Figure 5. 6PGD^−/− ablation results in enhanced non-oxidative PPP and accumulation of glycogen.

(A–C) Naive CD8⁺ T cells (CD3⁺CD8⁺CD44^lowCD62L^high) were isolated from 6PGD^−/− and 6PGD^fl/fl mice and stimulated with αCD3 + αCD28 mAbs and IL-2 (20 IU/mL) in the presence of D₇-glucose plus ¹³C₅, ¹⁵N₂-glutamine in (A) or ¹³C₆-glucose in (B) and (C). Isotope-labeling patterns of metabolites of cell extracts were analyzed by IC-UHRMS as fractional enrichment (A and C) or ¹H NMR (B). (A) Shows the conversion of D₇-glucose and ¹³C₅, ¹⁵N₂-glutamine into the metabolites of PPP and gluconeogenesis (GNG) via the Krebs cycle. (B and C) Tracks ¹³C₆-glucose incorporation, respectively, into glycogen as well as glucose-1-phosphate (G1P) and UDP-glucose (UDPG) as intermediates of glycogen biosynthesis.

(D) Accumulation of dark glycogen particles was evident in electron microscopy (EM) analysis of naive 6PGD^−/− CD8⁺ T cells compared with 6PGD^fl/fl after 4 days of stimulation. Measurements of scale bars are given in the figures.

(E) Schematic diagram indicating the site of GPI action.

(F) Accumulation of glycogen in 6PGD^−/− Cd8⁺ T cells after 48 h of culture evaluated in colorimetric method.

(G) 6PGD^fl/fl CD8⁺ T cells were stimulated for 48 h with αCD3 + αCD28 mAbs in the presence of either 6-AN, 6-AN + glycogen phosphorylase inhibitor (GPI), or DMSO and then glycogen content was assessed via colorimetric method. Results were generated from two independent experiments. Legend in x axis: 0 = unlabeled; Dx = sum of D₁ to D_x or Glc-derived species; C*Dx = sum of ¹³C-labeled species with 0-x number of D; N*Dx = sum of ¹⁵N-labeled species with 0-x number of D; C3NxDx = sum of ¹³C₃ with 0-x number of D and ¹⁵N, which could reflect incorporation of GNG product into the ribose unit of NADPH (cf. atom tracing diagram Figure S9). C*NxDx = sum of ¹³C-labeled species with 0-x number of ¹⁵N and D. G6P, glucose-6-phosphate; 6PG, 6-phosphogluconate; R5P, ribose-5-phosphate; S7P, sedoheptulose-7-phosphate; Gly3P, glyceraldehyde-3-phosphate; X5P, xylulose-5-phosphate; E4P, erythrose-4-phosphate; F6P, fructose-6-phosphate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; TK, transketolase; TA, transaldolase. Error bars represent ± SEM. **p < 0.01; ***p < 0.001.