Mechanism Study Of Macrophage Migration Inhibitory Factor Regulates GLUT4 Expression In Cardiomyocytes

Diabetes mellitus,as a chronic non-communicable diseases,has become the third most serious diseases followed by cancers and vasculopathy.Diabetes consists of insulin dependent type I and non insulin dependent type II.A large majority of patients with diabetes in Chinese are type II diabetes.The common features of the diabetes are hyperglycemia and insulin resistance.The greatest danger in diabetes is not itself,but is the cardiovascular complication caused by diabetes,just as diabetic cardiomyopathy.It has been reported that insulin resistance is the "cornerstone" of the diabetic cardiomyopathy.Insulin resistance is secreting deficiency or decreasing sensitivity from peripheral tissue to insulin,resulting in the peripheral tissues can not uptake and use the glucose fully.The myocardium would be dysfunction and it structure would be changed when insulin resistance in a long time,which will develop the diabetic cardiomyopathy.It has reported that(3-cells can release insulin and MIF at the same time.Macrophage migration inhibitory factor(MIF),a specific enzyme with multifunction,is a kind of important cytokine in regulating the inflammatory reaction and immune response.MIF can increase insulin release through pancreas β-cells,which can increase the glucose uptake.MIF also can decrease the glucose uptake in the adipocytes,which can increase the insulin resistance.What’s more,MIF still can increase glycolysis through myotubules.It has confirmed that MIF play a role in many cardiovascular diseases.The receptors and signal pathways of MIF are very complicated,but the mechanisms about MIF in Myocardial insulin resistance are unclear.Insulin resistance is mainly caused by insulin insufficient or target organs insensitive to it,which will cause that insulin signaling pathway can’t be activated fully in the downstream.So the glucose can’t be fully transported into the cells by the glucose transporters(GLUTs),then the glucose remain and its concentration elevate in blood.Keeping blood glucose levels in balance is mainly dependent on the glucose transporters(GLUTs).However,in the cardiomyocyte,glucose transporter 4(GLUT4)plays a major role in glucose transportion.insulin can work on insulin receptors and insulin receptor substrates to activate AKT and AS 160 signal pathways,which cause a serious responses included increasing expression of the GLUT4 and its transport speed.At present,in myocardium,whether MIF have an effect on GLUT4 expression or its function is unclear.In this thesis,the mRNA and protein levels of MIF and GLUT4 will be detected in diabetic mice myocardial tissue.In the neonatal mouse ventricular cells,GLUT4 expression and glucose uptake ability affected by MIF will be detected.In order to testify the transcription factors play roles in MIF upregulated GLUT4,so the siRNA and EMSA technology were operated.The signal pathways take part in MIF upregulated GLUT4 progress were detected by Western blot assays.Chapter Ⅰ:The expressions of MIF and GLUT4 in diabeticmyocardiumObjective:to identify the expressions of MIF and GLUT4 in diabetic myocardium.Methods:18w old male db/db and db/m mice(8 mice in each group),MIF and GLUT4 gene and protein levels by qPCR and Western blot assay.We also detected the expression of MIF by IHC assay.Results:qPCR and Western blot results show that MIF was significantly increased expression in db/db mice myocardium.However,the expression of GLUT4 was significantly decreased in them.We also confirmed the increased expression of MIF in db/db mice by IHC assay.Chapter Ⅱ:MIF upregulates GLUT4 expression in NMVCsObjective:to identify the effect of MIF on GLUT4 expression and glucose transport in myocardium.Methods:we separated primary neonatal mouse cardiomyocytes,cultured 48h,starved overnight,then exposed to different dose of MIF for 24h,Assay the changes of GLUT4 gene and protein levels in primary NMVCs by qPCR and western blot.We also collected the supernatant of the dose course MIF-treated NMVCs and detected the glucose concentration by ELISA assay.The changes of GLUT4 protein expression was observed in MIF-treated NMVCs in a time course study by fluorescence confocal assay.Results:qPCR and Western blot results showed that MIF can upregulate GLUT4 gene and protein expressions significantly in NMVCs.compared with the blank control group,the ELISA assay result showed that MIF can enhance the