Fig 2: Activation of mTOR by kynurenine (Kyn). a Jurkat human T cells were incubated with or without Kyn at the indicated concentrations of 0.5 and 1 mM for 24 h and analyzed by western blot. mTORC1 activity was assessed by the levels of phosphorylated substrates, p4E-BP1 and pS6K, relative to actin control. b Western blot analysis of PBL from 8 healthy subjects incubated with or without Kyn at the indicated concentrations of 0.1, 0.5, and 1 mM for 24 h. C, Flow cytometry of intracellular pS6RP levels in CD4, CD8, DN T-cell subsets of 8 healthy subjects. While top panels show representative western blots (a, b) and flow cytometry dot plots (c), bottom panels indicate cumulative analyses. *reflect p values <0.05

Fig 3: HL‐60 cell autophagy induced by ASK is dependent on a role of LKB1/AMPK and PI3K/Akt/mTOR signalling pathways. (A, B) P‐AMPK/AMPK, p‐Akt/Akt, PI3K (P85), p‐p70S6K/p70S6K, p‐Raptor/Raptor and p‐LKB1/LKB1 were assessed by immunoblotting after HL‐60 cells were exposed to various concentrations of ASK for 24 h. (C, D) P‐AMPK/AMPK, p‐Akt/Akt were assessed by immunoblotting after HL‐60 cells were exposed to 1.25 μM ASK for different times. (E) Akt (including Akt1, Akt2 and Akt3) and MAPKα1 mRNA expression were analysed by qRT‐PCR after HL‐60 cells were exposed to various concentrations of ASK for 24 h. (F) Akt and MAPKα1 mRNA expression were analysed by qRT‐PCR after HL‐60 cells were exposed to 1.25 μM ASK for different times. The results B, D, E and F are presented as Mean ± SD, n = 3. *p < 0.05, **p < 0.01

Fig 4: Effect on the viability and activation of the IR-β/Akt/p70S6K pathway of MCF-7 cells by stimulation with human sera from normal-weight and obese women with different hormonal states.A) Viability of MCF-7 cells exposed to sera from women with different BMIs and hormonal states. B) Western Blot of IR-β/Akt/p70S6K pathway members on MCF-7 cells exposed to sera from women with different BMIs and hormonal states. C) Group analysis of densitometric quantification stratified by serum condition of pIR-β (Tyr 1162–1163). D) Group analysis of densitometric quantification stratified by serum condition of pAkt (Ser 473). E) Group analysis of densitometric quantification stratified by serum condition of pp70S6K (Thr 389). F) Akt-1 expression levels on MCF-7 cells stimulated with human sera. G) Akt-2 expression levels on MCF-7 cells stimulated with human sera. H) Akt-3 expression levels on MCF-7 cells stimulated with human sera. For viability assay, the MCF-7 cells were treated with 10% inactivated fetal bovine serum (Ctr), 5% FBS, sera from normal-weight premenopausal women (NWSPre), sera from obese premenopausal women (OSPre), sera from obese postmenopausal women (OSPost) or sera from normal-weight postmenopausal women (NWSPost). For Western Blot test the cells were stimulated with recombinant human insulin (Ins) (0.5 UI/ml) (positive control for activation of the IR-β/Akt pathway) or 5% NWSPre, OSPre, OSPost or NWSPost. Labels of Western Blot as described in Fig 2. RT-PCR for Akt isoforms: Akt-1, Akt-2, Akt-3, and peptidylprolyl isomerase A (PPIA) as constitutive control. Western blot armed image. All experiments were carried out in triplicate (n = 9) for each serum evaluated. Comparison of means ** P <0.005, * P <0.05. ** Ctr or T0, ## 5% FBS or Ins, ++ NWSPre, $ $ OSPre, && OSPost.

Fig 5: T1R1/T1R3-mediated amino acids regulate ERK1/2 and mTORC1 through stimulation of Ca2+ in C2C12 myotubes. RNA was isolated from C2C12 myoblasts of different differentiation times for quantitative polymerase chain reaction (Q-PCR) analysis (A); Serum-starved C2C12 cells in Krebs-Ringer’s solution (KRBH) were treated with the indicated concentrations of BAPTA-AM 15 min prior to treatment with AA for 60 min, p-S6K1, p-mTOR protein levels were analyzed by Western blotting, and β-actin was used as a loading control (B); C2C12 myoblasts in KRBH were treated with the indicated concentrations of U0126 15 min prior to treatment with AA for 60 min. Immunoblots were performed with the indicated antibodies (C); C2C12 cells in KRBH were treated with the 1 mM/L IMP or 10 µM/L BAPTA-AM 15 min prior to treatment with AA for 60 min. Immunoblots were performed with the indicated antibodies (D). “+” means to add, “-” means not to add.