Three transduction pathways are involved in amino acid (AA) sensing in liver : the mammalian target of rapamycin (mTOR), adenosine monophosphate-activated protein kinase (AMPK) and general control non-depressible kinase 2 (GCN2). However, no study has investigated the involvement of these signaling pathways in hepatic AA sensing. To address the question of liver AA sensing and signaling in response to a high protein (HP) dietary supply, we investigated the changes in the phosphorylation state of hepatic mTOR (P-mTOR), AMPK (P-AMPK) and GCN2 (P-GCN2) by western blotting.. In rat fed a HP diet for 14 days, the hepatic P-AMPK and P-GCN2 were lower (P<0.001) and those of both the P-mTOR and eukaryotic initiation factor 4E-binding protein-1 phosphorylation (P-4E-BP1) were higher (P<0.01) compared to rats receiving a normal protein diet (NP). In hepatocytes in primary culture, high amino acid concentration decreased AMPK phosphorylation, whether insulin was present or not (P<0.01). Either amino acids or insulin can stimulate P-mTOR, but this is not sufficient for 4E-BP1 phosphorylation that requires both (P<0.01).As expected, branched chain amino acids (BCAA) or leucine stimulated the phosphorylation of mTOR but both insulin and BCAA or leucine are required for 4E-BP1 phosphorylation. GCN2 phosphorylation was reduced by both amino acids and insulin(P<0.01), suggesting for the first time that the translation-inhibitor GCN2 senses not only the amino acid deficiency but also the amino acid increase in the liver. The present findings demonstrate that amino acids and insulin exert a coordinated action on translation and involved mTOR, AMPK and GCN2 transduction pathways.