20-HETE对小鼠葡萄糖刺激胰岛素分泌反应的影响

为了考察20-羟基二十碳四烯酸(20-hydroxyeicosatetraenoic acids, 20-HETE)对葡萄糖刺激胰岛素分泌反应的影响,本研究选择CYP4F2转基因小鼠和小鼠胰岛素瘤INS-1E细胞作为研究材料,通过LCMS/MS检测WT和TG小鼠的胰腺20-HETE水平。通过IPGTT测定小鼠葡萄糖耐量,通过ELISA测定小鼠血浆C肽水平来检测胰岛素分泌。通过Western blotting、Real time PCR、免疫组化和免疫荧光来检测小鼠胰腺或INS-1E细胞中Glut2、GSK-3β(Ser9点)和AKT (Ser473点)的磷酸化水平。TG小鼠的20-HETE水平((7.26±2.03) ng/mg蛋白)显著高于WT小鼠((2.14±0.76) ng/mg蛋白)。在用20-HETE合成的选择性抑制剂HET0016处理后,TG小鼠((0.33±0.07) ng/mg蛋白)和WT小鼠((0.27±0.06) ng/mg蛋白)胰腺组织中的20-HETE水平均急剧降低。给予葡萄糖处理30 min后,TG小鼠的血糖水平均显著高于WT小鼠,而血浆C肽水平显著低于WT小鼠(p<0.05)。与WT小鼠相比,TG小鼠的胰腺组织中Glut2 m RNA和蛋白水平显著降低。与WT小鼠相比,CYP4F2转基因小鼠的GSK-3β和AKT磷酸化均显著降低。20-HETE处理可导致INS-1E细胞中AKT/GSK-3β磷酸化水平和Glut2表达水平显著降低(p<0.05)。此外,用17 mmol/L葡萄糖处理INS-1E细胞1 h,20-HETE处理组的胰岛素分泌显著降低。应用GSK-3β选择性抑制剂TWS119预处理INS-1E细胞3 h后,TWS119 (一种GSK-3β选择性抑制剂)预处理显著逆转了Glut2表达水平的降低以及胰岛素分泌的减少。20-HETE主要通过AKT/GSK-3β信号通路来下调Glut2的表达,进而减弱胰岛素分泌,导致胰岛素分泌功能障碍。

Effect of 20-HETE on Glucose-stimulated Insulin Secretion in Mice

To investigate the effects of 20-hydroxyeicosatetraenoic acids(20-HETE) on glucose-stimulated insulin secretion, CYP4 F2 transgenic mice and mouse insulinoma INS-1 E cells were selected as study materials, and pancreatic 20-HETE levels in WT and TG mice were detected by LC-MS/MS. Mice glucose tolerance was determined by IPGTT, and mouse plasma C-peptide levels were measured by ELISA to detect insulin secretion.Glut2, phosphorylation levels of GSK-3β(Ser9) and AKT(Ser473) in mouse pancreas or INS-1 E cells were detected by Western blotting, Real time PCR, immunohistochemistry and immunofluorescence. The 20-HETE level in TG mice((7.26±2.03) ng/mg protein) was significantly higher than that of WT mice((2.14±0.76) ng/mg protein), after treatment with HET0016(a selective inhibitor of 20-HETE synthesis), the 20-HETE levels in pancreatic tissue of TG mice((0.33±0.07) ng/mg protein) and WT mice((0.27±0.06) ng/mg protein) decreased dramatically. After 30 minutes of glucose treatment, the blood glucose level of TG mice was significantly higher than that of WT mice,and the plasma C-peptide level was significantly lower than that of WT mice(p<0.05). Glut2 mRNA and protein levels were significantly reduced in pancreatic tissue of TG mice compared to WT mice. Both GSK-3β and AKT phosphorylation were significantly reduced in CYP4 F2 transgenic mice compared to WT mice. 20-HETE treatment resulted in a significant decrease in AKT/GSK-3β phosphorylation and Glut2 expression levels in INS-1 E cells(p <0.05). In addition, INS-1 E cells were treated with 17 mmol/L glucose for 1 h, and insulin secretion was significantly reduced in the 20-HETE treated group. After pretreatment of INS-1 E cells with TWS119(a GSK-3βselective inhibitor) for 3 h, TWS119 pretreatment significantly reversed the decrease of Glut2 expression and the decrease of insulin secretion. 20-HETE mainly down-regulates the expression of Glut2 through AKT/GSK-3βsignaling pathway, which in turn weakens insulin secretion and leads to insulin secretion dysfunction.