Ca2+ signals promote GLUT4 exocytosis and reduce its endocytosis in muscle cells

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 GLUT4 myc, the Ca2+ ionophore ionomycin raised cytosolic Ca2+ and caused a gain in cell surface GLUT4 myc. 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 CaMKIIδ or AMPKα1/α2 partly reduced the ionomycin-induced gain in surface GLUT4 myc, as did peptidic or small molecule inhibitors of CaMKII (CN21) and AMPK (Compound C). Compared with the conventional isoenzyme PKC inhibitor Gö6976, the conventional plus novel PKC inhibitor Gö6983 lowered the ionomycin-induced gain in cell surface GLUT4 myc. Ionomycin stimulated GLUT4 myc exocytosis and inhibited its endocytosis in live cells. siRNA-mediated knockdown of CaMKIIδ or AMPKα1/α2 partly reversed ionomycin-induced GLUT4 myc exocytosis but did not prevent its reduced endocytosis. Compared with Gö6976, Gö6983 markedly reversed the slowing of GLUT4 myc endocytosis triggered by ionomycin. In summary, rapid Ca2+ influx into muscle cells accelerates GLUT4 myc exocytosis while slowing GLUT4 myc endocytosis. CaMKIIδ and AMPK stimulate GLUT4 myc exocytosis, whereas novel PKCs reduce endocytosis. These results identify how Ca2+-activated signals selectively regulate GLUT4 exocytosis and endocytosis in muscle cells.