glucose uptake capacity of the NMVCs.Fluorescence confocal results show that the expressions of GLUT4 in MIF-treated NMVCs were increasing in time-dependent.the most increases were at 12 h and 24 h versus the others groups.Chapter Ⅲ:Identications of transcription factors and signalingpathways mediating MIF-promoted GLUT4 expressionObjective:to identify the transcription factors and signal transduction pathways participating in MIF induce GLTU4 expression.Methods:In MIF treated NMVCs experiments,we have been detecting the transcription factors GATA6、KLF15、MYOD、PGC1、TRα、ZAC1、MEF2A、MEF2C、,MEF2D、AMPKα1、AMPKα2、pPPARr mRNA and protein levels by qRT-PCR and Western blot assay,and screening those transcription factors whose changed were significantly in the process of MIF upregulated GLUT4.We also detected those significantly changed transcription factors mRNA and protein levels in diabetic mice myocardium.The siRNA technology were used to knockdown those transcription factors,then assaying the mRNA and protein levels of the GLUT4 by qRT-PCR and Western blot,also detecting the glucose uptake capability of the NMVCs.The activations of those transcription factors in MIF-treated NMVCs was assayed by EMSA.The activation of AKT and AMPK signal pathways were assayed by Western blot in MIF-treated NMVCs and db/db mice myocardium.AKT、AMPK inhibitors LY294002、Compound C and MIF treated the NMVCs respectively.GLUT4 mRNA and protein levels were detected by qPCR and Western blot assay.Results:Based on the qRT-PCR and Western blot results,we found that transcription factors ZAC1,MEF2A,MEF2C,MEF2D genes and proteins increased significantly in MIF treated NMVCs,but they were reduced in diabetic myocardium.GLUT4 genes and proteins were decreased significantly when ZAC1、MEF2A、MEF2C、MEF2D were knockdown by the corresponding siRNAs.what’s more,the mRNA and protein levels of ZAC1 also decreased in MEF2A、MEF2C and MEF2D knockdown group.The glucose uptake capability of the NMVCs also reduced when the transcription factors ZAC1、MEF2A、MEF2C、MEF2D were knockdown by corresponding siRNA.EMSA results also show that the activations of the transcription factors ZAC1、MEF2 were increased in the nucleus of MIF-treated group versus the blank.According to Western blot results,we known that AKT、AMPK signal pathway in diabetic myocardium were inhibited,however,they were activated in MIF-treated NMVCs in a time-dependent.In order to observe the effect of the GLUT4 from AKT、AMPK signal pathways.So we treated the NMVCs with AKT、AMPK inhibitors LY294002、Compound C and MIF respectively.From the qPCR and WB results,GLUT4’s gene and protein levels were significantly decreased in AKT、AMPK inhibitors groups versus the positive group.Especially,in the both of AKT and AMPK inhibited group,the decreased of GLUT4 were the most significantly.SummaryIn the present study,we found that MIF was increased expression in db/db mice myocardium,but GLUT4 was decreased in them.In the neonatal mouse ventricular cell experiments,we found that MIF can increased GLUT4 expression significantly,it also can enhance the glucose uptake capability of the NMVCs.From the qPCR and Western blot assay the siRNA interfere expriments results,we found that the changes of ZAC1,MEF2A,MEF2C,MEF2D mRNA levels and protein levels were significantly increased in MIF-treated NMVCs.However,they were decreased in diabetic myocardium.The siRNAs technology were used to knockdown transcription factors ZAC1、MEF2A、MEF2C、MEF2D expressions,then qPCR and WB results show that the GLUT4 mRNA levels and proteins levels were decreased,and the glucose uptake capability of the NMVCs also was depressed.From EMSA experiment results,the transcript activity of ZAC1、MEF2 in MIF-treated NMVCs was increased obviously.From Western blot results,we found that MIF can activate AKT、AMPK signal pathways in NMVCs which were inactivated in diabetic mice.We identified that AKT、AMPK signal pathways were play roles in MIF upregulated GLUT4 expression progress.In conclusion,in our study,we found that MIF can increase the transcription factors ZAC 1、MEF2A、MEF2C、MEF2D expressions by activated AKT、AMPK signaling pathway,then those transcription factors binding to GLUT4 mRNA promoter zones to increase GLUT4 mRNA expression,and the GLUT4 protein level also increase.What’s more,transcription factors MEF2A、MEF2C、MEF2D also can regulate ZAC1 to increase GLUT4 expression,all of the reactions caused by MIF can result increasing the glucose transportion,finally.