围术期心肌胰岛素抵抗的发生及机制的研究
|
摘要
第一部分高胰岛素诱导大鼠离体心脏胰岛素抵抗模型的建立
目的观察10、20、30、50U/L浓度胰岛素对离体大鼠心肌葡萄糖代谢的影响，建立离体大鼠心肌胰岛素抵抗模型。
方法30只SD大鼠随机分为5组，建立Langendorff模型后，以含有不同浓度胰岛素的K-H液进行离体心脏灌注，时间为60min。其中对照组不含胰岛素，实验组为超生理浓度胰岛素，分别为10、20、30、50U/L。分别于灌注30min和60min时收集冠脉流出液样本，采用葡萄糖氧化法检测冠脉流出液中的葡萄糖浓度，计算心肌葡萄糖摄取量，以评估高浓度胰岛素对心肌组织葡萄糖消耗的影响。
结果灌流30min时，胰岛素刺激组大鼠心肌组织葡萄糖净摄取量分别为1．72±0．26mmol/L，1．59±0．23mmol/L，1．56±0．16mmol/L，1．39±0．27mmol/L，较对照组2．73±0．21 mmol/L明显减少。60min灌流结束时，心肌组织葡萄糖摄取进一步减少，胰岛素刺激组分别为1．59±0．21 mmol/L，1．49±0．18mmol/L，1．38±0．20mmol/L，1．23±0．22mmol/L，对照组为2．63±0．19mmol/L。结果表明，胰岛素刺激30min和60min时，胰岛素刺激组大鼠心肌组织对葡萄糖的摄取随着胰岛素浓度上升而逐渐减少，结果有显著意义(P＜0．05)。
结论10、20、30、50U/L胰岛素刺激离体大鼠心脏60min后，心肌组织对葡萄糖摄取减少，对胰岛素的作用出现抵抗。
第二部分胰岛素诱导离体心脏发生胰岛素抵抗时心肌损伤的研究
目的观察高浓度胰岛素刺激离体心脏发生胰岛素抵抗后心肌组织损伤的情况。
方法30只SD大鼠随机分为五组，建立Langendorff模型后，以含有不同浓度胰岛素的K-H液进行离体心脏灌注，时间为60min。其中对照组不含胰岛素，实验组为超生理浓度胰岛素，浓度分别为10、20、30、50U/L。观察心率(HR)，收缩期和舒张期左心室内压最大变化速率，于灌注60min时收集冠脉流出液样本，检测肌酸激酶(CK)，肌钙蛋白I(cTnI)含量；60min灌注完毕后留取心肌组织，TUNEL检测心肌细胞凋亡情况；光镜和电镜下分别观察心肌组织结构改变。
结果(1)灌注60min后，各胰岛素刺激组心脏左心室-dp/dt_(max)较对照组明显降低(P＜0．01)，左心室+dp/dt_(max)也呈降低趋势，但无统计学意义，而HR无明显改变，表明经过60min高浓度胰岛素刺激后，心脏受到损伤，心功能降低，以舒张功能受损为主。(2)60min灌流结束时，随着胰岛素刺激浓度增加，各胰岛素刺激组冠脉流出液中CK和cTnI蛋白含量较对照组明显增加(P＜0．01)。冠脉流出液中CK和cTnI的水平明显升高，表明心肌释放心肌酶和cTnI增加，心肌受到损伤。(3)TUNEL测定结果显示高胰岛素刺激组心肌组织阳性细胞数增加，AI值(凋亡指数)明显升高(P＜0．01)，心肌细胞凋亡增加。(4)光镜下可见10U/L胰岛素刺激组心肌纤维排列紊乱，水肿程度高于对照组，进一步观察超微结构可见10U/L．胰岛素刺激组心肌细胞肿胀，肌纤维排列紊乱，线粒体密度降低，部分出现肿胀，嵴断裂甚至消失，表明10U/L浓度胰岛素刺激离体心脏60min后，心肌发生病理改变。
结论10、20、30、50U/L胰岛素刺激离体大鼠心脏60min后，心肌发生胰岛素抵抗，心功能降低，释放心肌酶增加，心肌细胞凋亡增多，发生病理改变，心肌受到了损伤。
第三部分胰岛素诱导的离体心脏胰岛素抵抗机制的研究
目的从受体前、受体及受体后探讨高浓度胰岛素刺激离体大鼠心脏后心肌胰岛素抵抗的发生机制。
方法30只SD大鼠随机分为五组，建立Langendorff模型后，以含有不同浓度胰岛素的K-H液进行离体心脏灌注，时间为60min。其中对照组不含胰岛素，实验组为超生理浓度胰岛素，浓度分别为10、20、30、50U/L。60min灌注完毕后留取心肌组织，RT-PCR检测心肌组织胰岛素受体(InsR)和葡萄糖转运蛋白4(GLUT-4)mRNA表达；Western blot检测心肌组织胰岛素受体(InsR)和葡萄糖转运蛋白4(GLUT-4)表达；免疫组化检测心肌组织eNOS和ET-1表达。
结果(1)RT-PCR检测心肌组织胰岛素受体mRNA表达，结果显示胰岛素刺激组较对照组明显降低(P＜0．01)；Western blot结果显示InsR蛋白表达与mRNA变化一致，较对照组明显减少，其差异有极显著意义(P＜0．01)。表明高浓度胰岛素刺激离体大鼠心脏60min后，心肌组织InsR mRNA和蛋白表达均随胰岛素刺激浓度增高而降低。(2)胰岛素刺激组GLUT-4 mRNA较对照组明显降低(P＜0．01)；Wester blot检测发现蛋白含量也相应减少，差异有极显著意义(P＜0．01)。表明经过高浓度胰岛素60min刺激引起心肌组织GLUT-4的mRNA和蛋白表达下调，葡萄糖转运减少，心肌组织摄取葡萄糖降低。(3)eNOS和ET-1蛋白免疫组化检测结果，10U/L胰岛素刺激组eNOS蛋白表达低于对照组(P＜0．01)；而ET-1蛋白表达高于对照组(P＜0．01)。表明10U/L胰岛素刺激60min后，心肌组织eNOS表达降低而ET-1升高，eNOS与ET-1平衡被破坏，内皮功能出现障碍，冠脉收缩，血流量减少，引起胰岛素、葡萄糖向间质转运减少，代谢终产物堆积，加重心肌组织IR。
结论10、20、30、50U/L胰岛素刺激离体大鼠心脏60min后，心肌内皮发生功能障碍，心肌组织InsR和GLUT-4表达减少，受体前、受体及受体后均发生了改变，导致心肌发生胰岛素抵抗。
第四部分胰岛素诱导的体外培养心肌细胞胰岛素抵抗的研究
目的采用胰岛素诱导体外原代培养的心肌细胞的方法，建立胰岛素抵抗的心肌细胞模型，并观察10~(-6)mol/L胰岛素对心肌细胞胰岛素受体的影响及病理学改变。
方法采用胰蛋白酶消化法和差速贴壁法体外分离培养和纯化新生大鼠心室肌细胞，以不同浓度胰岛素干预24h，其中对照组不含胰岛素，实验组为超生理浓度胰岛素，分别为10~(-8)、10~(-7)、10~(-6)、10~(-5)mol/L；将干预后的心肌细胞用不含血清的培养基孵育24h，留取上清液，葡萄糖氧化法检测上清液中的葡萄糖含量；~3H-D-葡萄糖掺合实验检测10~(-6)mol/L胰岛素组和空白对照组心肌细胞的葡萄糖掺合率，以评估胰岛素敏感性；RT-PCR检测心肌细胞中胰岛素受体mRNA含量；Western blot检测心肌细胞中InsR蛋白含量；电镜观察心肌细胞超微结构变化。
结果(1)胰岛素刺激24h后，随着胰岛素浓度由10~(-8)mol/L增加到10~(-6)mol/L，各胰岛素刺激组心肌细胞葡萄糖消耗量减少，但当胰岛素刺激浓度由10~(-6)mol/L继续增加到10~(-5)mol/L时，葡萄糖消耗量减少不明显，表明以高浓度胰岛素刺激心肌细胞24h后，心肌细胞摄取利用葡萄糖减少，对胰岛素的作用产生了抗性，最佳干预浓度为10~(-6)mol/L。(2)加入胰岛素后，10~(-6)mmol/L胰岛素组细胞的~3H-D-葡萄糖掺入率在各种浓度的胰岛素刺激下均显著低于对照组细胞(P＜0．01)，并且10~(-6)mmol/L胰岛素组和对照组细胞的葡萄糖掺入率随着胰岛素浓度的升高而升高，呈浓度依赖性(3)10~(-6)mmol/L胰岛素组心肌细胞InsR mRNA和蛋白表达均减少，表明10~(-6)mmol/L胰岛素干预24h后心肌细胞IsnR表达下调(P＜0．01)。(4)电镜下可见10~(-6)mol/L胰岛素组心肌细胞明显肿胀，胞核固缩，部分胞质内出现空泡，线粒体肿胀，部分嵴断裂甚至消失，超微结构发生明显病理改变。
结论采用高胰岛素诱导培养法可以诱导心肌细胞发生胰岛素抵抗，最佳干预浓度为10~(-6)mmol/L，心肌细胞InsR mRNA和蛋白水平均下调，超微结构受到损伤。
PartⅠThe establishment of isolated rat heart insulinresistant model
Objective To investigate the effect of 10,20,30 and 50U/Linsulin on the myocardial glycometabolism of isolated rat heart andestablish the insulin-resistant model of isolated rat heart.
