TEMPORAL ACTIVATION OF P70 S6 KINASE AND AKT1 BY INSULIN - PI-3-KINASE-DEPENDENT AND PI-3-KINASE-INDEPENDENT MECHANISMS

Several studies have suggested that activation of p70 ribosomal S6 kin ase (p70 S6 kinase) by insulin may be mediated by the phosphatidylinos itol 3-kinase (PI 3-kinase)-Akt pathway. However, by temporal analysis of the activation of each kinase in L6 muscle cells, we report that t he activation of the two serine/threonine kinases (Akt and p70 S6 kina se) can be dissociated. Insulin stimulated p70 S6 kinase in intact cel ls in two phases. The first phase (5 min) of stimulation was fully inh ibited by wortmannin (IC50 = 20 nM) and LY-294002 (full inhibition at 5 mu M). After this early inhibition, p70 S6 kinase was gradually stim ulated by insulin in the presence of 100 nM wortmannin. After 30 min, the stimulation was 65% of the maximum attained in the absence of wort mannin. The IC50 of wortmannin for inhibition of this second phase was similar to 150 nM. In contrast, activation of Akt1 by insulin was com pletely inhibited bg 100 nM wortmannin at all time points investigated . Inhibition of mitogen-activated protein kinase/extracellular signal- regulated protein kinase kinase with PD-098059 (10 mu M) or treatment with the protein kinase C inhibitor bisindolylmaleimide (10 mu M) had no effect on the late phase of insulin stimulation of p70 S6 kinase. W e have previously shown that GLUT-1 protein synthesis in these cells i s stimulated by insulin via the mTOR-p70 S6 kinase pathway, based on i ts sensitivity to rapamycin. We therefore investigated whether the sig nals leading to GLUT-1 synthesis correlated with the early or late pha se of stimulation of p70 S6 kinase. GLUT-I synthesis was not inhibited by wortmannin (100 nM). In summary, insulin activates p70 ribosomal S 6 kinase in L6 muscle cells by two mechanisms, one dependent on and on e independent of the activation of PI 3-kinase. In addition, activatio n of Akt1 is fully inhibited by wortmannin, suggesting that Akt1 does not participate in the late activation of p70 S6 kinase. Wortmannin-se nsitive PI 3-kinases and Akt1 are not required for insulin stimulation of GLUT-1 protein biosynthesis.