The Role Of PKC In The Mechanism Of Contraction-regulated GLUT4Translocation In Skeletal Muscle Cells

Objective:To explore the roles of PKC isoforms in the mechanism of carbachol-stimulated GLUT4translocation in contractile mouse C2C12GLUT4myc skeletal muscle cells.Methods:On the basis of the previous work, we detected the phosphorylation of protein kinase C before and after carbachol stimulation by Western blotting. By using different protein kinase C inhibitors (bisindolylmaleimide I, G66983, Ro-31-8425and Go6976), cell surface GLUT4myc levels were measured by enzyme-linked immnosorbent assay (ELISA) to investigate the role of PKC on carbachol-stimulated GLUT4myc translocation.We further studied which isoforms of PKC is involved in carbachol-stimulated GLUT4myc translocation by using PKCδ specific inhibitor rottlerin, as well as PKCs peptidic inhibitor (Myr-EAVSLKPT) and PKCδ peptidic inhibitor (Myr-SFNSYELGSL), and cell surface GLUT4myc levels were measured by ELISA.Results:Our previous work found that, C2C12GLUT4myc myotube responsed to0.11mM carbachol stimulation. Carbachol increased cytoplasmic calcium concentration, and increased cell surface GLUT4myc level in C2C12GLUT4mmyc myotubes.In the first part of this study, Western blotting results show that carbachol stimulation increased phosphorylation of PKC in C2C12GLUT4myc myotubes. And ELISA results show that GLUT4myc translocation increased after0.1mM carbachol treatment for15minites.1μM BIM-I or Go6983(both are inhibitors of cPKC and nPKC inhibitors) inhibited72.7%and56.0%carbachol-stimulated GLUT4myc translocation respectively (p<0.05).1μM or3μM Ro-31-8425(cPKC and nPKC inhibitor) inhibited41.7%and54.4%carbachol-stimulated GLUT4myc translocation respectively (p<0.05) without affecting basal GLUT4myc level on the cell surface.1μM Go6976(cPKC inhibitor) had no effect on basal and carbachol-stimulated GLUT4myc translocation. In the second part of this study, we used chemical inhibitor for cPKC isoforms to further study which PKC subtype is involved in carbachol-stimulated GLUT4myc translocation. The results show that3μM and10μM of Rottlerin had no effect on carbachol-stimulated GLUT4myc translocation (p>0.01). In order to further confirm the results, we used3μM PKCε specific peptidic inhibitor and PKCδ peptidic inhibitor to preincubate C2C12GLUT4myc myotubes, we found that3μM PKCε peptidic inhibitor inhibited61.8±0.09%(p<0.01) carbachol-stimulated GLUT4myc translocation while3μM PKCδ peptidic inhibitor had no effect on carbachol-stimulated GLUTAmyc translocation.Conclusion:1. Carbachol increased phosphorylation of PKC in C2C12GLUT4myc myotubes. It indicates PKC response to carbachol.2. BIM-I, G66983and Ro-31-8425inhibited carbachol-stimulated GLUT4myc translocation, whereas the cPKC inhibitor Go6976had no effect. These show that nPKC mediated carbachol-stimulated GLUT4myc translocation in C2C12GLUT44myc myotubes, cPKC was not involved.3. The PKCδ specific chemical inhibitor Rottlerin had no effect on carbachol-stimulated GLUT4myc translocation while the PKCs peptidic inhibitor inhibited it, and the lack of effect of the PKCδ peptidic inhibitor was consistent with the inability of Rottlerin. These indicate PKCs mediating carbachol-regulated GLUT4myc translocation in C2C12GLUT44myc myotubes, PKCδ was not involved.