Methods Thirty Sprague-Dawley rats were randomized into fivegroups according to the concentration of insulin in the buffer,the concentration are 10,20,30 and 50U/L.The rats were killed and their hearts were taken out to be mounted onto a Langendorffperfusion apparatus to be perfused with Krebs-Henseleit buffer inthe presence of various concentration of insulin for 60min.Thecoronary effluent was collected at 30 and 60min,and theconcentration of glucose in the coronary effluent was determinedto calculate the glucose uptake and evaluate the glycometabolismof myocardial.
Results The glucose intake of myocardial are2.73±0.21,1.72±0.26,1.59±0.23,1.56±0.16,1.39±0.27mmol/Lat 30min,and2.63±0.19,1.59±0.21,1.49±0.18,1.38±0.20 and1.23±0.22mmol/Lat60min.Compared with the control group,the rat hearts stimulatedwith hyperinsulinism group had a significantly depression ofglucose uptake relied upon the level of insulin in the buffer(P＜0.05).
Conclusion Stimulated the isolated rat heart with 10,20,30 and50U/L insulin,the glucose uptake of myocardial depressed,and thephenomenon of myocardial insulin resistance significantly existed.
PartⅡThe study of myocardial damage on insulinresistance of isolatedheart
Objective To observe the damage of myocardial on isolated ratheart after stimulated with hyperinsulinism.
Methods Thirty Sprague- Dawley rats were randomized into fivegroups according to the concentration of insulin inthe buffer,the concentration are 10,20,30 and 50U/L.The rats were killedand their hearts were taken out to be mounted onto a Langendorffperfusion apparatus to be perfused with Krebs-Henseleit buffer inthe presence of various concentration of insulin for 60min.Heartrate and left ventricular±dp/dt_(max) were measured and recordedusing computerized data acquisition system.The coronary effluentwas collected at 60min,and the concentration of cardiac creatinekinase (CK) and cardial troponin I(cTn-I) were detected;theapoptosis index (AI) by TUNEL staining was caculated;and thestructural change of myocardial was observed by light and electronmicroscope.
Results (1)After stimulated 60min,left ventricular -dp/dt_(max)depressed compared with the control group (P＜0.01),but HR not,demonstrated that heart function cut down,especially left ventricular diastolic function.(2) The lever of CK and cTn-I inthe coronary effluent was significantly higher the controlgroup(P＜0.01) (3)The value of apoptosis increased in insulinstimulated group than those in the control group(P＜0.01),indicatedcardiomyocyte apoptosis decreated.(4)10U/L insulin groupmyocardium observed by light microscope,cardiac muscle fibersarrayed mussily and dropsied more than the control group,and inultramicrostructurethe density of chondriosome reduced,cristaecollapsed,even disappeared,the pathematology changes occurrencedon the myocardial structure.
Conclusion Stimulated the isolated rat heart with 10,20,30 and50U/L insulin for 60min,the myocardial insulin resistance existed,the heart function depressed,released more cardiac creatase,cardiomyocyte apoptosis increased,thestructure changed,and themyocardium got damaged.
PartⅢThe mechanism of myocardial insulin resistanceon isolated rat heart
Objective To investigate the mechanism of myocardialinsulinresistance on isolated rat heart after stimulated withhyperinsulinism from prereceptor,receptor and postreceptor.
Methods Thirty Sprague- Dawley rats were randomized into fivegroups according to the concentration of insulin in the buffer,theconcentration are 10,20,30 and 50U/L.The rats were killed andtheir hearts were taken out to be mounted onto a Langendorffperfusion apparatus to be perfusedwith Krebs-Henseleit buffer inthe presence of various concentration of insulin for 60min.Afterperfused for 60min,the myocardium was obtained,and the contentof insulin receptor (InsR) mRNAandglucosetransporter 4 (GLUT-4)mRNA by RT-PCR,InsR protein and GLUT-4 protein was detected byWestern blotting method,the eNOS and ET-1 by immunohistochemistry.
Results (1) Compared with the control group,the content of InsRmRNA and protein decreased (P＜0.01),demonstrated the expressionof InsR mRNA and protein were both down regulated after stimulatedwith hyperinsulinism for 60min.(2) The content of GLUT-4mRNAandprotein decreased either,compared to the control group(P＜0.01),demonstrated the expression of GLUT-4 mRNA and protein decreasedtoo,the myocardial glucose transport diminished and the glucose intake degrade.(3) The levels of eNOS protein descend while thelevels of ET-1 raise in 10U/L insulin group(P＜0.01),purportedendothelial dysfunction existed,the aeteria coronariacontracted,blood flow reduced,result in insulin and glucose transport tointerstitial substance decreased,metabolic product cumulated,aggraved myocardial insulin resistance.
Conclusion Stimulated the isolated rat heart with 10,20,30 and50U/L insulin for 60min,the myocardial endothelial function disord,and the expression of InsR and GLUT-4 decreased.The changesoccurred on prereceptor,receptor and postreceptor,and thephenomenon of myocardial insulin resistance significantly existed.
PartⅣThe study on i nsul in resistance on cardiocyteinduced with hyperinsulinsm
Objective To establish insulin resistant myocardial cell modelby induced primary cultured cardiocyte with hyperinsul insm,andinvest i gated the effect of 10~(-6)mol/L insulin on cardiocyte insulinreceptor and pathematology change.
Methods Cardiomyocytes of neonatal mice were in vitro harvested,culture and purified by trypsin digestion and differentialattachment technique.Cardiomyocytes were incubated in 10~(-8),10~(-7),10~(-6),10~(-5) mol/L insulin for 24 hours.The insulin inducedcardiomyocytes were incubated for another 24 hours by DMEM,finallythe culture fluid was obtained,and the concentration of glucosewas determined;~3H-D-glucose incorporation experiment was adopt toevaluate the insulin sensitivity of cardiomyocytes;the content ofmyocardial insulin receptor (InsR) mRNA was detected by RT-PCRmethod,and the InsR protein detected by Western blotting;inadditional,the structural change of myocardial cell was observedby electron microscope.
Results (1)When the concentration of insulin raised from 10~(-8)mol/Lto 10~(-6)mol/L,the glucose consumption of the cardiomyocytesdecreased gradually,but when the concentration raised to 10~(-5)mol/L,the glucose consumption decreased no more.It demonstrated thatafter stimulated with insulin for 24h,the cardiomyocytes glucoseintake reduced,insulinresistanceexisted,and the best interferedconcentration is 10~(-6)mol/L.(2) The amounts of incorporation of~3H-D-glucose in 10~(-6)mol/L insulin group cardiomyocytes were lower than the control group stimulated with various concentration ofinsulin,and the amounts of both groups raised upon theconcentration of insulin.(3) After stimulated thecardiomyocyteswith 10~(-6)mol/L insulin,the content of InsR mRNA and proteindecreased compared to the control group(P＜0.01).(4) 10~(-6)mol/Linsulin group cardiomyocytes changed significantly under theelectron microscope than the control group ones:dropsied more,andthe chondriosome reduced,cristaecollapsedor even disappeared.
Conclusion Stimulated cardiomyocytes with hyperinsulinism wasable to induce a state of insulin resistance,the best interferedconcentration is 10~(-6)mol/L,the insR mRNA and protein were both downregulated,and ultramicrostructure changed.
