胰岛素激活PI_3K-AKT通路而促进H9c2心肌细胞葡萄糖摄取  

Insulin promotes glucose uptake via PI_3K-AKT pathway in H9c2 myocardial cells

作　　者：叶林[1] 陆凯[2] 张冬颖[2] 覃数[2] 

机构地区：[1]重庆医科大学附属第一医院重症医学科,重庆400016 [2]重庆医科大学附属第一医院心血管内科,重庆400016

出　　处：《第三军医大学学报》2015年第6期538-542,共5页Journal of Third Military Medical University

摘　　要：目的探索胰岛素(Insulin)对H9c2心肌细胞葡萄糖摄取的影响及其与PI3K-AKT信号通路的关系。方法采用不同时间及不同浓度的胰岛素处理H9c2心肌细胞,确定胰岛素处理H9c2细胞的最佳浓度和最适时间。实验分空白对照组(NC组),2-NBDG组,Insulin组,Insulin+PI3K-AKT通路抑制剂LY294002组(LY294002组),Insulin+p38MAPK通路抑制剂BIRB796组(BIRB796组),使用荧光标记2-脱氧葡萄糖(2-NBDG)检测H9c2心肌细胞葡萄糖摄取能力,以流式细胞仪和荧光酶标仪检测心肌细胞对葡萄糖摄取的变化。通过间接免疫荧光法及激光共聚焦检测H9c2心肌细胞葡萄糖转运体-1(glucose tansporter-1,GLUT-1)及葡萄糖转运体-4(glucose tansporter-4,GLUT-4)的表达。结果 12-NBDG可用于检测H9c2心肌细胞葡萄糖摄取能力。2Insulin处理H9c2心肌细胞最适浓度和时间分别为200μmol/L和30 min。3Insulin组与对照组相比增加H9c2心肌细胞葡萄糖摄取(P<0.05);LY294002组与Insulin组相比降低心肌细胞葡萄糖摄取,两组间差异明显(P<0.05),BIRB796组较Insulin组无统计学差异(P>0.05),提示LY294002可抑制Insulin促进心肌细胞葡萄糖摄取,而BIRB796不能抑制上述作用。4 H9c2心肌细胞膜表达葡萄糖转运体1(GLUT1)和葡萄糖转运体4(GLUT4),Insulin处理H9c2心肌细胞30min后经激光共聚焦成像显示H9c2心肌细胞膜GLUT1和GLUT4表达增加(P<0.05)。结论胰岛素促进H9c2心肌细胞葡萄糖摄取的作用可能与PI3K-AKT通路相关,与p38MAPK通路不相关,胰岛素激活PI3K-AKT信号通路后可能促进H9c2心肌细胞膜上GLUT-1及GLUT-4的表达,从而使H9c2心肌细胞对葡萄糖摄取增加。Objective To explore the effect of insulin on glucose uptake in H9c2 myocardial cells and its relationship with PI3K-AKT pathway. Methods A fluorescence microplate reader was used to determine the optimal concentration and incubation time of insulin for H9c2 cells. The H9c2 cells were divided to 5 groups： a normal control group, a 2-[ N-（7-nitrobenz-2-oxa-1,3-diaxol-4-yl）amino]-2- deoxyglucose （2-NBDG） group, an insulin group, an insulin ＋ LY294002 （PI3K-AKT pathway inhibitor） group, and an insulin ＋ BIRB796 （p38MAPK pathway inhibitor） group. The glucose uptake was analyzed with the fluorescence microplate reader and a flow cytometer. The expression of glucose tansporter-1 （ GLUT1 ） and glucose tansporter-4 （GLUT4） in the H9c2 cells was detected by indirect immunofluorescence assay and confocal laser scanning analysis. Results 2-NBDG could be used to detect glucose uptake in H9c2 cells. The optimal concentration and incubation time of insulin were 200 μmol/L and 30 rain, respectively. Insulin could promote glucose uptake in the H9c2 cells （P 〈 0. 05 ）, and this effect was blocked by LY294002 （P 〈 0. 05 ） but not by BIRB796 （P 〉 0. 05 ）. GLUT1 and GLUT4 were expressed in the H9c2 cell membranes,and insulin increased the expressions of GLUTland GLUT4. Conclusion Insulin can increase the expression of GLUT1 and GLUT4 via PI3 K-AKT pathway rather than p38MAPK pathway, and then promote glucose uptake in the H9c2 ceils.

关 键 词：胰岛素 2-NBDG H9C2心肌细胞 葡萄糖摄取 PI3K-AKT p38MAPK GLUT1和GLUT4 

分 类 号：Q578[生物学—生物化学] R322.11[医药卫生—人体解剖和组织胚胎学]