The Effects Of PKC And RabGTPase On The Mechanism Underlying Calcium Signal-regulated GLUT4 Traffic In Skeletal Muscle Cells

Objective:To investigate the role of conventional PKC(cPKC), novel PKC(nPKC) and Rab GTPases on the mechanism underlying calcium signal-regulated glucose transpoter 4(GLUT4) traffic in rat L6 muscle cells stably expressing GLUT4 myc. Methods:In the first part, we treated L6-GLUT4 myc myoblasts with ionomycin(1 mM) to test the effect of ionomycin on the phosphorylation of PKC substrate by Western blot. We also tested the effects of conventional and novel PKCs inhibitor G?6983 and conventional PKCs inhibitor G?6976 on ionomycin-stimulated GLUT4 translocation and the phosphorylation of PKC substrate to explore the roles of cPKCs and nPKCs in ionomycin-stimulated GLUT4 translocation.In the second part, the expression of PKCα, PKCζ and PKCε were down-regulated by small interfering RNA, respectively. The cells were divided into basal(untreated) and ionomycin groups. The cell surface GLUT4 levels, endocytosis and exocytosis of GLUT4 were measured by ELISA to confirm which isoform of PKCs is involved in ionomycin-stimulated GLUT4 translocation.In the third part, the expression of Rab8 a, Rab13 and Rab14 were down-regulated by small interfering RNA, respectively. The cells were divided into basal(untreated) and ionomycin groups. Cell surface GLUT4 levels were measured by ELISA to confirm which Rab GTPases is involved in ionomycin-stimulated GLUT4 translocation. Results:Our previous work found that ionomycin increased intracellular Ca2+ concentration and markedly increased cell surface GLUT4 levels in L6-GLUT4 myc myoblasts.In the first part of this study, results show that ionomycin markedly increased phosphorylation of PKC substrate. Both G?6983 and G?6976 reduced ionomycin-induced gain of GLUT4 myc on cell surface(p<0.05, p<0.001, respectively) and lowered ionomycin-stimulated phosphorylation of PKC substrate. Ionomycin stimulated GLUT4 myc exocytosis and inhibited its endocytosis in live cells. Both G?6983 and G?6976 prevented ionomycin-reduced GLUT4 myc endocytosis(p<0.01, p<0.05, respectively). G?6983, but not G?6976, reversed ionomycin-enhanced GLUT4 myc exocytosis(p<0.001).In the second part of this study, results show that siPKCα and siPKCζ, but not siPKCε, reduced ionomycin-induced gain of GLUT4 myc on cell surface(p<0.001). Both siPKCζ and siPKCα prevented ionomycin-reduced GLUT4 myc endocytosis(p<0.001). siPKCζ reversed ionomycin-enhanced GLUT4 myc exocytosis(p<0.001).In the third part of this study, results show that siRab13, but not siRab8 a and siRab14, reduced ionomycin-induced gain of GLUT4 myc on cell surface(p<0.05). Conclusion: 1. Ionomycin can activate PKC. 2. Both cPKCs and nPKCs mediate ionomycin-stimulated GLUT4 translocation.PKCα and PKCζ are responsible for this function. 3. cPKCs prevent ionomycin-reduced GLUT4 endocytosis, as one of isoforms ofcPKCs, PKCα plays a role in this process. nPKCs mediate ionomycin-regulatedGLUT4 endocytosis and exocytosis, as one of isoforms of nPKCs, PKCζ is responsible for this function. 4. Rab13 mediates ionomycin-stimulated GLUT4 translocation.These results reveal that ionomycin promotes GLUT4 translocation in skeletal muscle by PKCα/ PKCζ- Rab13 signal transduction pathway.