Effect Of Tiliroside-derivatives On GLUT4Translocation In Skeletal Muscle Cells And Its Signal Mechanism

Objective:To explore the effects of tiliroside-derivatives which are potential antidiabetic compounds on GLUT4translocation and its cell signal mechanism in mouse C2C12-GLUT4myc and rat L6-GLUT4myc skeletal muscle cells.Methods:In the first part, we have synthesized7tiliroside-derivatives,(TDs)(TD-la; TD-1b; TD-1c; TD-2a; TD-2b; TD-3; TD-4) by organic chemistry methods in our previous reaserch. In this reaserch, C2C12-GLUT4myc myotubes were incubated with1μg/ml indicated derivatives, respectively, then cell surface GLUT4myc levels were measured by enzyme-linked immnosorbent assay (ELISA). L6-GLUT4myc myotubes were incubated with100nM insulin or with TD-3as indicated dose for24h or as indicated time, then, cell surface GLUT4myc levels were detected by ELISA. The cytotoxicity of tiliroside-derivatives was determined by3-(4,5-dmiemylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoli um (MTS) after24h incubation.In the second part, to investigate the mechanism of TD-3-stimulated GLUT4myc translocation, we detected the expression of GLUT4, GLUT1and AMPKa and the phosphorylation of5’-AMP-activated protein kinase (AMPK), Acetyl-CoA carboxylase (ACC) and protein kinase B (Akt) by western blotting, respectively. By using DT-3and insulin together, we explore if the effect of TD-3and insulin was additive. We also tested the effect of20uM Compound C (CC), the AMPK specfic inhibitor, on DT-3-stimulated GLUT4myc translocation to confirm whether AMPK involved in tiliroside-derivatives-mediated GLUT4myc translocation.Results:In the first part of this study, results show that TD-2a and TD-3significantly increased cell surface GLVT4myc levels in C2C12-GLUT4wyc myotubes after1and2h treatment. The maximal fold increase above basal was1.46±0.15-and1.61± 0.10-fold, respectively (p<0.05). In L6-GLUT4myc cells, TD-3enhanced GLUT4/nyc translocation in a dose-and time-dependent manner. However, the dose and time needed to increase surface GLUT4myc levels in L6is higher or longer than in C2C12cells. The different effects may be because of higher AMPK protein abundance in C2C12than L6-GLUT4myc cells. L6-GLUT4wyc cell viability was not affected by up to5μg/ml derivative TD-3.In the second part of this study, TD-3strongly phosphorylated AMPK and its substrate, ACC in both C2C12-and L6-GLUT4wyc skeletal muscle cells. However, Akt phosphorylation was not affected and the expression of GLUT4, GLUT1, Akt and AMPKa was also unchanged in both cells. The effect of TD-3and insulin was additive and AMPK specfic inhibitor Compound C inhibited25%TD-3-stimulated GLUT4myc translocation (p<0.05) without changing basal GLUTAmyc levels on the cell surface in C2C12-GLUT4myc cells.Conclusion:1. Tiliroside-derivative TD-3is the most effective compound of7derivatives.2. Tiliroside-derivative TD-3increases C2C12-GLUT4myc and L6-GLUT4myc skeletal muscle cells surface GLUT4myc levels.3. TD-3did not affect the expression of GLUT4, GLUT1and AMPKa in skeletal muscle cells.4. TD-3stimulates phosphorylation of AMPK and ACC of C2C12-GLUT4myc and L6-GLUT4myc myotubes, which indicates that AMPK response to TD-3.5. The effect of TD-3and insulin was additive, which indicates that the signal mechanism about TD-3-increased cells surface GLUTAmyc levels is different from insulin-increased.6. CC inhibits TD-3-stimulated GLUT4myc translocation, which indicates that AMPK might be involved in TD-3-increased GLVT4myc translocation in skeletal muscle cells.