Assessment of cell-signaling pathways in the regulation of mammalian target of rapamycin (mTOR) by amino acids in rat adipocytes

Enhanced phosphorylation of the ribosomal protein s6 kinase, p70(s6k), and the translational repressor, 4E-BP1, are associated with either insulin-ind uced or amino acid-induced protein synthesis. Hyperphosphorylation of p70(s 6k) and 4E-BP1 in response to insulin or amino acids is mediated through th e mammalian target of rapamycin (mTOR). In several cell lines, mTOR or its downstream targets can be regulated by phosphatidylinositol (PI) 3-kinase; protein kinases A, B, and C; heterotrimeric C-proteins; a PD98059-sensitive kinase or calcium; as well as by amino acids. Regulation by amino acids ap pears to involve detection of levels of charged t-RNA or t-RNA synthetase a ctivity and is sensitive to inhibition by amino acid alcohols. In the prese nt article, however, we show that the rapamycin-sensitive regulation of 4E- BP1 and p70(s6k) in freshly isolated rat adipocytes is not inhibited by eit her L-leucinol or L-histidinol. This finding is in agreement with other rec ent studies from our laboratory suggesting that the mechanism by which amin o acids regulate mTOR in freshly isolated adipocytes may be different than the mechanism found in a number of cell lines. Therefore we investigated th e possible role of growth factor-regulated and G-protein-regulated signalin g pathways in the rapamycin-sensitive, amino acid alcohol-insensitive actio ns of amino acids on 4E-BP1 phosphorylation. We found, in contrast to previ ously published results using 3T3-L1 adipocytes or other cell lines, that t he increase in 4E-BP1 phosphorylation promoted by amino acids was insensiti ve to agents that regulate protein kinase A, mobilize calcium, or inhibit p rotein kinase C. Furthermore, amino acid-induced 4E-BP1 phosphorylation was not blocked by pertussis toxin nor was it mimicked by the G-protein agonis ts fluoroaluminate or MAS-7. However,, amino acids failed to activate eithe r PI 3-kinase, protein kinase B, or mitogen-activated protein kinase and fa iled to promote tyrosine phosphorylation of cellular proteins, similar to o bservations made using cell lines. in summary, amino acids appear to use an amino acid alcohol-insensitive mechanism to regulate mTOR in freshly isola ted adipocytes. This mechanism is independent of cell-signaling pathways im plicated in the regulation of mTOR or its downstream targets in other cells . Overall, our study Emphasizes the need for caution when extending results obtained using established cell lines to the differentiated nondividing ce lls found in most tissues. (C) 2000 Wiley-Liss, Inc.