Effect of the AMP-Kinase Modulators AICAR, Metformin and Compound C on Insulin Secretion of INS-1E Rat Insulinoma Cells under Standard Cell Culture Conditions
Effect of the AMP-Kinase Modulators AICAR, Metformin and Compound C on Insulin Secretion of INS-1E Rat Insulinoma Cells under Standard Cell Culture Conditions
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DOI：
10.1159/000337589
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发表时间：
2012-01-01
影响因子：
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通讯作者：
Ritter, Markus
中科院分区：
文献类型：
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作者：
Langelueddecke, Christian;Jakab, Martin;Ritter, Markus
背景/目的：β细胞的功能受营养摄取和代谢的调节。细胞的代谢状态可用AMP、ADP和ATP的浓度比来表示。这些比率的相对变化调节胰岛素的释放。细胞内ATP浓度的增加导致K-ATP通道关闭和细胞膜去极化，从而触发刺激-分泌偶联（SSC）。除了K-ATP通道外，AMP依赖性蛋白激酶（AMPK）是多种细胞和组织中的主要细胞燃料传感器，也影响胰岛素分泌和β细胞存活。在之前的一项研究中，我们发现广泛使用的AMPK抑制剂化合物C可以抑制大鼠β细胞系INS-1 E的增殖并诱导细胞凋亡。因此，我们测试了AMPK激活剂（AICAR和二甲双胍）和化合物C在标准细胞培养条件（11 mM葡萄糖）下对INS-1 E细胞的AMPK磷酸化、胰岛素分泌、K-ATP通道电流、细胞膜电位、细胞内钙浓度、细胞凋亡和细胞周期分布的影响。方法：Western blotting、ELISA、膜片钳、钙离子成像和流式细胞术。结果：我们发现基础AMPK磷酸化被AICAR（1 mM）和二甲双胍（1 mM）增强，但不受化合物C（10 μ M）的影响。AICAR和化合物C均刺激基础胰岛素分泌，而二甲双胍没有作用。与AICAR（1 mM）的预孵育引起的抑制K-ATP电流，但没有显着改变的平均细胞膜电位（Vm）或阈值电位的电活动。急性给药AICAR（300 μ M）导致Vm的去极化，这不是由于基础或葡萄糖诱导的氯离子电导的抑制，并且不伴有细胞内钙（Ca-1）的升高。AICAR与甲苯磺丁脲合用时对K-ATP电流无相加性阻断作用。在24小时内施用的化合物C诱导半胱天冬酶活性阳性细胞百分比的增加，而施用48小时的AICAR（1 mM）没有效果。培养基中葡萄糖浓度< 3 mM时，细胞周期停滞，半胱天冬酶激活，细胞颗粒度增加。结论：我们的结论是，在标准细胞培养条件下，AMPK调节剂AICAR和化合物C，但不是二甲双胍，刺激胰岛素分泌AMPK的非依赖性机制。版权所有（C）2012 S. Karger AG，巴塞尔
Background/Aims: The function of beta-cells is regulated by nutrient uptake and metabolism. The cells' metabolic state can be expressed as concentration ratios of AMP, ADP and ATP. Relative changes in these ratios regulate insulin release. An increase in the intracellular ATP concentration causes closure of K-ATP channels and cell membrane depolarization, which triggers stimulus-secretion coupling (SSC). In addition to K-ATP channels, the AMP-dependent protein kinase (AMPK), a major cellular fuel sensor in a variety of cells and tissues, also affects insulin secretion and beta-cell survival. In a previous study we found that the widely used AMPK inhibitor compound C retards proliferation and induces apoptosis in the rat beta-cell line INS-1E. We therefore tested the effects of AMPK activators (AICAR and metformin), and compound C on AMPK phosphorylation, insulin secretion, K-ATP channel currents, cell membrane potential, intracellular calcium concentration, apoptosis and cell cycle distribution of INS-1E cells under standard cell culture conditions (11 mM glucose). Methods: Western blotting, ELISA, patch-clamp, calcium imaging and flow cytometry. Results: We found that basal AMPK phosphorylation is enhanced by AICAR (1 mM) and metformin (1 mM) but remained unaffected by compound C (10 mu M). Both AICAR and compound C stimulated basal insulin secretion whereas metformin had no effect. Pre-incubation with AICAR (1 mM) caused an inhibition of K-ATP currents but did not significantly alter the average cell membrane potential (Vm) or the threshold potential of electrical activity. Acute administration of AICAR (300 mu M) led to a depolarization of Vm, which was not due to an inhibition of the basal-or glucose-induced chloride conductance, and was not accompanied by elevations of intracellular calcium (Ca-i). AICAR had no additive blocking effect on K-ATP currents when applied together with tolbutamide. Compound C applied over 24 hours induced an increase in the percentage of cells positive for caspase activity, whereas AICAR (1 mM) applied for 48 hours was without effect. Medium glucose concentration < 3 mM caused cell cycle arrest, caspase activation and an increase of cell granularity. Conclusion: We conclude that under standard cell culture conditions the AMPK modulators AICAR and compound C, but not metformin, stimulate insulin secretion by AMPK-independent mechanisms. Copyright (C) 2012 S. Karger AG, Basel