和单相型（40%）热点。这些结果表明，PKA激活是必需的，但单独激活不足以促进复合性SG-SG融合。8-（4-氯苯硫基）-2'-O-甲基腺苷-3',5'-cAMP加N6-苯甲酰腺苷-3',5'-cAMP刺激完全重现了福斯高林的作用效果，包括增加胞吐位点的数量，其持续强烈型（42.6%）和逐步型（39.6%）热点模式相似，单相型热点较少（17.8%）。
EPAC和PKA调节不同的和共同的胞吐步骤以增强胰岛素胞吐作用，其中（a）EPAC激活促使SGs在PM处融合，从而增加PM胞吐位点的数量；（b）PKA和EPAC激活协同调节复合性和连续性胞吐作用中SG-SG融合。
