Fig. 5

Glutamine serves as a source of both carbon and nitrogen for lipid synthesis. a Schematic representation of the hypothesis that glutamine supplies both carbon and nitrogen for lipid synthesis in CD4⁺ T cells. b, c Stable isotope tracing of stimulated CD4⁺ T cells using ¹³C₅,¹⁵N₂ glutamine (five carbon atoms are labeled with ¹³C, and both nitrogen atoms are labeled with ¹⁵N) for measuring TCA intermediates (n = 3) (b) and palmitate (n = 8) (c). d Expression of enzymes related to glutaminolysis after CD4⁺ T cells were stimulated for 48 hours. e-g Quantification of metabolic intermediates in CD4⁺ T cells stimulated with anti-CD3 and anti-CD28 antibodies for 48 hours, followed by tracing for 4 hours with ¹³C₅,¹⁵N₂-glutamine in media (n = 3). The bar graphs represent the levels of ATP (e) and coenzyme A (f) and the labeled coenzyme A peak area normalized to the labeled ATP peak area (g). h ¹³C₅,¹⁵N₂-glutamine concentration in ¹³C₅,¹⁵N₂ glutamine-containing media after 4 hours of tracing in CD4⁺ T cells stimulated for 48 hours (n ≥ 6). i Glutamine consumption rate of CD4⁺ T cells stimulated for 48 hours with anti-CD3 and anti-CD28 antibodies (n ≥ 6). j Lipidomics of CD4⁺ T cells stimulated for 48 hours with anti-CD3 and anti-CD28 antibodies in glutamine-deficient media supplemented with 0.08 mM or 2 mM glutamine (n ≥ 4). k CellTrace Violet proliferation assay with CD4⁺ T cells stimulated with anti-CD3 and anti-CD28 antibodies for 72 hours in glutamine-deficient media supplemented with the indicated concentrations of glutamine. The data are representative of three (b-i) or four (d, j and k) independent experiments. The data are presented as the means ± SDs. Statistical analysis was performed via one-way ANOVA with the Bonferroni post hoc correction (h), the Mann‒Whitney U test (i and k), or an unpaired two-tailed t test for all other comparisons. For (b, c, e and f), statistical comparisons were conducted on the individual labeled isotopologues (colored asterisks correspond to the respective isotopologues) and the sum of labeled isotopologues (indicated by black asterisks). ns, not significant, *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001. CAR carnitine, Cer ceramide, PC phosphatidylcholine, PG phosphatidylglycerol, PS phosphatidylserine, CE cholesteryl ester, CL cardiolipin, FA fatty acid, LPC lysophosphatidylcholine, LPE lysophosphatidylethanolamine, LPI lysophosphatidylinositol, PE phosphatidyl-ethanolamine, PI phosphatidylinositol, SM sphingomyelin, TG triacylglycerol, DG diacylglycerol, NAE N-acyl ethanolamine, PA phosphatidic acid, MPE methylphosphatidyl-ethanolamine, Asp aspartate, Gly glycine, α-KG α-ketoglutarate, Aco aconitate, Mal malate, Cit citrate, Ac-CoA acetyl coenzyme A, C16:0-FA palmitate, C16:0-CoA palmitoyl CoA, C18:0-CoA, stearoyl CoA, C18:1-CoA oleoyl CoA