Fig. 7
Glutaminolysis is not important for sustaining OXPHOS in cytokine-activated NK cells. a–d NK cells were stimulated with IL-2/IL-12 for 18 h and then put in the presence or absence of glutamine (a, b) or in the presence or absence the glutaminase inhibitor BPTES (c, d) for 1 h prior to analysis of oxygen consumption rates (OCR) to assess rates of OXPHOS and maximal respiration. e, f Metabolic-tracing experiments were performed on NK cells stimulated with IL-2/IL-12 for 16 h in media with unlabelled glutamine and then further cultured for 0.5 h to 8 h in media containing 13C5-glutamine. Analysis of the isotopologue distribution (e) of citrate, fumarate and malate was performed or isotopic mean enrichment of citrate, fumarate and malate (f) was calculated. g Analysis of 13C incorporation into the TCA cycle intermediates citrate, succinate, fumarate and malate in 16 h IL-2/IL-12 activated NK cells cultured in 13C5-glutamine or 13C6-glucose for 30 min and 8 h. Data for the incorporation of 13C6-glucose in the TCA cycle intermediates have been previously published2 and are used here for direct comparison to the new data set of 13C5-glutamine incorporation in TCA cycle intermediates. h NK cells were activated for 18 h with IL-2/IL-12 before the analysis of NK cell oxygen consumption rate (OCR) to assess rates of OXPHOS and maximal respiration following the injection of BPTES or SB204990. i NK cells stimulated with IL-2/IL-12 for 18 h were analysed by GC-MS for total concentrations of citrate, succinate, fumarate and malate. Data are mean ± s.e.m. of 4 (a–i), 5 (d) 6 (e, f) or 3–6 (g, h) experiments or representative of 3–6 (c, h) or 4 (a) individual experiments. a–h Data were normalised to 200,000 cells. Statistical analysis was performed using Student’s t-test (b, g), a one-sample t-test (d, h), a one-way ANOVA with Tukey test (g, i) or a two-way ANOVA with Sidak test (f, g); *p < 0.05, **p < 0.01, ***p < 0.005, ns non-significant. Oligo oligomycin, 2DG 2-deoxyglucose, Anti A antimycin A, Rot rotenone, FCCP carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone
