The Effect Of Cytosolic Ca²⁺ On The Endocytosis And Exocytosis Of GLUT4in Skeletal Muscle Cells And The Signal Mechanism

Objective:To explore the effect of cytosolic Ca2+on the endocytosis and exocytosis of GLUT4and the involved signaling molecular in rat L6-GLUT4myc skeletal muscle cells.Methods:In the first part, Ca2+ionophore ionomycin increases intracellular Ca2+concentration and the elevation of Ca2+mediates exercise/contraction-stimulated glucose uptake in skeletal muscle. We treated L6-GLUT4myc myoblasts with1μM ionomycin to test the effect of ionomycin on the cell surface GLUT4myc level by enzyme-linked immnosorbent assay (ELISA) and on the phosphorylation of5’-AMP-activated protein kinase (AMPK) and calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) by Western blotting. Besides, we also test the effect of20μM AMPK specfic inhibitor Compound C (CC) and5μM CaMK Ⅱinhibitory peptide CN21(derived from CaMK Ⅱ N amino acids43-63) on ionomycin-stimulated GLUT4myc translocation and the phosphorylation of AMPK and CaMK Ⅱ, to confirm whether AMPK and CaMK Ⅱ are involved in ionomycin-mediated GLUT4myc translocation.In the second part, the level of cell surface GLUT4is maintained by a balance of endocytosis and exocytosis of the transporter, and increased by retarding endocytosis, stimulating exocytosis. By using CC and CN21, we explore if AMPK and CaMK Ⅱ are involved in the effect of ionomycin on GLUT4endocytosis and exocytosis, respectively.Results:In the first part of this study, results show that1μM ionomycin increased L6-GLUT4myc myoblasts surface GLUT4myc levels and phosphorylations of AMPK and CaMK Ⅱ; CC and CN21inhibited ionomycin-stimulated phosphorylation of AMPK and CaMK Ⅱ, and inhibited31%and30%ionomycin-stimulated GLUT4myc translocation respectively (p<0.05) without changing basal GLUT4myc levels on the cell surface.In the second part of this study, results show that AMPK inhibitor CC inhibited ionomycin-stimulated GLUT4myc exocytosis; CaMK Ⅱ inhibitory peptide CN21inhibited both ionomycin-stimulated GLUT4myc exocytosis and ionomycin-retarded GLUT4myc endocytosis.Conclusion:1. Ionomycin increases L6-GLUT4/myc myoblasts surface GLUT4myc levels, i.e. stimulates GLUT4myc translocation.2. Ionomycin stimulates phosphorylation of AMPK and CaMK Ⅱ of L6-GLUT4myc myoblasts, which indicates that AMPK and CaMK Ⅱ response to ionomycin and CC and CN21inhibite ionomycin-stimulated phosphorylations of AMPK or CaMK Ⅱ.3. CC and CN21inhibite ionomycin-stimulated GLUT4myc translocation in L6-GLUT4myc myoblasts, which indicates that AMPK and CaMK Ⅱ might be involved in ionomycin-increased GLUT4myc translocation in L6-GLUT4myc myoblasts.4. CC inhibtes ionomycin-stimulated GLUT4myc exocytosis, which indicates that AMPK is involved in ionomycin-regulated GLUT4myc exocytosis; CN21inhibites both ionomycin-stimulated GLUT4myc exocytosis and ionomycin-retarded GLUT4myc endocytosis, which indicates that CaMK II is involved in ionomycin-regulated GLUT4myc endocytosis and exocytosis.