Fig. 6.

Effect of amino acids and insulin on mammalian target of rapamycin (mTOR) signaling in hypothalamic cells. A: GT1–7 cells were cultured at 5% of basal amino acid concentrations for 7 h and then placed in media containing 100% amino acids. At defined time points following exposure to high amino acids, cells were collected for Western blot analysis of ribosomal protein S6 kinase (S6K1) phosphorylation. B: in similar fashion, GT1–7 cells cultured in normal levels of amino acids were treated with insulin (10 mM), cells were collected at similar time points, and total protein was used to assess phosphorylation of S6K1. C: to test whether amino acid-dependent activation of S6K1 requires mTOR signaling, GT1–7 cells incubated in low amino acid concentrations were treated for 20 min with either 100% amino acids or insulin (10 mM) in the presence or absence of the mTOR inhibitor rapamycin. Rapamycin treatment (50 nM) attenuated both amino acid- and insulin-induced increases in S6K1 phosphorylation (*P < 0.05).