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首页> 外文期刊>Cellular Physiology and Biochemistry >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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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

机译：在标准细胞培养条件下，AMP激酶调节剂AICAR，二甲双胍和化合物C对INS-1E大鼠胰岛素瘤细胞胰岛素分泌的影响

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Background/Aims The function of β-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 KsubATP/sub channels and cell membrane depolarization, which triggers stimulus-secretion coupling (SSC). In addition to KsubATP/sub channels, the AMP-dependent protein kinase (AMPK), a major cellular fuel sensor in a variety of cells and tissues, also affects insulin secretion and β-cell survival. In a previous study we found that the widely used AMPK inhibitor compound C retards proliferation and induces apoptosis in the rat β-cell line INS-1E. We therefore tested the effects of AMPK activators (AICAR and metformin), and compound C on AMPK phosphorylation, insulin secretion, KsubATP/sub 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 µM). Both AICAR and compound C stimulated basal insulin secretion whereas metformin had no effect. Pre-incubation with AICAR (1 mM) caused an inhibition of KsubATP/sub currents but did not significantly alter the average cell membrane potential (Vm) or the threshold potential of electrical activity. Acute administration of AICAR (300 µM) 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 (Casubi/sub). AICAR had no additive blocking effect on KsubATP/sub 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.
机译：背景/目的β细胞的功能由养分吸收和代谢调节。细胞的代谢状态可以表示为AMP，ADP和ATP的浓度比。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 通道的关闭和细胞膜去极化，从而触发刺激-分泌偶联（SSC）。除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。在先前的研究中，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-1E的增殖并诱导其凋亡。因此，我们测试了AMPK激活剂（AICAR和二甲双胍）和化合物C对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。在标准细胞培养条件下（11 mM葡萄糖）的INS-1E细胞。方法Western blotting，ELISA，膜片钳，钙成像和流式细胞仪。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 µM）的影响。 AICAR和化合物C均可刺激基础胰岛素分泌，而二甲双胍则无作用。用AICAR（1 mM）预温育可抑制K ATP 电流，但不会显着改变平均细胞膜电位（Vm）或电活动阈值电位。急性给予AICAR（300 µM）导致Vm去极化，这不是由于抑制了基础或葡萄糖诱导的氯化物电导，也没有伴随细胞内钙（Ca i ）。当与甲苯磺丁酰胺一起使用时，AICAR对K ATP 电流没有附加的阻滞作用。在24小时内施用的化合物C诱导半胱天冬酶活性呈阳性的细胞百分比增加，而AICAR（1 mM）施用48小时则没有效果。中度葡萄糖浓度<3 mM导致细胞周期停滞，胱天蛋白酶激活并增加细胞粒度。结论我们得出的结论是，在标准细胞培养条件下，AMPK调节剂AICAR和化合物C（而非二甲双胍）通过AMPK独立机制刺激胰岛素分泌。

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