GRP40介导游离脂肪酸对葡萄糖刺激的胰岛素分泌和β细胞内钙离子浓度的变化

GPR40 mediates the effects of FFAs on glucose stimulated insulin secretion and the change of intracellular calcium concentration in pancreatic β cells

目的探讨G蛋白耦联受体40（GPR40）是否介导游离脂肪酸（FFAs）短期、长期作用对小鼠胰岛NIT-1β细胞葡萄糖刺激的胰岛素分泌（GSIS）及胞内钙离子浓度的影响。方法利用siRNA技术抑制GPR40在NIT-1细胞的表达，ELISA法检测棕榈酸、油酸短期（2h）、长期（48h）干预对NIT-1细胞GSIS的影响，激光共聚焦显微镜下观察GSIS过程中细胞内钙离子的变化。结果FFAs短期干预促进空转染组、对照siRNA转染组GSIS水平（P〈0．01），而对GPR40 siRNA组GSIS无明显影响，该组细胞GSIS水平明显低于空转染组和对照siRNA转染组（P〈0．01）。FFAs长期干预明显抑制空转染组、对照siRNA转染组细胞GSIS水平，对GPR40 siRNA组GSIS无明显影响。空转染组、对照siRNA组GSIS水平低于GPR40 siRNA组（P〈0．05）。激光共聚焦显微镜结果显示，在GSIS过程中，FFAs短期干预后，GPR40 siRNA组胞内钙离子浓度峰值明显低于对照siRNA组（棕榈酸：5．10vs7．02，油酸：4．27vs6．21，均P〈0．05）；而FFAs长期干预后，GPR40 siRNA组胞内钙离子浓度峰值明显高于对照siRNA组（棕榈酸：3．24vs1．21，油酸：2．83vs1．18，均P〈0．05）。结论GPR40介导FFAs对GSIS和胞内钙离子浓度变化的短期刺激和长期抑制作用。

Objective To investigate whether G-protein coupled receptor 40（GPR40）mediates short-term and long-term effects of free fatty acids （FFAs） on glucose stimulated insulin secretion （GSIS） and the change of [ Ca^2＋]i in mouse insulinoma NIT-1 cells. Methods The expression of GPR40 in NIT-1 cells was inhibited by siRNA. The short-term and long-term effects of saturated （palmitate） or unsaturated （oleate） fatty acids on GSIS in NIT-1 cells transfected with GPR40 siRNA were examined. Insulin was measured by ELISA. The change in intracellular calcium concentration was observed by laser confocal scanning microscopy after NIT-1 cells were incubated with Fluo-3/AM. Results The short-term treatment of FFAs increased GSIS from NIT-1 cells transfected with control siRNA or mock（ P〈0.01 ） ,but not from cells transfected with GPR40 siRNA. The exposure of mock,control siRNA transfected cells to FFAs for 48 h resulted in attenuation of GSIS compared to nonexposed cells. In contrast,insulin secretion from cells transfected with GPR40 siRNA was not impaired by exposure to FFAs for 48 h. After incubation with FFAs for 2 h, the maximal fluorescence intensity in control siRNA transfected cells was higher compared with that in GPR40 siRNA transfected cells during the process of GSIS （P〈0.05）. However, after incubation with FFAs for 48 h, the maxiaml fluorescence intensity in control siRNA transfected cells was lower than that in GPR40 siRNA transfected cells during the process of GSIS （ P 〈 0.05 ）. Conclusion GPR40 is implicated in mediating the short-term and long-tern effects of FFAs on GSIS and the change of intracellular calcium concentration.