In several cellular systems, amino acids synergize with insulin in promoting protein synthesis through the activation of the protein kinases p70/S6-K and PHAS-1. Such activations are mediated by the upstream kinase: mammalian target of rapamycin (mTor).
In this work we have investigated the intracellular pathways involved in insulin-induced and amino acid-induced p70/S6-K activations in human endothelial cells.
In human umbilical vein endothelial cells, insulin induces the phosphorylation of p70/S6-K at 5 minutes decreasing thereafter, whereas amino acids alone or associated with insulin phosphorylate p70/S6-K at all the time points analyzed (60 minutes). Insulin and amino acids phosphorylate p70/S6-K by mTor-dependent and phosphotidylinositol 3-kinase-dependent mechanisms, whereas the mitogen-activated protein kinase pathway is involved only when p70/S6-K is activated by insulin.
Insulin induces the phosphorylation of Akt and extracellular signal-regulated protein kinase (ERK) 1/2, whereas amino acids did not. Moreover, amino acids suppress the phosphorylations induced by insulin. The inhibitory effects of amino acids are reverted by the mTor inhibitor rapamycin.
Insulin-induced phosphorylation of Akt (at 15 and 30 minutes) is not accompanied by the phosphorylation of the downstream kinase p70/S6-K, indicating the existence of a negative feedback at this level.
Our data demonstrate that at the level of human endothelial cells, amino acids synergize with insulin in the phosphorylation of the kinase that lies downstream mTor, as p70/S6-K, whereas they inhibit the upstream kinases Akt and extracellular signal-regulated protein kinase 1/2 when activated by insulin, by an mTor-dependent mechanism.
*Department of Pharmacological Sciences, University of Milan, via Balzaretti 9
†Istituto Scientifico Ospedale San Raffaele, via Olgettina 60
‡University Research Center: “Physical Exercise for Health and Wellness” Faculty of Exercise Sciences, via Olgettina 60, Milan, Italy
Reprints: Fabio Pellegatta, MD, PhD, Laboratory of lipid metabolism, Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20132 Milano, Italy (e-mail: email@example.com).
Received for publication December 19, 2005; accepted March 28, 2006