Protein kinase B Akt participates in GLUT4 translocation by insulin in L6 myoblasts

L6 myoblasts stably transfected with a GLUT4 cDNA harboring an exofacial my c epitope tag (L6-GLUT4myc myoblasts) were used to study the role of protei n kinase B alpha (PKB alpha)/Akt1 in the insulin-induced translocation of G LUT4 to the cell surface. Surface GLUT4myc was detected by immunofluorescen t labeling of the myc epitope in nonpermeabilized cells, Insulin induced a marked translocation of GLUT4myc to the plasma membrane within 20 min. This was prevented by transient transfection of a dominant inhibitory construct of phosphatidylinositol (PI) 3-kinase (Delta p85 alpha). Transiently trans fected cells were identified by cotransfection of green fluorescent protein . A constitutively active PKB alpha, created by fusion of a viral Gag prote in at its N terminus (GagPKB), increased the cell surface density of GLUT4m yc compared to that of neighboring nontransfected cells. A kinase-inactive, phosphorylation-deficient PKB alpha/Akt1 construct with the mutations K179 A (substitution of alanine for the lysine at position 179), T308A, and S473 A (AAA-PKB) behaved as a dominant-negative inhibitor of insulin-dependent a ctivation of cotransfected wild-type hemagglutinin (HA)-tagged PKB. Further more, AAA-PKB markedly inhibited the insulin-induced phosphorylation of cot ransfected BAD, demonstrating inhibition of the endogenous PKB/Akt, Under t he same conditions, AAA-PKB almost entirely blocked the insulin-dependent i ncrease in surface GLUT4myc. PKB alpha with alanine substitutions T308A and S473A (AA-PKB) or K179A (A-PKB) alone was a less potent inhibitor of insul in-dependent activation of wild-type HA-PKB or GLUT4myc translocation than was AAA-PKB. Cotransfection of AAA-PKB with a fourfold DNA excess of HA-PKB rescued insulin-stimulated GLUT4myc translocation, AAA-PKB did not prevent actin bundling (membrane ruffling), though this response was PI 3-kinase d ependent. Therefore, it is unlikely that AAA-PKB acted by inhibiting PI 3-k inase signaling. These results outline an important role for PKB alpha/Akt1 in the stimulation of glucose transport by insulin in muscle cells in cult ure.