The Role Of AMPK, CaMKII And PKC In The Mechanism Of Ca²⁺-regulated GLUT4Translocation In Skeletal Muscle Cell

Objective:In previous study, our group found that the Ca2+ionophore ionomycin elevated cytosolic Ca2+and stimulated glucose uptake through regulated intracellulare traffic of glucose transporter4(GLUT4) in skeletal muscle cell, ionomycin enhanced GLUT4exocytosis and inhibited GLUT4endocytosis. To confirm the detail signaling mechanism, our purpose is to study the function of AMPK, CaMKII and PKC in ionomycin regulated GLUT4endocytosis and exocytosis.Methods:Use1μM of final concentration ionomycin treatment L6-GLUT4myc myoblast which is stable over expressing GLUT4myc in L6, test the cell surface GLUT4myc level by enzyme-linked immnosorbent assay (ELISA), the phosphorylation of different proteins, the efficiency of knockdown and the effect of chemical inhibitor on the phosphorylation of protein were measured by western blot. siRNA-mediated knockdown to silence the purpose proteins, and then study whether AMPK and CaMKII involved in the ionomycin-regulated GLUT4myc endocytosis and exocytosis. Apply the conventional plus novel PKC inhibitor Go6983or the conventional PKC inhibitor Go6976, study if PKC invole in the GLUT4translocation.Results:1μM of final concentration ionomycin activated calcium calmodulin kinase II (CaMKII), AMP-activated protein kinase (AMPK) and protein kinase Cs (PKCs) at5min and10min, but not Akt. Apply siRNA-mediated mothod to silence CaMKIIδ, inhibited ionomycin stimulated GLUT4myc exocytosis but no effect on GLUT4myc endocytosis, and then reached to lower ionomycin stimulated cell surface GLUT4myc number. Apply siRNA-mediated mothod to silence AMPKα1/a2or use small molecule inhibitor of AMPK (Compound C) did not effect on ionomycin inhibited GLUT4myc endocytosis. The inhibitor of CaMKK (STO609), inhibited ionomycin raised GLUT4exocytosis, but did not influent the rate of GLUT4my enodocytosis, and then reached to reduce ionomycin-induced gain in surface GLUT4wyc. The conventional plus novel PKC inhibitor Go6983lowered the ionomycin-induced gain in cell surface GLUT4myc. The ionomycin stimulated PKC substrate phosporylation was reduced by conventional PKC inhibitor Go6976or conventional plus novel PKC inhibitor Go6983. siRNA-mediated mothod to silence CaMKII8, decreased the total protein expression of CaMKII, reduced the basal and ionomycin stimulated CaMKII T286phosphorylation, the basal and ionomycin stimulated AMPK T172phosphorylation no change. Inhibited the protein expression of AMPK, decreased the protein expression level, the basal and ionomycin stimulated AMPK T172phosphorylation decreased, but the fold change of CaMKII T286phosphorylation no difference. The chemical inhibitor STO609of CaMKK pre-incubate cell reduced the basal CaMKII T286phosporylation, but the fold of ionomycin elevated CaMKII T286phosporylation no significant difference. The inhibitor of PKC Go6976(conventional) and Go6983(conventional and novel) no effect on the basal and ionomycin stimulated AMPK and CaMKII.Conclusion:1. Ionomycin increased the cell surface number of GLUT4myc in L6-GLUT4myc skeletal muscle cell, stimulated AMPK, CaMKII, PKCs phosporylation.2. CaMIIδ involved in ionomycin regulated GLUT4myc exocytosis.3. AMPK and CaMII8did not involved in ionomycin GLUT4myc endoocytosis.4. The conventional plus novel PKC inhibitor Go6983involved in ionomycin regulated GLUT4myc traffic.5. The activity of AMPK which is stimulated by ionomycin is CaMKII-independent.6. The activity of CaMKII which is stimulated by ionomycin is AMPK-independent or CaMKK-independent.7. Ionomycin stimulated PKC no effect on the CaMKII and AMPK signaling pathway.These results identify how Ca2+-activated signals selectively regulate GLUT4myc exocytosis and endocytosis in muscle cells.