Study The Role Of Tiam1 In AICAR-regulated Glucose Metabolism In Muscle Cells

Objective:Skeletal muscle is the largest tissue in regulating postprandial glucose uptake.Insulin-promoted glucose uptake in skeletal muscle plays a key role in the development of insulin resistance and type 2 diabetes.Insulin-independent way to promote the glucose uptake in skeletal muscle can effectively prevent and treat type 2 diabetes.Exercise or AMPK activator AICAR activating AMPK stimulate glucose uptake in skeletal muscle.In addition,Rac1 is also involved in the regulation of skeletal muscle glucose uptake by exercise.Our previous work found that AICAR activated Rac1 in C2C12 skeletal muscle cells.Rac1 inhibitor II significantly inhibited AICAR-stimualted GLUT4 translocation,suggesting that Rac1 may mediate the effect of AICAR.Rac is a small G protein which is regulated by Rac guanosine exchange factors(Rac-GEFs).Both Tiam1 and VAV2 can act as Rac-GEFs to activate Rac1.This study investigated if AICAR activate Rac1 via Tiam1 and/or VAV2 and if Tiam1 and VAV2 mediate AICAR-promoted glucose uptake in skeletal muscle cells.Content:The first part: C2C12 myotubes were treated with AICAR.Protein expression and phosphorylation of Tiam1 and VAV2 were detected by western blot.The second part: Up-regulate or down-regulate of AMPK or Tiam1 by chemical inhibitors,adenovirus or siRNA to investigate the relationship between AMPK and Tiam1: 1.C2C12 myoblasts were transfected with consistive activate AMPK adenovirus(Ad-AMPK-CA),then Tiam1 protein was detected by western blot;2.The AMPK inhibitor Compound C and siRNA were used to inhibit AMPK,then Tiam1 protein was detected by western blot.3.C2C12 myotubes were transfected with siTiam1,then AMPK phosphorylation was detected by western blot.The third part: C2C12 myotubes were transfected with siTiam1,then 2-NBDG glucose uptake was meausured.Methods:Cultured C2C12 cells were differentiated into mature contractile myotubes.C2C12 myotubes were treated with AICAR.Western Blot detected the protein expression of Tiam1 and VAV2.Adenovirus interference,chemical inhibitors and siRNA of AMPK or Tiam1 were used to study the the relationship between AMPK and Tiam1.The expression of Tiam1 was down-regulated by small interfering RNA.Glucose uptake was meausured with 2-NBDG.Results:1.The expression of Tiam1 was increased upon 3 h AICAR treatment,but the protein expression and tyrosine phosphorylation of VAV2 had no change.2.Ad-AMPK-CA increased the protein expression of Tiam1.AICAR-upregulated Tiam1 level was inhibited by Compound C and siAMPK.siTiam1 had no effect on AICAR-stimulated AMPK phosphorylation.3.siTiam1 significantly inhibited AICAR-simulated glucose uptake.Conclusion:AMPK-Tiam1-Rac1 signaling pathway mediates AICAR-stimualted glucose uptake in skeletal muscle cells.