目的观察10、20、30、50U/L浓度胰岛素对离体大鼠心肌葡萄糖代谢的影响，建立离体大鼠心肌胰岛素抵抗模型。
方法30只SD大鼠随机分为5组，建立Langendorff模型后，以含有不同浓度胰岛素的K-H液进行离体心脏灌注，时间为60min。其中对照组不含胰岛素，实验组为超生理浓度胰岛素，分别为10、20、30、50U/L。分别于灌注30min和60min时收集冠脉流出液样本，采用葡萄糖氧化法检测冠脉流出液中的葡萄糖浓度，计算心肌葡萄糖摄取量，以评估高浓度胰岛素对心肌组织葡萄糖消耗的影响。
结果灌流30min时，胰岛素刺激组大鼠心肌组织葡萄糖净摄取量分别为1．72±0．26mmol/L，1．59±0．23mmol/L，1．56±0．16mmol/L，1．39±0．27mmol/L，较对照组2．73±0．21 mmol/L明显减少。60min灌流结束时，心肌组织葡萄糖摄取进一步减少，胰岛素刺激组分别为1．59±0．21 mmol/L，1．49±0．18mmol/L，1．38±0．20mmol/L，1．23±0．22mmol/L，对照组为2．63±0．19mmol/L。结果表明，胰岛素刺激30min和60min时，胰岛素刺激组大鼠心肌组织对葡萄糖的摄取随着胰岛素浓度上升而逐渐减少，结果有显著意义(P＜0．05)。
结论10、20、30、50U/L胰岛素刺激离体大鼠心脏60min后，心肌组织对葡萄糖摄取减少，对胰岛素的作用出现抵抗。
第二部分胰岛素诱导离体心脏发生胰岛素抵抗时心肌损伤的研究
目的观察高浓度胰岛素刺激离体心脏发生胰岛素抵抗后心肌组织损伤的情况。
方法30只SD大鼠随机分为五组，建立Langendorff模型后，以含有不同浓度胰岛素的K-H液进行离体心脏灌注，时间为60min。其中对照组不含胰岛素，实验组为超生理浓度胰岛素，浓度分别为10、20、30、50U/L。观察心率(HR)，收缩期和舒张期左心室内压最大变化速率，于灌注60min时收集冠脉流出液样本，检测肌酸激酶(CK)，肌钙蛋白I(cTnI)含量；60min灌注完毕后留取心肌组织，TUNEL检测心肌细胞凋亡情况；光镜和电镜下分别观察心肌组织结构改变。
结果(1)灌注60min后，各胰岛素刺激组心脏左心室-dp/dt_(max)较对照组明显降低(P＜0．01)，左心室+dp/dt_(max)也呈降低趋势，但无统计学意义，而HR无明显改变，表明经过60min高浓度胰岛素刺激后，心脏受到损伤，心功能降低，以舒张功能受损为主。(2)60min灌流结束时，随着胰岛素刺激浓度增加，各胰岛素刺激组冠脉流出液中CK和cTnI蛋白含量较对照组明显增加(P＜0．01)。冠脉流出液中CK和cTnI的水平明显升高，表明心肌释放心肌酶和cTnI增加，心肌受到损伤。(3)TUNEL测定结果显示高胰岛素刺激组心肌组织阳性细胞数增加，AI值(凋亡指数)明显升高(P＜0．01)，心肌细胞凋亡增加。(4)光镜下可见10U/L胰岛素刺激组心肌纤维排列紊乱，水肿程度高于对照组，进一步观察超微结构可见10U/L．胰岛素刺激组心肌细胞肿胀，肌纤维排列紊乱，线粒体密度降低，部分出现肿胀，嵴断裂甚至消失，表明10U/L浓度胰岛素刺激离体心脏60min后，心肌发生病理改变。
结论10、20、30、50U/L胰岛素刺激离体大鼠心脏60min后，心肌发生胰岛素抵抗，心功能降低，释放心肌酶增加，心肌细胞凋亡增多，发生病理改变，心肌受到了损伤。
第三部分胰岛素诱导的离体心脏胰岛素抵抗机制的研究
目的从受体前、受体及受体后探讨高浓度胰岛素刺激离体大鼠心脏后心肌胰岛素抵抗的发生机制。
方法30只SD大鼠随机分为五组，建立Langendorff模型后，以含有不同浓度胰岛素的K-H液进行离体心脏灌注，时间为60min。其中对照组不含胰岛素，实验组为超生理浓度胰岛素，浓度分别为10、20、30、50U/L。60min灌注完毕后留取心肌组织，RT-PCR检测心肌组织胰岛素受体(InsR)和葡萄糖转运蛋白4(GLUT-4)mRNA表达；Western blot检测心肌组织胰岛素受体(InsR)和葡萄糖转运蛋白4(GLUT-4)表达；免疫组化检测心肌组织eNOS和ET-1表达。
结果(1)RT-PCR检测心肌组织胰岛素受体mRNA表达，结果显示胰岛素刺激组较对照组明显降低(P＜0．01)；Western blot结果显示InsR蛋白表达与mRNA变化一致，较对照组明显减少，其差异有极显著意义(P＜0．01)。表明高浓度胰岛素刺激离体大鼠心脏60min后，心肌组织InsR mRNA和蛋白表达均随胰岛素刺激浓度增高而降低。(2)胰岛素刺激组GLUT-4 mRNA较对照组明显降低(P＜0．01)；Wester blot检测发现蛋白含量也相应减少，差异有极显著意义(P＜0．01)。表明经过高浓度胰岛素60min刺激引起心肌组织GLUT-4的mRNA和蛋白表达下调，葡萄糖转运减少，心肌组织摄取葡萄糖降低。(3)eNOS和ET-1蛋白免疫组化检测结果，10U/L胰岛素刺激组eNOS蛋白表达低于对照组(P＜0．01)；而ET-1蛋白表达高于对照组(P＜0．01)。表明10U/L胰岛素刺激60min后，心肌组织eNOS表达降低而ET-1升高，eNOS与ET-1平衡被破坏，内皮功能出现障碍，冠脉收缩，血流量减少，引起胰岛素、葡萄糖向间质转运减少，代谢终产物堆积，加重心肌组织IR。
结论10、20、30、50U/L胰岛素刺激离体大鼠心脏60min后，心肌内皮发生功能障碍，心肌组织InsR和GLUT-4表达减少，受体前、受体及受体后均发生了改变，导致心肌发生胰岛素抵抗。
第四部分胰岛素诱导的体外培养心肌细胞胰岛素抵抗的研究
目的采用胰岛素诱导体外原代培养的心肌细胞的方法，建立胰岛素抵抗的心肌细胞模型，并观察10~(-6)mol/L胰岛素对心肌细胞胰岛素受体的影响及病理学改变。
方法采用胰蛋白酶消化法和差速贴壁法体外分离培养和纯化新生大鼠心室肌细胞，以不同浓度胰岛素干预24h，其中对照组不含胰岛素，实验组为超生理浓度胰岛素，分别为10~(-8)、10~(-7)、10~(-6)、10~(-5)mol/L；将干预后的心肌细胞用不含血清的培养基孵育24h，留取上清液，葡萄糖氧化法检测上清液中的葡萄糖含量；~3H-D-葡萄糖掺合实验检测10~(-6)mol/L胰岛素组和空白对照组心肌细胞的葡萄糖掺合率，以评估胰岛素敏感性；RT-PCR检测心肌细胞中胰岛素受体mRNA含量；Western blot检测心肌细胞中InsR蛋白含量；电镜观察心肌细胞超微结构变化。
结果(1)胰岛素刺激24h后，随着胰岛素浓度由10~(-8)mol/L增加到10~(-6)mol/L，各胰岛素刺激组心肌细胞葡萄糖消耗量减少，但当胰岛素刺激浓度由10~(-6)mol/L继续增加到10~(-5)mol/L时，葡萄糖消耗量减少不明显，表明以高浓度胰岛素刺激心肌细胞24h后，心肌细胞摄取利用葡萄糖减少，对胰岛素的作用产生了抗性，最佳干预浓度为10~(-6)mol/L。(2)加入胰岛素后，10~(-6)mmol/L胰岛素组细胞的~3H-D-葡萄糖掺入率在各种浓度的胰岛素刺激下均显著低于对照组细胞(P＜0．01)，并且10~(-6)mmol/L胰岛素组和对照组细胞的葡萄糖掺入率随着胰岛素浓度的升高而升高，呈浓度依赖性(3)10~(-6)mmol/L胰岛素组心肌细胞InsR mRNA和蛋白表达均减少，表明10~(-6)mmol/L胰岛素干预24h后心肌细胞IsnR表达下调(P＜0．01)。(4)电镜下可见10~(-6)mol/L胰岛素组心肌细胞明显肿胀，胞核固缩，部分胞质内出现空泡，线粒体肿胀，部分嵴断裂甚至消失，超微结构发生明显病理改变。
结论采用高胰岛素诱导培养法可以诱导心肌细胞发生胰岛素抵抗，最佳干预浓度为10~(-6)mmol/L，心肌细胞InsR mRNA和蛋白水平均下调，超微结构受到损伤。
PartⅠThe establishment of isolated rat heart insulinresistant model
Objective To investigate the effect of 10,20,30 and 50U/Linsulin on the myocardial glycometabolism of isolated rat heart andestablish the insulin-resistant model of isolated rat heart.
Methods Thirty Sprague-Dawley rats were randomized into fivegroups according to the concentration of insulin in the buffer,the concentration are 10,20,30 and 50U/L.The rats were killed and their hearts were taken out to be mounted onto a Langendorffperfusion apparatus to be perfused with Krebs-Henseleit buffer inthe presence of various concentration of insulin for 60min.Thecoronary effluent was collected at 30 and 60min,and theconcentration of glucose in the coronary effluent was determinedto calculate the glucose uptake and evaluate the glycometabolismof myocardial.
