葡萄糖转运蛋白4及其下游信号分子在高糖刺激下肾小球系膜细胞中的作用

目的 探讨高糖和胰岛素对肾小球系膜细胞（GMC）葡萄糖转运蛋白4（GLUT4）和Cbl相关蛋白（CAP）的mRNA表达及细胞骨架纤维状肌动蛋白F-actin 的影响，探讨糖尿病肾病发生发展中GLUT4 及其下游分子F-actin和CAP的重要作用。 方法 将细胞分为8组：正常对照组、生理浓度胰岛素（10－9 mol/L）组、低浓度胰岛素（10－8 mol/L）组、高浓度胰岛素（10－6 mol/L）组、高糖（30 mmol/L）组、甘露醇组（25 mmol/L甘露醇＋5 mmol/L葡萄糖）、高糖加高浓度胰岛素组、高糖加生理浓度胰岛素组。采用RT-PCR法和免疫组化法，观察不同情况下GMC中GLUT4蛋白和mRNA以及CAP mRNA 的表达及其变化。Rhodamine-phalloidin染色和激光共聚焦显微镜观察F-actin形态及荧光强度。 结果 正常对照组GMC中GLUT4蛋白和mRNA以及CAP mRNA有一定表达，而生理浓度胰岛素组与正常对照组差异均无统计学意义。高糖组GLUT4蛋白（P < 0.01)和mRNA（P < 0.05）以及CAP mRNA（P < 0.01)表达均显著减少，F-actin解聚增加（P < 0.01）；而甘露醇组以上各指标与对照组差异均无统计学意义。低浓度胰岛素组和高浓度胰岛素组GLUT4 mRNA表达分别为生理浓度胰岛素组的2.06倍和2.66倍，GLUT4蛋白表达分别为对照组的1.93倍和2.83倍，CAP mRNA表达分别为对照组的1.91倍和2.15倍，F-actin荧光强度分别为对照组的1.296倍及1.224倍，均呈一定的浓度依赖性。高糖加高浓度胰岛素组GLUT4 mRNA表达为高糖组的2.15倍（P < 0.05），GLUT4蛋白表达为高糖组的2.08倍（P < 0.01），CAP mRNA表达为高糖组的2.14倍（P < 0.01），F-actin荧光强度为高糖组的1.838倍（P < 0.01）。GLUT4 mRNA与CAP mRNA呈正相关（r = 0.905，P = 0.002）；GLUT4与F-actin呈正相关（r = 0.929，P = 0.001）。 结论 （1）正常GMC中GLUT4 mRNA与蛋白、CAP mRNA有一定表达。（2）高糖可抑制GLUT4的蛋白和mRNA以及CAP mRNA表达，促进F-actin解聚。（3）胰岛素能部分拮抗高糖导致系膜细胞中GLUT4的蛋白和mRNA以及CAP mRNA表达的下调作用。（4）GLUT4、CAP和F-actin是糖尿病肾病发生发展的重要影响因子之一。

Effects of high glucose and insulin on expression of glucose transporter 4, Cbl-associated protein and cytoskeleton protein F-actin in rat glomerular mesangial cells in vitro

Objective To investigate the effects of high glucose and insulin on the expression of glucose transporter 4 (GLUT4), Cbl-associated protein (CAP) and cytoskeleton protein F-actin of glomerular mesangial cells (GMCs), in order to explore the function of GLUT4, Cbl-associated protein and F-actin in the pathogenesis and development of diabetic nephropathy (DN). Methods Cultured 1097 rat glomerular mesangial cells were divided into 8 groups: control, 10-9 mol/L insulin, 10-8 mol/L insulin, 10-6 mol/L insulin, high glucose (30 mmol/L), mannitol (25 mmol/L mannitol+5 mmol/L glucose), high glucose plus 10-6 mol/L insulin, high glucose plus 10-9 mol/L insulin. Expression of CAP mRNA and GLUT4 was measured by RT-PCR and immunohistochemistry method. F-actin was stained by rhodamine-pholloidin and the fluorescent intensity was calculated by image analysis system. Results The expression of GLUT4 mRNA and protein, CAP mRNA was found in normal giomerular mesangial cells (control), and there was no significant difference in 10-9 mol/L insulin group. The expression of GLUT4 mRNA (P<0.05) and protein (P<0.01), CAP mRNA (P<0.01) level was decreased in high glucose group compared with that of control group, but there was no significant difference in mannitol group. The expression of GLUT4 and CAP mRNA up-regulated with the increase of concentration of insulin. The expressions of GLUT4 mRNA in 10-8 mol/L insulin and 10-6 mol/L insulin groups were 2.06-fold and 2.66-fold of 10-9 mol/L insulin group, of GLUT4 protein were 1.93-fold and 2.83-fold of control, and of CAP mRNA were 1.91-fold and 2.15-fold of control, respectively. The expressions of GLUT4 mRNA, GLUT4 protein, CAP mRNA in high glucose plus insulin group were 2.15-fold, 2.08-fold, 2.14-fold of high glucose group respectively. High glucose decreased the fluorescent intensity of F-actin to 44.5% (P<0.01). 10-8 mol/L insulin and 10-6 mol/L insulin groups increased to 1.224-fold (P<0.05), 1.296-fold (P<0.01) in a concentration-dependent manner. The spearman correlation coefficient between GLUT4 and F-actin was 0.929 (P=0.001), between GLUT4 mRNA and CAP mRNA was 0.905 (P=0.002). Conclusions (1) A certain expression of GLUT4 mRNA and protein, CAP mRNA from GMC is found in normal glomerular mesangial cells. (2) High glucose can inhibit the expression of GLUT4 and CAP mRNA significantly, and facilitate the depolymerization of F-aetin. (3) Insulin can reverse down-regnlation of GLUT4 and CAP mRNA caused by high glucose. (4) GLUT4, CAP and F-actin are important factors in the development of DN.