Fig. 3: GPT2 activity drives reductive glutamine carboxylation to sustain glycolysis in SDH-deficient cells.

A Intracellular levels of α-ketoglutarate in Sdhb^fl/fl and Sdhb^Δ/Δ cells infected with lentiviruses expressing either a non-targeting control shRNA (shNTC) or one shRNA sequence targeting Gpt2. Data are presented as mean ± s.e.m. of n = 4 replicates. **P < 0.01 (two-tailed Student’s t-test). B Number of Gpt2-silenced Sdhb^fl/fl and Sdhb^Δ/Δ cells measured after 96 h of culture in the presence/absence of the highest subtoxic concentration of α-ketoglutarate (α-KG, 0.5 mM), 20 mM aspartate (ASP) or 100 uM NAD⁺ in the medium. Data were presented as mean ± s.e.m. of n = 4 wells/replicates. *P < 0.05; **P < 0.01; ***P < 0.001 (two-tailed Student’s t-test). C Labeling pattern of the indicated metabolites in cells incubated with U-¹³C₅-glutamine. MDH1 malate dehydrogenase 1, GAPDH glyceraldehyde dehydrogenase. D Levels of ¹³C₅-citrate and ¹³C₃-malate measured in Gpt2-silenced Sdhb^fl/fl and Sdhb^Δ/Δ cells cultured for 24 h in a medium containing U-¹³C₅-glutamine. Data were presented as mean ± s.e.m. of n = 4 replicates. *P < 0.05; ***P < 0.001 (two-tailed Student’s t-test). E Levels of TCA cycle-derived metabolites ¹³C₅-citrate and ¹³C₃-malate in Sdhb^fl/fl and Sdhb^Δ/Δ cells cultured for 24 h in the presence/absence of 100 µM UK5099 in a medium containing U-¹³C₅-glutamine. Data were presented as mean ± s.e.m. of n = 3 replicates. **P < 0.01; ***P < 0.001 (two-tailed Student’s t-test), ns not significant. F Determination of intracellular NAD⁺ to NADH ratio in Gpt2-silenced Sdhb^fl/fl and Sdhb^Δ/Δ cells. Data were presented as mean ± s.e.m. of n = 3 replicates. *P < 0.01 (two-tailed Student’s t-test). G Heatmap depicting the intracellular levels of the indicated glycolytic intermediates in Gpt2-silenced Sdhb^fl/fl and Sdhb^Δ/Δ cells. Data were presented as row normalized Z-scores of peak area/ug proteins of n = 4 replicates. H LC-MS-mediated determination of glucose consumption and lactate secretion rates of the indicated cells cultured for 48 h. Data were presented as mean ± s.e.m of n = 6 technical replicates. *P < 0.05; ***P < 0.001 (two-tailed Student’s t-test). I Intracellular levels of ATP in Gpt2-silenced Sdhb^fl/fl and Sdhb^Δ/Δ cells. Data were presented as mean ± s.e.m. of n = 4 replicates. *P < 0.05 (two-tailed Student’s t-test).