Results The glucose intake of myocardial are2.73±0.21,1.72±0.26,1.59±0.23,1.56±0.16,1.39±0.27mmol/Lat 30min,and2.63±0.19,1.59±0.21,1.49±0.18,1.38±0.20 and1.23±0.22mmol/Lat60min.Compared with the control group,the rat hearts stimulatedwith hyperinsulinism group had a significantly depression ofglucose uptake relied upon the level of insulin in the buffer(P＜0.05).
Conclusion Stimulated the isolated rat heart with 10,20,30 and50U/L insulin,the glucose uptake of myocardial depressed,and thephenomenon of myocardial insulin resistance significantly existed.
PartⅡThe study of myocardial damage on insulinresistance of isolatedheart
Objective To observe the damage of myocardial on isolated ratheart after stimulated with hyperinsulinism.
Methods Thirty Sprague- Dawley rats were randomized into fivegroups according to the concentration of insulin inthe buffer,the concentration are 10,20,30 and 50U/L.The rats were killedand their hearts were taken out to be mounted onto a Langendorffperfusion apparatus to be perfused with Krebs-Henseleit buffer inthe presence of various concentration of insulin for 60min.Heartrate and left ventricular±dp/dt_(max) were measured and recordedusing computerized data acquisition system.The coronary effluentwas collected at 60min,and the concentration of cardiac creatinekinase (CK) and cardial troponin I(cTn-I) were detected;theapoptosis index (AI) by TUNEL staining was caculated;and thestructural change of myocardial was observed by light and electronmicroscope.
Results (1)After stimulated 60min,left ventricular -dp/dt_(max)depressed compared with the control group (P＜0.01),but HR not,demonstrated that heart function cut down,especially left ventricular diastolic function.(2) The lever of CK and cTn-I inthe coronary effluent was significantly higher the controlgroup(P＜0.01) (3)The value of apoptosis increased in insulinstimulated group than those in the control group(P＜0.01),indicatedcardiomyocyte apoptosis decreated.(4)10U/L insulin groupmyocardium observed by light microscope,cardiac muscle fibersarrayed mussily and dropsied more than the control group,and inultramicrostructurethe density of chondriosome reduced,cristaecollapsed,even disappeared,the pathematology changes occurrencedon the myocardial structure.
Conclusion Stimulated the isolated rat heart with 10,20,30 and50U/L insulin for 60min,the myocardial insulin resistance existed,the heart function depressed,released more cardiac creatase,cardiomyocyte apoptosis increased,thestructure changed,and themyocardium got damaged.
PartⅢThe mechanism of myocardial insulin resistanceon isolated rat heart
Objective To investigate the mechanism of myocardialinsulinresistance on isolated rat heart after stimulated withhyperinsulinism from prereceptor,receptor and postreceptor.
Methods Thirty Sprague- Dawley rats were randomized into fivegroups according to the concentration of insulin in the buffer,theconcentration are 10,20,30 and 50U/L.The rats were killed andtheir hearts were taken out to be mounted onto a Langendorffperfusion apparatus to be perfusedwith Krebs-Henseleit buffer inthe presence of various concentration of insulin for 60min.Afterperfused for 60min,the myocardium was obtained,and the contentof insulin receptor (InsR) mRNAandglucosetransporter 4 (GLUT-4)mRNA by RT-PCR,InsR protein and GLUT-4 protein was detected byWestern blotting method,the eNOS and ET-1 by immunohistochemistry.
Results (1) Compared with the control group,the content of InsRmRNA and protein decreased (P＜0.01),demonstrated the expressionof InsR mRNA and protein were both down regulated after stimulatedwith hyperinsulinism for 60min.(2) The content of GLUT-4mRNAandprotein decreased either,compared to the control group(P＜0.01),demonstrated the expression of GLUT-4 mRNA and protein decreasedtoo,the myocardial glucose transport diminished and the glucose intake degrade.(3) The levels of eNOS protein descend while thelevels of ET-1 raise in 10U/L insulin group(P＜0.01),purportedendothelial dysfunction existed,the aeteria coronariacontracted,blood flow reduced,result in insulin and glucose transport tointerstitial substance decreased,metabolic product cumulated,aggraved myocardial insulin resistance.
Conclusion Stimulated the isolated rat heart with 10,20,30 and50U/L insulin for 60min,the myocardial endothelial function disord,and the expression of InsR and GLUT-4 decreased.The changesoccurred on prereceptor,receptor and postreceptor,and thephenomenon of myocardial insulin resistance significantly existed.
PartⅣThe study on i nsul in resistance on cardiocyteinduced with hyperinsulinsm
Objective To establish insulin resistant myocardial cell modelby induced primary cultured cardiocyte with hyperinsul insm,andinvest i gated the effect of 10~(-6)mol/L insulin on cardiocyte insulinreceptor and pathematology change.
Methods Cardiomyocytes of neonatal mice were in vitro harvested,culture and purified by trypsin digestion and differentialattachment technique.Cardiomyocytes were incubated in 10~(-8),10~(-7),10~(-6),10~(-5) mol/L insulin for 24 hours.The insulin inducedcardiomyocytes were incubated for another 24 hours by DMEM,finallythe culture fluid was obtained,and the concentration of glucosewas determined;~3H-D-glucose incorporation experiment was adopt toevaluate the insulin sensitivity of cardiomyocytes;the content ofmyocardial insulin receptor (InsR) mRNA was detected by RT-PCRmethod,and the InsR protein detected by Western blotting;inadditional,the structural change of myocardial cell was observedby electron microscope.
Results (1)When the concentration of insulin raised from 10~(-8)mol/Lto 10~(-6)mol/L,the glucose consumption of the cardiomyocytesdecreased gradually,but when the concentration raised to 10~(-5)mol/L,the glucose consumption decreased no more.It demonstrated thatafter stimulated with insulin for 24h,the cardiomyocytes glucoseintake reduced,insulinresistanceexisted,and the best interferedconcentration is 10~(-6)mol/L.(2) The amounts of incorporation of~3H-D-glucose in 10~(-6)mol/L insulin group cardiomyocytes were lower than the control group stimulated with various concentration ofinsulin,and the amounts of both groups raised upon theconcentration of insulin.(3) After stimulated thecardiomyocyteswith 10~(-6)mol/L insulin,the content of InsR mRNA and proteindecreased compared to the control group(P＜0.01).(4) 10~(-6)mol/Linsulin group cardiomyocytes changed significantly under theelectron microscope than the control group ones:dropsied more,andthe chondriosome reduced,cristaecollapsedor even disappeared.
Conclusion Stimulated cardiomyocytes with hyperinsulinism wasable to induce a state of insulin resistance,the best interferedconcentration is 10~(-6)mol/L,the insR mRNA and protein were both downregulated,and ultramicrostructure changed.
引文
1.Reaven GM.Role of insulin resistance in human disease.Diabetes,1988,37(12):1595-1607.
2.Stem MP.Diabetes and cardiovascular disease,the 'common soil hypothesis'.Diabetes,1995,44(4):396-374.
3.Engin A,Bozkurt BS,Ersoy E,et al.Stress hyperglycemia in minimally invasive surgery.Surg Laparosc Endosc,1998,8(6):435-437.
4.Valerio G,Franzese A,Carlin E et al.A high prevalence of stress Hyperglycaemia in children with febrile seizures and traumatic injures.Acta paediatr,2001,90(6):618-622.
5.Thorell A,Marin P,Smith U,et al.Insulin resistance after abdominal Surgery.British Journal of Surgey,1994,8(11):59-63.
6.Lamarche B,Tchemof A,Mauriege P,et al.Fasting insulin and apolipoprotein B levels and low-density lipoprotein particle size as risk factors for ischemic heart disease.JAMA,1998,279:1955-1961.
7．贾恩志，陈士伟，戚光跃，等．胰岛素抵抗与心血管危险因素聚集的流行病学研究．中华医学杂志，2002，82(17)：1183-1187．
1.Gravlee GP,Davis RF,Utley JR,et al.Cardiopulmonary bypass:principles and practice.Baltimore:Williams& wilkins,1993:513-519.
2．隋东虎，石应康，王儒蓉，等．体外循环缺血-再灌注后心肌胰岛素抵抗现象的初步研究．．中国胸心血管外科临床杂，2001，8：28-31．
3.Talke H,Schubert GE.Enzymatic urea determination in the blood and serum in the Warburg optical tese.Klin Wochenschr,1965,43:174-175.
4.Tiffany,TO,Jansen JM,Burtis CA,et al.Enzymatic kinetic rate and end-point analyses of substrate,by use of a GeMSAEC fast analyzer.Clin Chem,1972,18(8):829-840.
5.Reaven GM.Role of insulin resistance in human disease.Diabetes,1988,37:1595-1607.
6.Stern MP.Diabetes and cardiovascular disease,the 'common soil hypothesis'.Diabetes,1995,44(4):396-374.
7.Thorell A,Nygren J ,Ljungqvist O,et al.Insulin resistance:a marker of surgical stress.Curr Opin Nutr Metab Care ,1999,2:69-78.
