Leucine deprivation stimulates PRLR expression by activating GCN2 and decreasing mTOR/S6K1 signaling. A and B: Gcn2+/+ or Gcn2−/− mice were fed a control (+) leu or (−) leu diet for 7 days. C and D: Mice were infected with Ad-CA-S6K1 (+ Ad-CA-S6K1) or control GFP Ad (− Ad-CA-S6K1) via tail-vein injection and were fed
a (−) leu diet for 7 days. E: Gcn2−/− mice were intraperitoneally injected with rapamycin (Rapa) at a dose of 1 mg/kg body weight (+ Rapa) or vehicle alone (−
Rapa) once a day while fed (−) leu diet for 7 days. Data are the mean ± SEM and are representative of at least two independent
in vivo experiments with the number of mice as indicated (n = 5–7 per group each time in A–D; n = 5 per group each time in E). Statistical significance was calculated using a two-tailed Student t test for the effects of (−) leu vs. control diet (*P < 0.05) and the effects of Gcn2+/+ mice fed (−) leu diet vs. Gcn2−/− mice fed (−) leu diet (&P < 0.05) in A and B, with vs. without Ad-CA-S6K1 under (−) leu treatment (*P < 0.05) in C and D, with vs. without rapamycin under (−) leu treatment in E (*P < 0.05). A and C: Prlr mRNA. B, D, and E: PRLR and p-S6 (ser235/236) protein (top, Western blot; bottom, quantitative measurements of PRLR relative to actin). F: Working model. t, total.