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. 2021 Dec 23;10:e72593. doi: 10.7554/eLife.72593

Figure 2. TCA cycle inhibition impairs glutamine-derived proline and aspartate synthesis but promotes cytosolic GSH synthesis.

(A) Schematic diagram highlighting U-13C-glutamine tracing into distinct metabolic modules. (B) U-13C-glutamine tracing into proline (m + 5 labeling intensity and total isotopologue fraction distribution). (C) U-13C-glutamine tracing into aspartate, asparagine and alanine (m + 4 and m + 3 labeling intensity and total isotopologue fraction distribution). (D) U-13C-glutamine tracing into glutathione synthesis pathway (m + 5 and m + 10 labeling intensity and total isotopologue fraction distribution) (B–D) show DMSO, FHIN-1 (20 μM)-, AA5 (1 μM)- and TTFA (500 μM)-treated Fh1fl/fl cells (24 h timepoint) (n = 5 independent biological replicates). Data are mean ± standard error of mean (SEM). p Value determined by ordinary one-way ANOVA, corrected for multiple comparisons using Tukey statistical hypothesis testing. (E) Proline and aspartate level fold change in mitochondrial and cytosol fractions in FHIN-1 and AA5-treated versus DMSO-treated Fh1fl/fl cells (24 h timepoint) (n = 3 independent biological replicates). Data are mean ± standard error of mean (SEM). p Values determined by unpaired two-tailed t-test. p < 0.05*; p < 0.01**; p < 0.001***.

Figure 2.

Figure 2—figure supplement 1. Glutamine fate tracing and compartment-specific metabolomics upon acute TCA cycle inhibition.

Figure 2—figure supplement 1.

(A) Schematic diagram highlighting U-13C-glutamine tracing into the TCA cycle and reductive carboxylation. (B) U-13C-glutamine tracing into α-KG, succinate, fumarate, malate, citrate, cis-aconitate and 2-HG (total isotopologue fraction distribution) in DMSO, FHIN-1 (20 μM), AA5 (1 μM), and TTFA (500 μM)-treated Fh1fl/fl cells (24 hr timepoint) (n = 5 independent biological replicates). (C) U-13C-glutamine tracing into α-KG, succinate, fumarate, malate, citrate, cis-aconitate, and 2-HG (m + 5 and m + 3 labeling intensity) in DMSO, FHIN-1 (20 μM), AA5 (1 μM) and TTFA (500 μM)-treated Fh1fl/fl cells (24 hr timepoint) (n = 5 independent biological replicates). Data are mean ± SEM. p Value determined by ordinary one-way ANOVA, corrected for multiple comparisons using Tukey statistical hypothesis testing. p < 0.05*; p < 0.01**; p < 0.001***. (D) Glutamine, glutamate, GSH, GSSG, alanine, asparagine level fold changes and succGSH relative intensity in mitochondrial and cytosol fractions in FHIN-1 (20 μM)- and AA5(1 μM)-treated versus DMSO-treated Fh1fl/fl cells (24 hr timepoint) (n = 3 independent biological replicates). Data are mean ± SEM. p values determined by unpaired two-tailed t-test. p < 0.05*; p < 0.01**; p < 0.001***.