The Role Of AMPK In The Mechanism Of Myocardial Insulin Resistance During Myocardial Ischemia-reperfusion Injury In Dogs Cardiopulmonary Bypass

Objective: To detect the changes of the expression and activity of AMPK during myocardial ischemia-reperfusion injury in dogs cardiopulmonary bypass,and investigate the role of AMPK in the mechanism of myocardial insulin resistance(IR)during myocardial ischemia-reperfusion injury.Methods: 24 healthy mongrel dogs were randomly assigned to four groups.Group I(n=6)no aortic cross-clamping;Group II(n=6)received aortic cross-clamping for 60 min;Group III(n=6)received aortic cross-clamping for 60 min with AICAR(AMPK activator),Group IV(n=6)received aortic cross-clamping for 60 min with compound C(AMPK inhibitor).Before bypass(T1)and after the aortic opening,at 15 min(T2),30 min(T3),90 min(T4)respectively,a left subclavian artery blood,coronary sinus blood were colleceted.moreover,before bypass(T1),as well as aortic cross-clamping at 60min(T1-2),and after the aortic opening at 15 min(T2),30 min(T3),90 min(T4)respectively,the left ventricular myocardium were harvested.Next,plasma glucose was detected by enzymatic measurement.Myocardial glucose intake and the net extraction rate of glucose were calculated.Plasma free fatty acids concentration was measured by oxidase colorimetic measurement.Insulin and glucagon were determined by the radioimmunoassay.The changes of the insulin resistance index(IRI)at each time point were analyzed and compared.The changes of cardiac hemodynamics were monitored in perioperative period.The expressions of AMPK and Glut4 mRNA were detected by real-time PCR.Finally,the protein expression of P-AMPK,T-AMPK and Glut4 were detected by immunohistochem-istry.Results: 1.Myocardial glucose metabolism: After myocardial ischemia-reperfusion,compared with that in groupⅠand before bypass,plasma glucose concentration increased to various degrees in groupⅡ,group Ⅲ and group Ⅳ respectively(p<0.01),reached the peak after being reperfused 15 min later(p<0.01).Compared with that in groupⅡ,plasma glucose concentration increased slightly,lasted shortly and recovered fastly in group Ⅲ(P<0.01),plasma glucose concentration increased more significantly in group Ⅳ,lasted longer and recovered slower(p<0.01).Compared with that in group Ⅲ,plasma glucose concentration increased more significantly,lasted longer and recovered slower in group Ⅳ(P<0.01).Meanwhile the net extraction and net extraction ratio of myocardium glucose decreased significantly to various degrees in groupⅡ,groupⅢ and group Ⅳ respectively(p<0.01).Compared with that in groupⅡ,the net extraction and net extraction ratio of myocardium glucose decreased to lower degree,lasted shortly and recovered fastly in group Ⅲ(p<0.01),the net extraction and net extraction ratio of myocardium glucose decreased more significantly,lasted longer and recovered slower in group Ⅳ(p<0.05 or p<0.01).Compared with that in group Ⅲ,the net extraction and net extraction ratio of myocardium glucose decreased more significantly,lasted longer and recovered slower in group Ⅳ(P<0.01 or P<0.05).2.Plasma insulin and glucagon: After myocardial ischemia-reperfusion,compared with that in the groupⅠand before bypass,plasma insulin and glucagon concentration decreased to various degrees in groupⅡ,groupⅢ and groupⅣ respectively(p<0.05 or p<0.01);Compared with that in groupⅡ,plasma insulin and glucagon concentration increased slightly,lasted shortly and recovered fastly in groupⅢ(P<0.01 or P<0.05),plasma insulin and glucagon concentration increased more significantly,lasted longer and recorvered slower in groupⅣ(p<0.05 or p<0.01).Comparing with that in groupⅢ,plasma insulin and glucagon concentration increased more significantly,lasted longer and recorvered slower in groupⅣ(P<0.01 or P<0.05).3.Insulin resistance index: After myocardial ischemia-reperfusion,comparing with that in the groupⅠand before bypass,insulin resistance index increased to various degrees in groupⅡ,groupⅢ and groupⅣ(p<0.01);Compared with that in groupⅡ,insulin resistance index increased slightly,lasted shortly and recovered fastly in groupⅢ(p<0.01),insulin