Enhanced Rap1 Activation and Insulin Secretagogue Properties of an Acetoxymethyl Ester of an Epac-selective Cyclic AMP Analog in Rat INS-1 Cells STUDIES WITH 8-pCPT-2′-O-Me-cAMP-AM
Enhanced Rap1 Activation and Insulin Secretagogue Properties of an Acetoxymethyl Ester of an Epac-selective Cyclic AMP Analog in Rat INS-1 Cells STUDIES WITH 8-pCPT-2′-O-Me-cAMP-AM
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DOI：
10.1074/jbc.m900166200
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发表时间：
2009-04-17
影响因子：
4.8
通讯作者：
Holz, George G.
中科院分区：
文献类型：
--
作者：
Chepurny, Oleg G.;Leech, Colin A.;Holz, George G.
为了确定介导cAMP的胰岛素促分泌作用的环核苷酸结合蛋白的身份，在胰腺β细胞系（大鼠INS-1细胞）中评价了由cAMP直接激活的交换蛋白（Epac）和蛋白激酶A（PKA）的可能贡献。Rap 1激活，CREB磷酸化和PKA依赖性基因表达的测定与活细胞成像和基于荧光共振能量转移的cAMP传感器（Epac 1-cams）的高通量筛选相结合，以验证cAMP类似物的乙酰氧基甲基酯（AM-酯）优先激活Epac或PKA的选择性。将INS-1细胞分别暴露于8-pCPT-2 ′-O-Me-cAMP-AM或Bt（2）cAMP-AM后，Epac或PKA被选择性激活。这两种cAMP类似物均表现出剂量依赖性和葡萄糖代谢依赖性作用以刺激胰岛素分泌，并且当每一种与另一种共同给药时，观察到超累加效应。因为与8-pCPT-2 ′-O-Me-cAMPAM相比，响应于饱和浓度（10 μ M）的Bt（2）cAMP-AM分泌的胰岛素多2.4倍，并且因为Bt（2）cAMP-AM的作用而不是8-pCPT-2 ′-O-Me-cAMP-AM的作用几乎被3 μ M PKA抑制剂H-89消除，所以得出结论，对于INS-1细胞，PKA作为支持胰岛素分泌的主要cAMP结合蛋白。出乎意料的是，10-100 μ M的8-pCPT-2 ′-O-Me-cAMP的非AM-酯不能刺激胰岛素分泌，并且是INS-1细胞中Rap 1的弱激活剂。此外，10 μ M的8-pCPT-2 ′-O-Me-cAMP的AM-酯刺激小鼠胰岛的胰岛素分泌，而非AM-酯则没有。因此，8-pCPT-2 ′-O-Me-cAMP在胰岛素分泌细胞中的膜通透性很低，从而限制了其生物活性。得出结论，需要通过使用该cAMP类似物的AM-酯来重新评估先前记录8-pCPT-2 '-O-Me-cAMP在β细胞或其他细胞类型中作用失败的报告。
To ascertain the identities of cyclic nucleotide-binding proteins that mediate the insulin secretagogue action of cAMP, the possible contributions of the exchange protein directly activated by cAMP (Epac) and protein kinase A (PKA) were evaluated in a pancreatic beta cell line (rat INS-1 cells). Assays of Rap1 activation, CREB phosphorylation, and PKA-dependent gene expression were performed in combination with live cell imaging and high throughput screening of afluorescence resonance energy transfer-based cAMP sensor (Epac1-camps) to validate the selectivity with which acetoxymethyl esters(AM-esters) of cAMP analogs preferentially activate Epac or PKA. Selective activation of Epac or PKA was achieved following exposure of INS-1 cells to 8-pCPT-2'-O-Me-cAMP-AM or Bt(2)cAMP-AM, respectively. Both cAMP analogs exerted dose-dependent and glucose metabolism-dependent actions to stimulate insulin secretion, and when each was co-administered with the other, a supra-additive effect was observed. Because 2.4-fold more insulin was secreted in response to a saturating concentration (10 mu M) of Bt(2)cAMP-AM as compared with 8-pCPT-2'-O-Me-cAMPAM, and because the action of Bt(2)cAMP-AM but not 8-pCPT-2'-O-Me-cAMP-AM was nearly abrogated by treatment with 3 mu M of the PKA inhibitor H-89, it is concluded that for INS-1 cells, it is PKA that acts as the dominant cAMP-binding protein in support of insulin secretion. Unexpectedly, 10-100 mu M of the non-AM-ester of 8-pCPT-2'-O-Me-cAMP failed to stimulate insulin secretion and was a weak activator of Rap1 in INS-1 cells. Moreover, 10 mu M of the AM-ester of 8-pCPT-2'-O-Me-cAMP stimulated insulin secretion from mouse islets, whereas the non-AM-ester did not. Thus, themembrane permeability of 8-pCPT-2'-O-Me-cAMP ininsulin-secreting cells is so low as to limit its biological activity. It is concluded that prior reports documenting the failure of 8-pCPT-2'-O-Me-cAMP to act in beta cells, or other cell types, need to be re-evaluated through the use of the AM-ester of this cAMP analog.