8.Braden H,Cheema-Dbadlis,Mazerc D,et al.Hyperglycemia during normothermic cardiopulmonary bypass.Ann Thorac Surg,1998,65(6):1588.
9.Rapp-Kesek D,Stridsberg M,Andersson LG,et al.Insulin-resistance after cardiopulmonary bypass in the elderly patient.Scand Cardiovasc J,2007,41:102-108.
10.Langendorff O.Untersuchungen am uberlebenden saugetierherz.Arch Ges Physiol,1895,61:291-316.
11．黑飞龙，房秀生．围体外循环期应激激素变化及胰岛素抵抗研究．中华胸心血管外科杂志，1999，10(5)：301-302．
12.DeFronzo RA,Ferrannini E.Insulin resistance:A multifaceted syndrome responsible for NIDDM,obesity,hypertension,dyslipidemia,and atherosclerotic cardiovascular disease.Diabetes Care,1991,14(3):173-194.
1.Thrainsdottir IS,Aspelund T,Thorgeirss0n G,et al.The association between glucose abnormalities and heart failure in the population- based Reykjavik study.Diabetes Care,2005,28(3):612-616.
2.Hamby RI,Zoneraich S,Sherman L.Diabetic cardiomyopathy.JAMA,1974,229(13):1749-1754.
3.Fischer M,Baessler A,Hense HW,et al.Prevalence of left ventricular diastolic dysfunction in the community.Results from a Doppler echocardiographic- based survey of a population sample.Eur Heart J,2003,24(4):320-328.
4.Srikanthan P,Hsueh W.Preventing heart failure in patients with diabetes.Med Clin NorthAm,2004,88(5):1237-1256.
5.Wilson Tang WH,Maros A,Young JB.Ischemic heart disease and congestive heart failure in diabetic patients.Med Clin North Am,2004,88(4):1037-1061.
6.Engin A,Bozkurt BS,Ersoy E,et al.Stress hyperglycemia in minimally invasive surgery.Surg Laparosc Endosc,1998,8(6):435-437.
7.Valerio G,Franzese A,Carlin E et al.A high prevalenceof stress Hyperglycaemia in children with febrile seizures and traumatic injures.Acta paediatr,2001,90(6):618-622.
8．彭新丽．2型糖尿病患者血脂、载脂蛋白、脂蛋白、尿微量白蛋白及心肌酶变化的探讨．中华现代内科学杂志，2006，8：918．
9.Diamant M,Lamb H J,Smit JW.Diabetic cardiomyopathy in uncomplicated type 2 diabetes is associated with the metabolic syndrome and systemic inflammation.Diabetologia,2005,48:1669-1670.
10.Korrald C,Elvenes OP,Myrmel T,et al.Myocardial substrate metabolism influences left ventricular energtics in vivo.Am J physiol,2000,278:H1345-H1351.
11.Longnus SI,Wambolt RB,Barr RL,et al.Regulation of myocardial fatty acid oxidation by sustrate supply.Am J Physiol,2001,281:H1561-H1567.
12. Lopaschuk GD. Abnormal mechanical functionin diabete: relationship to altered myocardial carbohydrate/ lipid metabolism. Coron Artery Dis, 1996, 7: 116-123.
1.Foti M,Moukil MA,Dudoqn0n P,et al.Insulin and IGF-1 receptor trafficking and signaling.Novartis Found Symp,2004,262:125-141.
2.Cheatham B,Kahn CR.Insulin action and their insulins signaling network.Endocr Rev,1995,16(2):117-142.
3.Moller D,Flier JS.Insulin resistance mechanisms,syndromes and implications.N Engl J Med,1991,325(13):938-948.
4．杜智勇，史忠，杨天德，等．体外循环心内直视术中胰岛素受体 TPK 活性的变化及意义．体外循环杂志，2002，4：81-87
5.Van Echten G Eckel J,Reinauer H.Down-regulation of insulin receptors in the heart:Studies on primary cultred adult cardiac myocytes.Biochem Biophys Acta,1986,886(3):468-473.
6．杜智勇，史忠．应激与胰岛素抵抗．国外医学-麻醉学与复苏分册，2001，22(1)：10-13．
7.Strommer L,Wickbom M,Wang F,et al.Early impairment of insulin secretion in rats after surgical trauma.Eur J Endocrinol,2002,147:825-833.
8.Hoffman RP.Sympathetic mechanisms of hypoglycemic counter-regulation.Curr Diabetes Rev.2007,3(3):185-193.
9.Katz EB,Burcelin R,Tsao TS,et al.The metabolic corrsequences of alterd glucose transporter expression in transgenic mice.Mol Med,1996,74:639-652.
10.Zorzano A,Fandos C,Palaci M,et al.Role of plasma membrane transporers in muscle metabolism.Biochem J,2000,349(3):667-688.
11.Zierath JR,Krook A,Wallberg- Henriksson H.Insulin action in skeletal muscle from patients with NIDDM.Mol Cell Biochem,1998,182:153-160.
12.Abel ED.Glucose transport in the heart.Front Biosci,2004,9:201-215.
13.Czech MP,Coruera S.Signaling mechanisms that regulate glucose transport.J Biol Chem,1999,274:1865-1868.
14.Mather K,Anderson TJ,Verma S.Insulin action in the vasculature:physioloty and pathophysiology.J Vasc Rec,2001,38(5):415-422.
15．柴伟栋，陈家伟，沈捷，等．葡萄糖、胰岛素对血管内皮细胞功能的影响．中国微循环杂志，2003，7(4)：204-207．
16.Vincent MA,Montagnani M,Quon MJ.Molecular and physiologic actions of insulin related to production of nitric oxide in vascular endothelium.Curr Diab Rep,2003,3:279-188.
17.Wilkes J J,Hevener A,Olefsky J.Chronic endothelin-1 treatment leads to insulin resistance in vivo.Diabetes,2003,52:1904-1999.
18.Helmut OS,Ginger B,Ann J,et al.Insulin-induced skeletal muscle vasodilation is nitric oxide dependent.J Clin Invest,1994,94:1172-1179.
19.Davda RK,stepniakow ski KT,Lu G,et al.Oleic acid inhibits endothelialnitric oxide synthase by a protein kinase C-independent mechanism.Hypertension,1995,26(5):764-770.
1. Russell DS, Gherzi R, Johnson EL, et al. The protein- tyrosine kinase activity of the insulin receptor is necessary for insulin- mediated receptor down- regulation. J Biol Chem, 1987, 262(24): 11833-11840.
2. Hari J, Roth RA. Defective internalization of insulin and its receptor in cells expressing mutated insulin receptors lacking kinase activity. J Biol Chem, 1987, 262(32): 15341-15344.
3. Senn JJ, Klover PJ, Nowark IA, et al. Interleukin- 6 induces cellular insulin resistance in hepatocytes. Diabetes, 2002, 51(12): 3391- 3399.
4. Bernal-Mizrachi C, Weng S, Feng C, et al. Dexamethasone induction of hypertension and diabetes is PPAR- alpha dependent in LDL receptor null mice. Nat Med, 2003,9(8): 1069-1075.
5. Hah J, Jo I, Chakrabarti R, et al. Demonstration of an insulin - insensitive storage pool of glucose transporters in rat hepatocytes and HepG2 cells. J Cell Physiol, 1992, 152(1): 56-63.
6. Melin B, Cherqui G, Blivet MJ, et al. Dual effect of metformin in cultured rat hepatocytes: potentiation of insulin action and prevention of insulin- induced resistance. Metabolism, 1990, 39(10): 1089 -1095.
7. DeFronzo RA, Ferrannini E. Insulin resistance: A multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Diabetes Care, 1991,14(3): 173 -194.
8. Scuteri A, Di- Bernardo MG, De- Propris AM, et al. Insulin effects on the left ventricular in older hypertensive subjects. J Am Geriatr Soc, 1999, 47(6): 727 - 729.
9. Foti M, Moukil MA, Dudoqnon P, et al. Insulin and IGF- 1 receptor trafficking and signaling. Novartis Found Symp, 2004, 262: 125 - 141.
10. Moller D, Flier JS, Insulin resistance mechanisms, syndromes and implications. N Engl J Med, 1991, 325(13): 938 - 948.
11.Van Echten G Eckel J,Reinauer H.Down- regulation of insulin receptors in the heart:Studies on primary cultred adult cardiac myocytes.Biochem Biophys Acta,1986,886(3):468-473.
12．杜智勇，史忠．应激与胰岛素抵抗．国外医学-麻醉学与复苏分册，2001，22(1)：10-13．
13.Diamant M,Lamb H J,Smit JW.Diabetic cardiomyopathy in uncomplicated type 2 diabetes is associated with the metabolic syndrome and systemic inflammation.Diabetologia,2005,48:1669-1670.
14.Lopaschuk GD.Abnormal mechanical functionin diabete:relationship to altered myocardial carbohydrate/lipid metabolism.Coron Artery Dis,1996,7:116-123.
