Abstract

Elevating cytosolic Ca2+ stimulates glucose uptake in skeletal muscle, but how Ca2+ affects intracellular traffic of GLUT4 is unknown. In tissue, changes in Ca2+ leading to contraction preclude analysis of the impact of individual, Ca2+ -derived signals. In L6 muscle cells stably expressing GLUT4myc, the Ca2+ ionophore ionomycin raised cytosolic Ca2+ and caused a gain in cell surface GLUT4myc. Extra- and intracellular Ca2+ chelators (EGTA, BAPTA-AM) reversed this response. Ionomycin activated calcium calmodulin kinase II (CaMKII), AMPK, and PKCs, but not Akt. Silencing CaMKII delta or AMPK alpha 1/alpha 2 partly reduced the ionomycin-induced gain in surface GLUT4myc, as did peptidic or small molecule inhibitors of CaMKII (CN21) and AMPK (Compound C). Compared with the conventional isoenzyme PKC inhibitor Go6976, the conventional plus novel PKC inhibitor Go6983 lowered the ionomycin-induced gain in cell surface GLUT4myc. Ionomycin stimulated GLUT4myc exocytosis and inhibited its endocytosis in live cells. siRNA-mediated knockdown of CaMKII delta or AMPK alpha 1/alpha 2 partly reversed ionomycin-induced GLUT4myc exocytosis but did not prevent its reduced endocytosis. Compared with Go6976, Go6983 markedly reversed the slowing of GLUT4myc endocytosis triggered by ionomycin. In summary, rapid Ca2+ influx into muscle cells accelerates GLUT4myc exocytosis while slowing GLUT4myc endocytosis. CaMKII delta and AMPK stimulate GLUT4myc exocytosis, whereas novel PKCs reduce endocytosis. These results identify how Ca2+ -activated signals selectively regulate GLUT4 exocytosis and endocytosis in muscle cells.