resistance index increased more significantly,lasted longer and recorvered slower in groupⅣ(p<0.05).Compared with that in groupⅢ,insulin resistance index increased more significantly,lasted longer and recorvered slower in groupⅣ(P<0.01).4.The cardiac hemodynamics and heart function: After myocardial ischemia-reperfusion,compared with that in the groupⅠand prior bypass,LVSP and ±dp/dtmax decreased significantly(P<0.05 or P< 0.01)and LVEDP increased significantly in groupⅡ,groupⅢ and groupⅣ(P<0.05 or P< 0.01);Compared with that in groupⅡ,LVSP and ±dp/dtmax decreased slightly(P<0.05 or P< 0.01),LVEDP increased slightly,lasted shortly and recovered fastly in groupⅢ(P<0.05 or P< 0.01);LVSP and ±dp/dtmax in groupⅣ decreased more significantly(P<0.05 or P< 0.01),LVEDP increased more significantly,lasted longer and recovered slower(P<0.05 or P<0.01);Comparing with that in groupⅢ,LVSP and ±dp/dtmax decreased more significantly(P< 0.01),LVEDP increased more significantly,lasted longer and recovered slower in GroupⅣ,(P<0.01).5.Total expression volume of myocardial AMPK mRNA and Glut4 mRNA: After myocardial ischemia-reperfusion,compared with that in the groupⅠand before bypass,the expression of myocardial AMPKmRNA decreased significantly in groupⅡand groupⅣ respectively(P<0.05 or P<0.01);Compared with that in groupⅡ,the myocardial AMPK mRNA in groupⅢ was higher than in groupⅡ(P<0.05 or P< 0.01 or P>0.05),the expression of myocardial AMPK mRNA decreased significantly in groupⅣ(P<0.01).Compared with that in groupⅢ,the expression of myocardial AMPK mRNA decreased more significantly in GroupⅣ,(P<0.01).After myocardial ischemia–reperfusion,the expression of myocardial Glut4 mRNA decreased to various degrees in groupⅡ、groupⅢand groupⅣ(P<0.05 or P< 0.01);Compared with those in groupⅡ,the expression of myocardial Glut4 mRNA higher than groupⅡ(P<0.01),the expression of myocardial Glut4 mRNA decreased significantly in groupⅣ(P<0.01).Compared with that in groupⅢ,the expression of myocardial Glut4 mRNA decreased more significantly in GroupⅣ(P<0.01).6.Total expression volume of myocardial AMPK protein and Glut4 protein: After myocardial ischemia-reperfusion,compared with that in the groupⅠand prior bypass,the expression of myocardial T-AMPK protein and P-AMPK protein decreased significantly in groupⅡand groupⅣ respectively(P<0.05 or P<0.01);Compared with that in groupⅡ,the expression of myocardial P-AMPK protein in groupⅢ was higher than in groupⅡ(P<0.05 or P<0.01),but the change of the expression of myocardial T-AMPK protein was no significant difference in groupⅢ(P>0.05);the expression of myocardial P-AMPK protein decreased significantly in groupⅣ(P<0.01),the expression of myocardial T-AMPK protein decreased,but there was significant difference(P>0.05).Compared with that in groupⅢ,the expression of myocardial P-AMPK protein decreased more significantly in GroupⅣ(P<0.01),the expression of myocardial T-AMPK protein decreased,but there was significant difference(P>0.05).After myocardial ischemia –reperfusion,the expression of myocardial Glut4 protein decreased to various degrees in groupⅡ、groupⅢand groupⅣ(P<0.01);Compared with those in groupⅡ,the expression of myocardial Glut4 protein was higher than groupⅡ(P<0.01),the expression of myocardial Glut4 protein decreased more significantly in groupⅣ(P<0.01).Compared with those in groupⅢ,the expression of myocardial Glut4 protein decreased more significantly in GroupⅣ(P<0.01).Conclusion: 1.In the process of CPB,P-AMPK is the active form of AMPK,and plays an important role in the maintenance of homeostasis of myocardial energy metabolism during myocardial ischemia-reperfusion injury.2.In the process of myocardial ischemia-reperfusion injury during cardiopulmonary bypass,P-AMPK is an important transductive pathway factor,mediating the expression and translocation of Glut4 protein,regulating glucose uptake and utilization.the decrease in total amount of the expression of P-AMPK phosphorylation and T-AMPK protein may be the most important molecular mechanism causing the disorder of Glut4 expression in the process of myocardial IR during myocardial ischemia-reperfusion injury.