1. Reaven GM. Role of insulin resistance in human disease. Diabetes, 1988, 37(12): 1595- 1607.
2. Stern MP. Diabetes and cardiovascular disease, the 'common soil hypothesis'. Diabetes, 1995,44(4): 396 -374.
3. Thorell A , Nygren J ,Ljungqvist O , et al . Insulin resistance : a marker of surgical stress. Curr Opin Nutr Metab Care ,1999 , 2 :69 - 78.
4. Salim A, Hadjizacharia P, Dubose J, et al. Persistent hyperglycemia in severe traumatic brain injury: an independent predictor of outcome. Am Sung, 2009, 75(1): 25-29.
5. Krinsley J S. Association between hyperglycemia and increased hospital mortality in a heterogeneous population of critically ill patients. Mayo Clin Proc, 2003, 78: 1417-1478.
6. Engin A,Bozkurt BS, Ersoy E, et al. Stress hyperglycemia in minimally invasive surgery. Surg Laparosc Endosc, 1998, 8(6): 435-437.
7. Valerio G, Franzese A, Carlin E et al. A high prevalence of stress Hyperglycaemia in children with febrile seizures and traumatic injures. Acta paediatr, 2001, 90(6):618-622.
8. Thorell A, Marin P, Smith U, et al. Insulin resistance after abdominal Surgery. British Journal of Surgey, 1994,8(11): 59 -63.
9. Hager P, Hagman B, Wikstrom AC, et al. CRF- receptor 1 blockade attenuates acute posttraumatic hyperglycermia in rats. J of Surg Res, 2004, 119: 72 - 79.
10. Kuroda M, Honnor RC, Cushman SW, et al. Regulation of insulin stimulated glucose transport in the isolated ratadipocyte: CAMP independent effects of lipolytic and antilipolytic agents. J Biol Chem, 1987, 262(1): 245 -253.
11. Mescheryakov A V, Kozlov I A, Dementyeva I I, et al. Glucose metabolism and insulin activity during cardiac surgery. J Cardiothorac Anesth. 1989; 3(5): 536 - 543.
12. Cuthbertson DP: Observations on disturbance of metabolism produced by injury to the limbs:Quat.J.Med,1932,25:233-246.
13.Strommer L,Wickbom M,Wang F,et al.Early impairment of insulin secretion in rats after surgical trauma.Eur J Endocrinol,2002,147:825-833.
14.McPheed S J,et al.Pathophisiology of Disease.(3~(rd) version).Mc Graw-Hill,2000,434-436.
15.Dohan FC,Lukens FDW.Experimental diabetes produced by the administration of glucose.Endocrinology,1948,42:244-262.
16.Leahy JL,Weir GC,Evolution of abnormal insulin secretory responses during 48h in vivo hyperglycemia.Diabets,1988,37:217-222.
17.Randle P J,Garland PB,Hales CN,et al.The glucose fatty acid cycle:its role in insulin sensistivity and the metabolic disturbances of diabetes mellitus.Lancet,1963,1:785-789.
18.Cheatham B,Kahn CR.Insulin action and their insulins signaling network.Endocr Rev,1995,16(2):117-142.
19.Moller D,Flier JS,Insulin resistance mechanisms,syndromes and implications.N Engl J Med,1991,325(13):938-948.
20．杜智勇，史忠，杨天德，等．体外循环心内直视术中胰岛素受体 TPK 活性的变化及意义．体外循环杂志，2002，4：81-87．
21．杜智勇，史忠．应激与胰岛素抵抗．国外医学-麻醉学与复苏分册，2001，22(1)：10-13．
22．毛旭虎，许霖水．胰岛素受体酪氨酸蛋白激酶在严重烫伤大鼠胰岛素抵抗中作用的研究．第三军医大学学报，1996，18：1-3．
23．张波，许霖水，吴喜贵．烧伤后胰岛素受体信号传到缺陷机理的研究．第三军医大学学报，1997，19(1)：4-6．
24.Bang P,Nygren J,Thorell A,et al.Postoperative induction of insulin- like growth factor binding protein- 3 proteolytic activity:relationto insulin and insulin sensitivity.J Clin Endocrirnol Metab,1998,83(7):2509-2515.
25. Black PR, David M, Brooks C, et al. Mechanisms of insulin resistance following injury. Ann Surg, 1982, 196(4): 420.
26. Cusi K, Maezono K, Oaman A, et al. Insulin. resistance differentially affects the pi3-kinase- and MAP kinase- mediated signaling in human muscle. J Clin Invest, 2000, 105:311-320.
27. Gao Z, zuberi A, Quon MJ, et al. Aspririn inhibits serine phosphorylation of insulin receptor substrate 1 in rumor necrosis factor- treated cells through targeting multiple serine kinases. J Boil Chem, 2003, 278: 24944 - 24950.
28. Le Marchand- Brustel Y, Gual P, Gremeaux T, et al. Fatty acid - induced insulin resistance: role of insulin receptor substrate 1 serine phosphorylation in the retroregulation of insulin signaling. Biochem Soc Trans, 2003, 31(Pt6): 1152-1156.
29. Ozes ON, Akca H, Mayo LD, et al. A phosphatidylinositol 3 - Kinase/ Akt/ mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate- 1. Proc Natl Acad Sci USA, 2001, 98: 4640- 4645.
30. Zorzano A, Fandos C, Palaci M, et al. Role of plasma membrane transporers in muscle metabolism. Biochem J, 2000, 349(3): 667- 688.
31. Yu ZW, Buren J, Enerback S, et al. Insulin can enhance GLUT4 gene expression in 3T3- F442A cells and this effect is mimicked by vanadate but counteracted by cAMP and high glucose- potential implications for insulin resistance. Biochim Biophys Acta, 2001, 1535(2): 174- 185.
32. Rea S, James DE. Moving GLUT4: the biogenesis and trafficking of GLUT4 storage vesicles. Diabetes, 1997,46: 1667-1677.
33. McKcown NM, Mcigs JB, Liu S, et al. Carbohydrate nutrition insulin resistance and the prevalence of the metabolic syndrome in the Framingham offspring cohort. Diabetes Care, 2004, 27(2): 538- 546.
34. Zierath JR, Krook A, Wallberg-Henriksson H. Insulin action in skeletal muscle from patients with NIDDM. Mol Cll Biochem, 1998, 182: 153-160.
35. Liu LS, Spelleken M, Rohrig K, et al. Tumor necrosis factor - a acutely inhibits insulin signaling in human adipocytes, implication of the p80 tumor necraosis factor receptor. Diabetes, 1998,47:515-522.
36. Le Marchand- Brustel Y, Gual P, Gremeaux T, et al. Fatty acid- induced insulin resistance: role of insulin receptor substrate 1 serine phosphorylation in the retroregulation of insulin signaling. Biochem Soc Trans, 2003, 31: 1152 - 1156.
37. Gao Z, Zuberi A, Quon MJ, et al. Aspirin inhibits serine phosphorylation of insulin receptor substrate 1 in tumor necrosis factor- treated cells through targeting multiple serine kinases. J Boil Chem, 2003, 278: 24944 - 24950.
38. Cheung AT, Ree D, Kolls JK, et al. An in vivo model for elucidation of the mechanism of tumor necrosis factor- a- induced insulin resistance: Evidence for differential regulation of insulin signaling by TNF-α. Endocrinology, 1998, 139: 4928-4935.
39. Xu J, Kim HT, Ma Y, et al. Trauma and hemorrhage- induced acute hepatic insulin resistance: dominant role of tumor necrosis factor- alpha. Endocrinology, 2008, 149(5):2369-2382.
40. Gupta A, Ten S, Anhalt H. Serum levels of solubole tumor necrosis factor- alpha receptor 2 are linked to insulin resistance and glucose intolerance in children. J Pediatr Endocrinol Metab, 2005, 18(1): 75-82.
2.Stem MP.Diabetes and cardiovascular disease,the 'common soil hypothesis'.Diabetes,1995,44(4):396-374.
3.Engin A,Bozkurt BS,Ersoy E,et al.Stress hyperglycemia in minimally invasive surgery.Surg Laparosc Endosc,1998,8(6):435-437.
4.Valerio G,Franzese A,Carlin E et al.A high prevalence of stress Hyperglycaemia in children with febrile seizures and traumatic injures.Acta paediatr,2001,90(6):618-622.
5.Thorell A,Marin P,Smith U,et al.Insulin resistance after abdominal Surgery.British Journal of Surgey,1994,8(11):59-63.
6.Lamarche B,Tchemof A,Mauriege P,et al.Fasting insulin and apolipoprotein B levels and low-density lipoprotein particle size as risk factors for ischemic heart disease.JAMA,1998,279:1955-1961.
7．贾恩志，陈士伟，戚光跃，等．胰岛素抵抗与心血管危险因素聚集的流行病学研究．中华医学杂志，2002，82(17)：1183-1187．
1.Gravlee GP,Davis RF,Utley JR,et al.Cardiopulmonary bypass:principles and practice.Baltimore:Williams& wilkins,1993:513-519.
