Study On Improving Effects And Mechanisms Of Coumarins On Insulin Resistance In Vitro And In Vivo

Objective: Our study aims to investigate the improving effects and its mechanisms of coumarins on insulin resistance(IR)in vitro and in vivo.To establish IR model of C2C12 skeletal muscle cells induced by dexamethasone in order to screen coumarins with the potential of improving the ability of glucose uptake,futhermore,to explore in vivo therapeutic effect of coumarins on IR in mice triggered by dexamethasone.Methods: 1.In vitro:(1)C2C12 cells were cultured in DMEM medium containing 10% FBS,then they were induced differentiation into myotubes with DMEM containing 2% HS.Proliferation and differentiation were studied with morphological assay and Hoechst 33342 staining;(2)Cells were treated 24 hours with different concentrations of dexamethasone,then the optimum concentration of dexamethasone for IR modeling was determined by MTT assay and GOD-POD method;(3)Cells were incubated with three concentrations(low,medium,high)of coumarin derivants,the vitality of cells was determined by MTT assay;(4)Cells were incubated with non-toxic coumarin derivants for 24 hours,GOD-POD method was used to detect cells glucose uptake,to evaluate whether coumarins have insulin-likeness or insulin-sensitizing effect,thus we screened the coumarins which had the potential of improving IR.2.In vivo experiments were carried out to explore the protective effects of coumarins on IR in mice.1 mg/kg dexamethasone(i.m.)was chosen to induce mice IR.Metformin as the positive drug,mice were given coumarins which was screened in the cell experiments:(1)Mice body weight and blood glucose were determined every other week;(2)Testing glucose tolerance of mice;(3)Histopathological observations of skeletal muscle tissue with HE staining were carried out to study the effects of coumarins on IR.3.In molecular level,the therapeutic mechanisms of coumarins on IR was studied.Expression of proteins total GLUT4、membrane GLUT4、p-Akt(Ser473)、Akt、p-AMPK(Thr172)、AMPK、p-TBC1D1(Ser237)and TBC1D1 in skeletal muscle were determined by western blot.Results: Coumarin,fraxetin,esculin and osthole could effectively protect C2C12 cell against IR induced by dexamethasone,fraxetin,esculin and osthole were screened in cell experiments for the following in vivo experiments:(1)Normal C2C12 cells were spindle,several C2C12 cells fused into a cell during differentiation,the new cells presented polarity,they paralleled regularly in the same directionin and formed into myotubes,and Hoechst 33342 staining pictures showed there existed multiple nucleus in myotubes;(2)Cells were treated 24 hours with 0.1 μM dexamethasone,the uptake of glucose was significantly reduced,however,there was no obvious effect on cell growth,thus we established cell IR model;(3)Isopsoralen,aesculetin and high concentration osthole all had significant influence on vitality of C2C12 cell,however,other coumarins and 25 μM Osthole affected C2C12 cell vitality indistinctively;(4)Coumarin,fraxetin,esculin and osthole all played roles in insulin-mimetic or insulin-sensitizing,they improved IR induced by dexamethasone remarkably.2.Fraxetin(40 mg/kg/day),esculin(40 mg/kg/day)and osthole(20 mg/kg/day)all relieved mice IR:(1)Esculin,fraxetin and osthole all increased IR mice weight,significantly reduced mice blood sugar,improved glucose tolerance notably;(2)Esculin,fraxetin and osthole all improved skeletal muscle atrophy,reduced nuclei shift;3.Esculin,fraxetin and osthole all had the effects of activating associated proteins in insulin signaling pathway,thereby improving IR:(1)Fraxetin promoted GLUT4 translocation to the plasma membrane mediated by activation of Akt,thus reduce IR mice blood glucose;(2)Esculin and osthole all had the potential to activate Akt and AMPK,further activated its downstream substrate TBC1D1,promoted GLUT4 translocation to the plasma membrane,finally improved IR in mice.Conclusion: Esculin,fraxetin and osthole could promote the glucose uptake of insulin resistance cells induced by 0.1 μM dexamethasone,reduce blood sugar and improve glucose tolerance of IR mice induced by dexamethasone;probably by activation of Akt,promotion of GLUT4 translocation,thus improvement of insulin resistance.Esculin and osthole also could activate AMPK,thereby activate TBC1D1 promote GLUT4 translocation to membrane and,consequently improve IR.