Fig. 3

A quantitative view of mitochondrial and cytosolic fluxes in the TCA cycle and citrate metabolism under normoxia. a–d Mass-isotopomer labeling kinetics of citrate in mitochondria (a) and cytosol (b), and malate in mitochondria (c) and cytosol (d) when feeding HeLa cells with [U-¹³C]-glutamine under standard normoxic conditions. e Measured isotopic labeling ratio in HeLa cells for citrate m + 5 /m + 4 and malate m + 3 /m + 4 in media (red) in comparison to the expected labeling via computational simulation, considering the measured labeling kinetics of citrate and malate in mitochondria (green) and cytosol (orange). f Gibbs free energy of mitochondrial (IDH2/3) and cytosolic (IDH1) IDH isozymes (in the oxidative direction) in HeLa cells under normoxia (green) and hypoxia (red). g Mitochondrial and cytosolic fluxes, showing percentage from citrate synthase flux (which is 0.48 mM h⁻¹). Arrow represents the direction of net flux; number represents net flux in the direction of the arrow and number in parenthesis correspond to the backward flux. Confidence intervals for estimated fluxes are shown in Supplementary Data 8. h The measured mass-isotopomer distribution of palmitate when feeding cells with [U-¹³C]-glutamine (green) is consistent with the simulated fit (orange). For the simulation, acetyl-CoA labeling was assumed to follow a binomial distribution with a probability of 7.1% having m + 2 labeling form. i Validation of the method based on knock-down of IDH1 or IDH2 genes and following citrate isotopic labeling after feeding cells with [U-¹³C]-glutamine. Upon IDH1 knockdown, the ratios between citrate m + 5 and m + 4 in mitochondria and cytosol are similar, indicating that all reductive glutamine flux occurred in mitochondria (where citrate m + 4 is produced from malate m + 4). Meanwhile, IDH2 knockdown resulted in a higher citrate m + 5 to m + 4 ratio in cytosol, indicating that reductive IDH1 remains active. n.s. not significant. *P < 0.05 and **P < 0.01 by two-sample t-test. Data are mean ± SD, n = 3 independent biological replicates