2．隋东虎，石应康，王儒蓉，等．体外循环缺血-再灌注后心肌胰岛素抵抗现象的初步研究．．中国胸心血管外科临床杂，2001，8：28-31．
3.Talke H,Schubert GE.Enzymatic urea determination in the blood and serum in the Warburg optical tese.Klin Wochenschr,1965,43:174-175.
4.Tiffany,TO,Jansen JM,Burtis CA,et al.Enzymatic kinetic rate and end-point analyses of substrate,by use of a GeMSAEC fast analyzer.Clin Chem,1972,18(8):829-840.
5.Reaven GM.Role of insulin resistance in human disease.Diabetes,1988,37:1595-1607.
6.Stern MP.Diabetes and cardiovascular disease,the 'common soil hypothesis'.Diabetes,1995,44(4):396-374.
7.Thorell A,Nygren J ,Ljungqvist O,et al.Insulin resistance:a marker of surgical stress.Curr Opin Nutr Metab Care ,1999,2:69-78.
8.Braden H,Cheema-Dbadlis,Mazerc D,et al.Hyperglycemia during normothermic cardiopulmonary bypass.Ann Thorac Surg,1998,65(6):1588.
9.Rapp-Kesek D,Stridsberg M,Andersson LG,et al.Insulin-resistance after cardiopulmonary bypass in the elderly patient.Scand Cardiovasc J,2007,41:102-108.
10.Langendorff O.Untersuchungen am uberlebenden saugetierherz.Arch Ges Physiol,1895,61:291-316.
11．黑飞龙，房秀生．围体外循环期应激激素变化及胰岛素抵抗研究．中华胸心血管外科杂志，1999，10(5)：301-302．
12.DeFronzo RA,Ferrannini E.Insulin resistance:A multifaceted syndrome responsible for NIDDM,obesity,hypertension,dyslipidemia,and atherosclerotic cardiovascular disease.Diabetes Care,1991,14(3):173-194.
1.Thrainsdottir IS,Aspelund T,Thorgeirss0n G,et al.The association between glucose abnormalities and heart failure in the population- based Reykjavik study.Diabetes Care,2005,28(3):612-616.
2.Hamby RI,Zoneraich S,Sherman L.Diabetic cardiomyopathy.JAMA,1974,229(13):1749-1754.
3.Fischer M,Baessler A,Hense HW,et al.Prevalence of left ventricular diastolic dysfunction in the community.Results from a Doppler echocardiographic- based survey of a population sample.Eur Heart J,2003,24(4):320-328.
4.Srikanthan P,Hsueh W.Preventing heart failure in patients with diabetes.Med Clin NorthAm,2004,88(5):1237-1256.
5.Wilson Tang WH,Maros A,Young JB.Ischemic heart disease and congestive heart failure in diabetic patients.Med Clin North Am,2004,88(4):1037-1061.
6.Engin A,Bozkurt BS,Ersoy E,et al.Stress hyperglycemia in minimally invasive surgery.Surg Laparosc Endosc,1998,8(6):435-437.
7.Valerio G,Franzese A,Carlin E et al.A high prevalenceof stress Hyperglycaemia in children with febrile seizures and traumatic injures.Acta paediatr,2001,90(6):618-622.
8．彭新丽．2型糖尿病患者血脂、载脂蛋白、脂蛋白、尿微量白蛋白及心肌酶变化的探讨．中华现代内科学杂志，2006，8：918．
9.Diamant M,Lamb H J,Smit JW.Diabetic cardiomyopathy in uncomplicated type 2 diabetes is associated with the metabolic syndrome and systemic inflammation.Diabetologia,2005,48:1669-1670.
10.Korrald C,Elvenes OP,Myrmel T,et al.Myocardial substrate metabolism influences left ventricular energtics in vivo.Am J physiol,2000,278:H1345-H1351.
11.Longnus SI,Wambolt RB,Barr RL,et al.Regulation of myocardial fatty acid oxidation by sustrate supply.Am J Physiol,2001,281:H1561-H1567.
12. Lopaschuk GD. Abnormal mechanical functionin diabete: relationship to altered myocardial carbohydrate/ lipid metabolism. Coron Artery Dis, 1996, 7: 116-123.
1.Foti M,Moukil MA,Dudoqn0n P,et al.Insulin and IGF-1 receptor trafficking and signaling.Novartis Found Symp,2004,262:125-141.
2.Cheatham B,Kahn CR.Insulin action and their insulins signaling network.Endocr Rev,1995,16(2):117-142.
3.Moller D,Flier JS.Insulin resistance mechanisms,syndromes and implications.N Engl J Med,1991,325(13):938-948.
4．杜智勇，史忠，杨天德，等．体外循环心内直视术中胰岛素受体 TPK 活性的变化及意义．体外循环杂志，2002，4：81-87
5.Van Echten G Eckel J,Reinauer H.Down-regulation of insulin receptors in the heart:Studies on primary cultred adult cardiac myocytes.Biochem Biophys Acta,1986,886(3):468-473.
6．杜智勇，史忠．应激与胰岛素抵抗．国外医学-麻醉学与复苏分册，2001，22(1)：10-13．
7.Strommer L,Wickbom M,Wang F,et al.Early impairment of insulin secretion in rats after surgical trauma.Eur J Endocrinol,2002,147:825-833.
8.Hoffman RP.Sympathetic mechanisms of hypoglycemic counter-regulation.Curr Diabetes Rev.2007,3(3):185-193.
9.Katz EB,Burcelin R,Tsao TS,et al.The metabolic corrsequences of alterd glucose transporter expression in transgenic mice.Mol Med,1996,74:639-652.
10.Zorzano A,Fandos C,Palaci M,et al.Role of plasma membrane transporers in muscle metabolism.Biochem J,2000,349(3):667-688.
11.Zierath JR,Krook A,Wallberg- Henriksson H.Insulin action in skeletal muscle from patients with NIDDM.Mol Cell Biochem,1998,182:153-160.
12.Abel ED.Glucose transport in the heart.Front Biosci,2004,9:201-215.
13.Czech MP,Coruera S.Signaling mechanisms that regulate glucose transport.J Biol Chem,1999,274:1865-1868.
14.Mather K,Anderson TJ,Verma S.Insulin action in the vasculature:physioloty and pathophysiology.J Vasc Rec,2001,38(5):415-422.
15．柴伟栋，陈家伟，沈捷，等．葡萄糖、胰岛素对血管内皮细胞功能的影响．中国微循环杂志，2003，7(4)：204-207．
16.Vincent MA,Montagnani M,Quon MJ.Molecular and physiologic actions of insulin related to production of nitric oxide in vascular endothelium.Curr Diab Rep,2003,3:279-188.
17.Wilkes J J,Hevener A,Olefsky J.Chronic endothelin-1 treatment leads to insulin resistance in vivo.Diabetes,2003,52:1904-1999.
18.Helmut OS,Ginger B,Ann J,et al.Insulin-induced skeletal muscle vasodilation is nitric oxide dependent.J Clin Invest,1994,94:1172-1179.
19.Davda RK,stepniakow ski KT,Lu G,et al.Oleic acid inhibits endothelialnitric oxide synthase by a protein kinase C-independent mechanism.Hypertension,1995,26(5):764-770.
1. Russell DS, Gherzi R, Johnson EL, et al. The protein- tyrosine kinase activity of the insulin receptor is necessary for insulin- mediated receptor down- regulation. J Biol Chem, 1987, 262(24): 11833-11840.
2. Hari J, Roth RA. Defective internalization of insulin and its receptor in cells expressing mutated insulin receptors lacking kinase activity. J Biol Chem, 1987, 262(32): 15341-15344.
3. Senn JJ, Klover PJ, Nowark IA, et al. Interleukin- 6 induces cellular insulin resistance in hepatocytes. Diabetes, 2002, 51(12): 3391- 3399.
4. Bernal-Mizrachi C, Weng S, Feng C, et al. Dexamethasone induction of hypertension and diabetes is PPAR- alpha dependent in LDL receptor null mice. Nat Med, 2003,9(8): 1069-1075.
5. Hah J, Jo I, Chakrabarti R, et al. Demonstration of an insulin - insensitive storage pool of glucose transporters in rat hepatocytes and HepG2 cells. J Cell Physiol, 1992, 152(1): 56-63.
6. Melin B, Cherqui G, Blivet MJ, et al. Dual effect of metformin in cultured rat hepatocytes: potentiation of insulin action and prevention of insulin- induced resistance. Metabolism, 1990, 39(10): 1089 -1095.
7. DeFronzo RA, Ferrannini E. Insulin resistance: A multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. Diabetes Care, 1991,14(3): 173 -194.
8. Scuteri A, Di- Bernardo MG, De- Propris AM, et al. Insulin effects on the left ventricular in older hypertensive subjects. J Am Geriatr Soc, 1999, 47(6): 727 - 729.
