Fig. 4
SIRT5 enhances glutamine-driven TCA cycle metabolite abundances in colorectal cancer cells. a Heat map representing significantly different metabolites after SIRT5 deletion in HCT116 cells. Blue (red) indicates the relative down (up) regulation levels of TCA cycle and glutaminolysis intermediates compared with cells treated with the control siRNA; n = 6. b Schematic model of glutamine metabolism in cancer cells. Red circles represent carbons derived from [U-13C5] glutamine, and black circles are unlabeled. The black arrows indicate oxidative carboxylation flux from glutamine. c Glutamine uptake was determined in HCT116 and LoVo cells. At 48 h post transfection with SIRT5 siRNAs, the cells were placed in fresh medium. Metabolite levels were measured after 9 h of culture and normalized to the cell number. The results were normalized to the control siRNA. Data are the mean ± SD of five independent samples. Student’s t-test. N.S. = not significant for the indicated comparison. d–h Mass isotopologue distributions of glutamate (d); TCA cycle metabolites including α-KG (e), succinate, fumarate, malate, citrate, and isocitrate (f); glutamine-derived aspartate and asparagine (g); and pyruvate and lactate (h) in LoVo cells treated with the control siRNA or SIRT5 siRNAs. Cells were cultured in [U-13C5] glutamine for 24 h before metabolites extraction and GC-MS analysis; n = 3, data in d–h are shown as the mean ± SD. Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. N.S. = not significant for the indicated comparison. i LoVo cells stably expressing control vector, SIRT5 WT, and SIRT5 H158Y were cultured with [U-13C5] glutamine for 24 h before metabolite extraction and GC-MS analysis. Mass isotopologues of α-KG were identified by GC-MS. j The fraction of m + 4 fumarate, malate, citrate, isocitrate, aspartate, and asparagine in LoVo cells stably expressing the control vector, SIRT5 WT, and SIRT5 H158Y were measured by GC-MS; n = 3. Data in i and j are shown as the mean ± SD. ANOVA with Tukey’s test. *P < 0.05, ***P < 0.001
