Rab8a Regulates The GLUT4 Translocation In C2C12 Myoblasts

OBJECTIVE: To investigate the regulation of Rabs especially Rab8 A protein on GLUT4 translocation in C2C12 myoblasts.To validate insulin sensitivity in C2C12 myoblasts,establish and optimize the transfection condition,screen of colocalization of Rab proteins and GLUT4,and then investigate the role of Rab proteins in GLUT4 translocation.METHODS:(1)C2C12 myoblasts were cultured with DMEM containing 10% fetal bovine serum,the proliferation status being detected by cell morphology through phase contrast microscopy,and transfection condition was optimized.(2)C2C12 myoblasts were lipotransfected with HA-GLUT4-mCherry to explore the effects of insulin stimulation on GLUT4 translocation in C2C12 myoblasts and determine the insulin sensitivity of C2C12 cells.(3)Cells were transfected of HA-GLUT4-GFP/mCherry and Rab-RFP/GFP to label intracellular Rab proteins and GLUT4 protein,and colocalization was observed with TIRFM microscope,screening Rab protein which can affect the GLUT4 translocation in C2C12 myoblasts.(4)IRAP-PHluorin can reveal the cell fusing with the plasma membrane in insulin-stimulated state,which is a PH-sensitive GFP mutant that only fluoresces as a molecular probe when exposed to a nonacidic environment.Because the PH inside GLUT4 vesicles is acidic and that outside of cells is neutral,when GLUT4 vesicles fuse with the PM,exposing IRAP-PHluorin extracellularly,the probe fluoresces brightly.C2C12 cells were co-expressed with Rab8A-mCherry and IRAP-PHluorin to determine the role of Rab8 A while GLUT4 vesicles undergoing fusion with the plasma membrane with TIRFM microscopy.(5)Rab8A-WT-mCherry,Rab8A-T22N-GFP or Rab8A-Q67L-GFP were co-expressed with Myc-GLUT4-BFP in C2C12 myoblasts,after 24 h,performed immunofluorescence staining of Myc on the cell membrane,and then examined the effect of Rab8 A mutants on the GLUT4 translocation in C2C12 myoblastswith TIRFM microscopy.(6)Knockdown efficiency of Rab8 A was detected by Western Blot.After optimization of protein knockout conditions,the three different conditions(Rab8A,Rab8A-knockdown,Rab8A-rescue)of GLUT4 translocation efficiency were observed by TIRFM microscope,and then the role of Rab proteins in GLUT4 translocation was determined.RESULTS:(1)Cells were cultured with DMEM with 10% fetal bovine serum,normal myoblasts were spindle-shaped after adherent.After digestion,cell transfection efficiency of suspended state is higher than that of adherent state,and the optimal ratio of liposome to plasmidwas 5:1.(2)After insulin stimulation the GLUT4 translocation percentage was 1.8 times more than that in basal.(3)After Rab protein and GLUT4 were transfected with fluorescence labeling,it is obvious that there is a large area of overlap between GLUT4 and Rab8 A,Rab13,Rab14,but not Rab10 or Rab 11 with TIRFM microscopy.(4)Rab8A is present before the GLUT4 fusioning,and then spread to the outside of the cell after GLUT4 vesicle fusion.(5)Overexpression of Rab8 A mutants have different effects on the GLUT4 translocation in C2C12 myoblasts,overexpression of Rab8A-Q67 L enhanced GLUT4 translocation,but the expression of Rab8A-T22 N reduced GLUT4 translocation.(6)After optimization of knockout efficiency,the ratio of siRNAto RNA iMax was 4:1 and protein knockdown efficiency reached 80%.Rab8 A knockdown reduce insulin-stimulation induced GLUT4 traffic to the plasma membrane,nevertheless,it can be rescued by re-expression of wild-type Rab8A-mCherry.CONCLUSION:(1)C2C12 myoblasts proliferated fast in vitro,suspended cells were used for transfection after digestionand the ratio of lipo to plasmid is 5:1.(2)Insulin stimulation can enhance the GLUT4 translocation in C2C12 myoblasts.C2C12 myoblasts with insulin sensitivity are suitable for the follow-up study.(3)Endogenous Rab8 A,Rab13 and Rab14 colocalize with GLUT4 in C2C12 myoblasts,but not Rab10 and Rab11.(4)Rab8A plays an important role while GLUT4 fusing with the cell membrane in C2C12 myoblasts.(5)Overexpression of Rab8 A mutants have different effects on the GLUT4 translocation in C2C12 myoblasts.Rab8A-Q67 L promotes the GLUT4 translocation,while Rab8A-T22 N inhibited GLUT4 translocation.(6)Rab8A knockdown reduce insulin-stimulation induced GLUT4 traffic to the plasma membrane,which can be rescued by re-expression of Rab8A-mCherry.The Rab8 A can regulate C2C12 intracellular translocation of GLUT4 mainly through the regulation of GLUT4 fusioning to the membrane.