Fig. 7

Nutrient activates mTOR signaling by accumulating BCAAs. a The conversion of Leucine-1,2-¹³C₂ to ¹³C-labeled citrate was traced by targeted liquid-chromatography tandem mass spectrometry (LC–MS/MS) in HEK293T cells cultured in low (1 mM) and high (25 mM) glucose media, and in ECHS1 overexpressing HEK293T cells cultured in high glucose media. b The BCAA levels in HEK293T cells and ECHS1 knocked down HEK293T cells were determined (n = 4). BCAA levels were normalized to those of HEK293T cells. c BCAA levels of HEK293T cells, NAM treated HEK293T cells, and NAM treated HEK293T cells with overexpression of ECHS1^K101R were determined by GC–MS (n = 4). All levels were normalized to those of HEK293T cells. The expression of ECHS1^K/R was confirmed by western blot. d BCAAs levels in the liver of 129/C57BL6 mice, Sirt3 knockout 129/C57BL6 mice, and ECHS1^K101R overexpressing Sirt3 knockout 129/C57BL6 mice were determined (n = 6). ECHS1^K/R expression was achieved by tail vein injection of the ECHS1^K/R plasmid, and the expression was confirmed by western blot. BCAA levels were normalized to those in the liver of isogenic control mice. e The levels of P-T389 on S6K were determined in HEK293T cells with and without ECHS1 knockdown. f The levels of P-T389 on S6K were determined in HEK293T cells with and without ECHS1 overexpression. g The levels of P-T389 on S6K were determined in the hepatocytes of Sirt3 KO and its isogenic control mice. h The effects of shSIRT3 knockdown (for knockdown effects see Figure 4) on P-T389 of S6K were detected in HEK293T cells cultured with or without BCAA supplementation in the medium. i The cell size of Chang cells and ECHS1 or SIRT3 knocked down Chang cells were determined by measuring forward side scatter (FSC) in the presence or absence of rapamycin. j The percentage of Chang cells and ECHS1 or SIRT3 knocked down Chang cells in the G2/M phase was determined by fluorescence-activated cell sorter (FACS) in the presence or absence of rapamycin. For all figures, mean values with SD are reported. NS not significant; *P < 0.05; **P < 0.01; ***P < 0.001