磷脂酰肌醇-3激酶在犬体外循环缺血再灌注心肌胰岛素抵抗发生机制中的作用

Role of phosphatidylinositol 3 kinase in myocardial insulin resistance induced by canine cardiopulmonary bypass ischemia reperfusion

目的 检测在犬体外循环（CPB）缺血再灌注心肌胰岛素抵抗过程中磷脂酰基醇3-激酶（PI3K）的表达,观察心肌细胞是否通过PI3K来诱导葡萄糖转运蛋白-4（Glut-4）的转运,探讨体外循环心肌胰岛素抵抗的受体后发生机制.方法 健康杂种犬24条,随机均分为4组（n=6）,组Ⅰ主动脉阻断30 min;组Ⅱ主动脉阻断120 min;组Ⅲ主动脉阻断30 min＋胰岛素（PI3K激活剂）;组Ⅳ主动脉阻断30 min＋渥曼青霉素（PI3K抑制剂）.分别于CPB转流前、开放主动脉后15、45、75 min时采集心肌组织标本.测定PI3K及Glut-4的变化.结果 心肌细胞Glut-4外膜百分比：于再灌注15 min,4组外膜百分比值分别由转流前（18.68±6.76）％、（19.76±3.41）％、（17.43±5.36）％、（18.43±4.98）％下降至（3.88±1.43）％、（1.89±1.27）％、（4.2l±1.59）％、（0.65±1.32）％,组Ⅰ、Ⅱ、Ⅲ于再灌注45 min有不同程度缓慢上升,组Ⅳ持续下降,至再灌注75 min达最低;但与转流前比较,4组差异有统计学意义（P＜0.01）;与组Ⅰ比较,再灌注后组Ⅱ、组Ⅳ外膜百分比值下降明显,持续时间长（P＜0.05）,组Ⅲ外膜百分比值上升（P＜0.05）.PI3K表达变化：缺血再灌注后心肌细胞PI3K表达不同程度下降;于再灌注75 min,4组分别由转流前21.23 ±2.58、22.73土2.45、20.19±37.30、21.76±2.74下降至18.53±1.86、15.44±9.27、24.00±1.90、1.06±0.43;与组Ⅰ比较,组ⅣPI3K表达下降明显（P＜0.01）,而组ⅢPI3K表达增强（P＜0.05）.结论 CPB缺血再灌注后,PI3K作为主要的信号传导因子,刺激心肌细胞Glut-4从胞质内转运至包膜上,完成对葡萄糖的摄取,即缺血再灌注心肌胰岛素抵抗过程中心肌细胞Glut-4的转运主要通过PI3K途径实现.

Objective To investigate the expression of phosphatidylinositol 3 kinase （PI3K） in the process of myocardial insulin resistance induced by extracorporal circulation ischemia repeffusion,making clear whether myocardial cells induce the transfer of glucose transporter protein-4 （Glut-4） by way of PI3K,and further probing into the mechanism of insulin resistance post-receptor in the course of cardiopulmonary bypass （CPB） ischemia.Methods Twenty-four mongrel dogs were randomly divided into 4 groups：group Ⅰ undergoing 30-min aortic occlusion; group Ⅱ undergoing 120 min aortic occlusion; group Ⅲ undergoing 30 min aortic occlusion plus insulin-PI3K activator; group Ⅳ undergoing 30 min aortic occlusion plus wortmannin-PI3K inhibitor.Samples of cardiac tissue were simultaneously collected before CPB,and 15,45 and 75 min after cross-clamp removal.The activity of PI3K and the change of myocardial cell envelope and cytoplastic Glut-4 were detected.Results The Glut-4 membrane percentage in myocardial cells were decreased in groups 1-4 from （18.68 ±6.76）％,（19.76 ±3.41）％,（17.43 ±5.36）％ and （18.43 ±4.98）％ before CPB to （3.88±1.43）％,（1.89± 1.27）％,（4.21 ± 1.59）％ and （0.65 ± 1.32）％ at the 15th min after ischemia-reperfusion,and went up slowly to various degrees at the 75th minte after the ischemia-reperfusion （P ＜ 0.01）.The membrane percentage of Glut-4 in group Ⅳ,in contrast,was continuously decreased till reaching the nadir at the 75th min （P ＜ 0.05）.The membrane percentage of Glut-4 in group Ⅱ,in comparison with group Ⅰ,went down strikingly in and lasted a longer time （P ＜ 0.05）.The PI3K in the cardiac myocytes was declined to various extents after the ischemia reperfusion.The expression of PI3K was reduced in group Ⅰ-Ⅳ from 21.23 ± 2.58,22.73 ± 2.45,20.19 ±37.30 and 21.76 ±2.74 before CPB to 18.53 ± 1.86,15.44±9.27,24.00 ± 1.90 and 1.06 ± 0.43 at the 75th min after ischemia-reperfusion.The expression of PI3K in group Ⅳ was reduced notably in myocardial cells compared to group Ⅰ （P ＜ 0.01）,whereas that in group Ⅲ was increased （P ＜ 0.05）.Conclusion After the CPB ischemia-reperfusion,PI3K,as a key signal transduction factor,stimulates the transfer of Glut-4 in myocardial cellsfrom the cytoplasma to the envelope,thus assimilating glucose,namely,Glut-4 in myocardial cells is mainly transferred by way of PI3K in the process of myocardial insulin resistance induced by ischemia reperfusion.