Effects And Mechanisms Of Exendin-4Onglocose Uptaking In H9c2Myocardial Cells

Part I Study on2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose used for glucose uptake in H9c2cellsObjective: To assess the possibility of a deoxyglucose analog(2-NBDG) as a fluorescence probe to detect the glucose uptake in H9c2cells.Method: Glucose transporter1(GLUT1) and Glucose transporter4(GLUT4) proteins in H9c2cells were identified byindirect immunofluorescence (IIF) assays. H9c2cells were incubated with2-NBDG at a cohort concentration for different periods and the mostsuitable concentration and exposure period were determined byfluorescence microplate reader. H9c2cellswere incubated with100μmol/L2-NBDG for30min with or without D-Glu, a potent competitive inhibitorof2-NBDG. After the incubation, the2-NBDG uptake was observed andrecorded by confocal laser scanning microscope (CLSM) and flowcytometry (FCM). Result:(1): Overexpressed GLUT1and GLUT4proteins were discovered inH9c2cells. Used the2-NBDG to detecte cell glucose uptake and confirmedthat insulin can promote glucose uptake in H9c2cells.(2)：The most suitable concentration and exposure period for H9c2cells incubation were100μmol/L2-NBDG and30minutes. Afterincubated with2-NBDG with or without D-Glu, bright green fluorescenceof the2-NBDG in H9c2cells could be observed under CLSM. Theintracellular accumulation of2-NBDG fluorescence decreased to27%(CLSM)(P<0.01) and46.7%(FCM)(P<0.01) in groups of co-incubatingwith D-Glu.(3)：Used the2-NBDG to detecte cell glucose uptake and confirmedthat insulin can promote glucose uptake in H9c2cells.Conclusion:2-NBDG is quickly transported by GLUT1and GLUT4and accumulated in the H9c2cells, which can be inhibited by D-Glu.2-NBDG may be used as an optical probe for glucose uptake in H9c2cells. Part I I Exendin-4Increases Myocardial Glucose Uptake viap38MAP Kinase-Mediated in H9c2myocardial cellsObjective: To study the effects and mechanisms of exendin-4onglucose uptaking in H9c2myocardial cells.Method: Use different concentrations and different incubation ofEx-4to deal with H9c2myocardial cells.To explore the relationship ofEx-4’s dose-effect and the time-effect, Use the fluores cencemicroplatereader to determinate the optimal concentration and time of Ex-4in H9c2cells. We divide H9c2cells to different group: normal control group (NC),Ex-4group, BIRB796group,Insulin-group. Used2-NBDG to detect theglucose uptake in H9c2cells by fluorescence microplate reader and flowcytometry (FCM). GLP-1receptor (GLP-1R) in H9c2cells were identifiedby indirect immunofluorescence (IIF) assays.Result:1. Overexpressed GLP-1Rproteins were discovered in H9c2cells.2. The most suitable concentration and exposure period for H9c2cellsincubation were200μmol/L EX-4and45minutes.3. Used the2-NBDG to detecte cell glucose uptake and confirmed thatEx-4(200uM) and Insulin(200uM) can promote glucose uptake in H9c2cells(p＜0.05). 4. EX-4can effectively promote the H9c2myocardial cells glucoseuptake by flow cytometry (FCM), this effect can be blocked by thep38MAPK pathway inhibitor BIRB796(p＜0.05).Conclusion: Exendin-4increases myocardial glucose uptake via p38MAP Kinase mediated in H9c2cells,and independent of its insulinotropiceffects.