9. Foti M, Moukil MA, Dudoqnon P, et al. Insulin and IGF- 1 receptor trafficking and signaling. Novartis Found Symp, 2004, 262: 125 - 141.
10. Moller D, Flier JS, Insulin resistance mechanisms, syndromes and implications. N Engl J Med, 1991, 325(13): 938 - 948.
11.Van Echten G Eckel J,Reinauer H.Down- regulation of insulin receptors in the heart:Studies on primary cultred adult cardiac myocytes.Biochem Biophys Acta,1986,886(3):468-473.
12．杜智勇，史忠．应激与胰岛素抵抗．国外医学-麻醉学与复苏分册，2001，22(1)：10-13．
13.Diamant M,Lamb H J,Smit JW.Diabetic cardiomyopathy in uncomplicated type 2 diabetes is associated with the metabolic syndrome and systemic inflammation.Diabetologia,2005,48:1669-1670.
14.Lopaschuk GD.Abnormal mechanical functionin diabete:relationship to altered myocardial carbohydrate/lipid metabolism.Coron Artery Dis,1996,7:116-123.
1. Reaven GM. Role of insulin resistance in human disease. Diabetes, 1988, 37(12): 1595- 1607.
2. Stern MP. Diabetes and cardiovascular disease, the 'common soil hypothesis'. Diabetes, 1995,44(4): 396 -374.
3. Thorell A , Nygren J ,Ljungqvist O , et al . Insulin resistance : a marker of surgical stress. Curr Opin Nutr Metab Care ,1999 , 2 :69 - 78.
4. Salim A, Hadjizacharia P, Dubose J, et al. Persistent hyperglycemia in severe traumatic brain injury: an independent predictor of outcome. Am Sung, 2009, 75(1): 25-29.
5. Krinsley J S. Association between hyperglycemia and increased hospital mortality in a heterogeneous population of critically ill patients. Mayo Clin Proc, 2003, 78: 1417-1478.
6. Engin A,Bozkurt BS, Ersoy E, et al. Stress hyperglycemia in minimally invasive surgery. Surg Laparosc Endosc, 1998, 8(6): 435-437.
7. Valerio G, Franzese A, Carlin E et al. A high prevalence of stress Hyperglycaemia in children with febrile seizures and traumatic injures. Acta paediatr, 2001, 90(6):618-622.
8. Thorell A, Marin P, Smith U, et al. Insulin resistance after abdominal Surgery. British Journal of Surgey, 1994,8(11): 59 -63.
9. Hager P, Hagman B, Wikstrom AC, et al. CRF- receptor 1 blockade attenuates acute posttraumatic hyperglycermia in rats. J of Surg Res, 2004, 119: 72 - 79.
10. Kuroda M, Honnor RC, Cushman SW, et al. Regulation of insulin stimulated glucose transport in the isolated ratadipocyte: CAMP independent effects of lipolytic and antilipolytic agents. J Biol Chem, 1987, 262(1): 245 -253.
11. Mescheryakov A V, Kozlov I A, Dementyeva I I, et al. Glucose metabolism and insulin activity during cardiac surgery. J Cardiothorac Anesth. 1989; 3(5): 536 - 543.
12. Cuthbertson DP: Observations on disturbance of metabolism produced by injury to the limbs:Quat.J.Med,1932,25:233-246.
13.Strommer L,Wickbom M,Wang F,et al.Early impairment of insulin secretion in rats after surgical trauma.Eur J Endocrinol,2002,147:825-833.
14.McPheed S J,et al.Pathophisiology of Disease.(3~(rd) version).Mc Graw-Hill,2000,434-436.
15.Dohan FC,Lukens FDW.Experimental diabetes produced by the administration of glucose.Endocrinology,1948,42:244-262.
16.Leahy JL,Weir GC,Evolution of abnormal insulin secretory responses during 48h in vivo hyperglycemia.Diabets,1988,37:217-222.
17.Randle P J,Garland PB,Hales CN,et al.The glucose fatty acid cycle:its role in insulin sensistivity and the metabolic disturbances of diabetes mellitus.Lancet,1963,1:785-789.
18.Cheatham B,Kahn CR.Insulin action and their insulins signaling network.Endocr Rev,1995,16(2):117-142.
19.Moller D,Flier JS,Insulin resistance mechanisms,syndromes and implications.N Engl J Med,1991,325(13):938-948.
20．杜智勇，史忠，杨天德，等．体外循环心内直视术中胰岛素受体 TPK 活性的变化及意义．体外循环杂志，2002，4：81-87．
21．杜智勇，史忠．应激与胰岛素抵抗．国外医学-麻醉学与复苏分册，2001，22(1)：10-13．
22．毛旭虎，许霖水．胰岛素受体酪氨酸蛋白激酶在严重烫伤大鼠胰岛素抵抗中作用的研究．第三军医大学学报，1996，18：1-3．
23．张波，许霖水，吴喜贵．烧伤后胰岛素受体信号传到缺陷机理的研究．第三军医大学学报，1997，19(1)：4-6．
24.Bang P,Nygren J,Thorell A,et al.Postoperative induction of insulin- like growth factor binding protein- 3 proteolytic activity:relationto insulin and insulin sensitivity.J Clin Endocrirnol Metab,1998,83(7):2509-2515.
25. Black PR, David M, Brooks C, et al. Mechanisms of insulin resistance following injury. Ann Surg, 1982, 196(4): 420.
26. Cusi K, Maezono K, Oaman A, et al. Insulin. resistance differentially affects the pi3-kinase- and MAP kinase- mediated signaling in human muscle. J Clin Invest, 2000, 105:311-320.
27. Gao Z, zuberi A, Quon MJ, et al. Aspririn inhibits serine phosphorylation of insulin receptor substrate 1 in rumor necrosis factor- treated cells through targeting multiple serine kinases. J Boil Chem, 2003, 278: 24944 - 24950.
28. Le Marchand- Brustel Y, Gual P, Gremeaux T, et al. Fatty acid - induced insulin resistance: role of insulin receptor substrate 1 serine phosphorylation in the retroregulation of insulin signaling. Biochem Soc Trans, 2003, 31(Pt6): 1152-1156.
29. Ozes ON, Akca H, Mayo LD, et al. A phosphatidylinositol 3 - Kinase/ Akt/ mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate- 1. Proc Natl Acad Sci USA, 2001, 98: 4640- 4645.
30. Zorzano A, Fandos C, Palaci M, et al. Role of plasma membrane transporers in muscle metabolism. Biochem J, 2000, 349(3): 667- 688.
31. Yu ZW, Buren J, Enerback S, et al. Insulin can enhance GLUT4 gene expression in 3T3- F442A cells and this effect is mimicked by vanadate but counteracted by cAMP and high glucose- potential implications for insulin resistance. Biochim Biophys Acta, 2001, 1535(2): 174- 185.
32. Rea S, James DE. Moving GLUT4: the biogenesis and trafficking of GLUT4 storage vesicles. Diabetes, 1997,46: 1667-1677.
33. McKcown NM, Mcigs JB, Liu S, et al. Carbohydrate nutrition insulin resistance and the prevalence of the metabolic syndrome in the Framingham offspring cohort. Diabetes Care, 2004, 27(2): 538- 546.
34. Zierath JR, Krook A, Wallberg-Henriksson H. Insulin action in skeletal muscle from patients with NIDDM. Mol Cll Biochem, 1998, 182: 153-160.
35. Liu LS, Spelleken M, Rohrig K, et al. Tumor necrosis factor - a acutely inhibits insulin signaling in human adipocytes, implication of the p80 tumor necraosis factor receptor. Diabetes, 1998,47:515-522.
36. Le Marchand- Brustel Y, Gual P, Gremeaux T, et al. Fatty acid- induced insulin resistance: role of insulin receptor substrate 1 serine phosphorylation in the retroregulation of insulin signaling. Biochem Soc Trans, 2003, 31: 1152 - 1156.
37. Gao Z, Zuberi A, Quon MJ, et al. Aspirin inhibits serine phosphorylation of insulin receptor substrate 1 in tumor necrosis factor- treated cells through targeting multiple serine kinases. J Boil Chem, 2003, 278: 24944 - 24950.
38. Cheung AT, Ree D, Kolls JK, et al. An in vivo model for elucidation of the mechanism of tumor necrosis factor- a- induced insulin resistance: Evidence for differential regulation of insulin signaling by TNF-α. Endocrinology, 1998, 139: 4928-4935.
39. Xu J, Kim HT, Ma Y, et al. Trauma and hemorrhage- induced acute hepatic insulin resistance: dominant role of tumor necrosis factor- alpha. Endocrinology, 2008, 149(5):2369-2382.
40. Gupta A, Ten S, Anhalt H. Serum levels of solubole tumor necrosis factor- alpha receptor 2 are linked to insulin resistance and glucose intolerance in children. J Pediatr Endocrinol Metab, 2005, 18(1): 75-82.