Figure 6.
GlcNAc treatment promotes increased glutamine consumption. (A) IL-3-dependent cells were withdrawn from glucose in the presence of 0, 7.5, or 15 mM GlcNAc for 3 d. IL-3 was present in all conditions. Glutamine consumption from the medium was measured by BioProfile Flex automated metabolite analyzer (mean ± SD of triplicates). (B) After 24 h of glucose starvation, 15 mM glucose or 15 mM GlcNAc was added to cells. IL-3 was present throughout the experiment. At 6, 12, 24, and 72 h after metabolite addition, a portion of the cells was harvested and the glutamine transport capacity of the cells was determined by measuring glutamine uptake over 5 min, as described in the Materials and Methods. (C) Cells were starved of glucose for 24 h, and then either 15 mM glucose or 15 mM GlcNAc was added to cells in the presence or absence of Jaki. After an additional 24 h, cells were harvested and 14C-glutamine uptake over 5 min was measured (mean ± SD of triplicates). IL-3 was present in all conditions. (D) Cells were starved of glucose for 24 h, and then treated in the presence or absence of 15 mM GlcNAc and 60 μM DON. After 48 h, cell size was measured. IL-3 was present in all conditions. (E) Cells were starved of glucose for 24 h, then 15 mM glucose or 15 mM GlcNAc was added to cells. IL-3 was present throughout the experiment. RNA was isolated at 0, 12, 24, and 48 h after metabolite addition, and gene expression was analyzed by quantitative RT–PCR. Data were normalized to 18S rRNA. Results are representative of two independent experiments. (F) Cells were starved of glucose for 24 h, and then glucose or GlcNAc was added for an additional 24 h, in the presence or absence of Jaki. IL-3 was present in all conditions. RNA was isolated from triplicate wells and gene expression was determined by quantitative RT–PCR, normalized to 18S rRNA (mean ± SD). For all indicated panels, P < 0.05 (*), P < 0.005 (**), and P < 0.0005 (***).