The hexosamine biosynthetic pathway couples growth factor-induced glutamine uptake to glucose metabolism

Abstract

Glucose and glutamine serve as the two primary carbon sources in proliferating cells, and uptake of both nutrients is directed by growth factor signaling. Although either glucose or glutamine can potentially support mitochondrial tricarboxylic acid (TCA) cycle integrity and ATP production, we found that glucose deprivation led to a marked reduction in glutamine uptake and progressive cellular atrophy in multiple mammalian cell types. Despite the continuous presence of growth factor and an abundant supply of extracellular glutamine, interleukin-3 (IL-3)-dependent cells were unable to maintain TCA cycle metabolite pools or receptor-dependent signal transduction when deprived of glucose. This was due at least in part to down-regulation of IL-3 receptor α (IL-3Rα) surface expression in the absence of glucose. Treatment of glucose-starved cells with N-acetylglucosamine (GlcNAc) to maintain hexosamine biosynthesis restored mitochondrial metabolism and cell growth by promoting IL-3-dependent glutamine uptake and metabolism. Thus, glucose metabolism through the hexosamine biosynthetic pathway is required to sustain sufficient growth factor signaling and glutamine uptake to support cell growth and survival.

Figure 1.

Glutamine consumption is reduced in the absence of glucose. (A) IL-3-dependent bax^−/−bak^−/− cells and K562 cells were cultured in the presence or absence of glucose for 2 or 3 d and cell size was measured in femtoliters (fL) each day (mean ± SD of triplicates). Primary bone marrow cells were cultured in the presence or absence of glucose for 4 d and size was measured in femtoliters (mean ± SD of triplicates). (B) Transport of ¹⁴C-glutamine over 5 min was measured (mean ± SD of triplicates) after the following treatments: IL-3-dependent cells were cultured in the presence or absence of glucose or IL-3 for 2 d, K562 cells were cultured in the presence or absence of glucose for 2 d, and primary bone marrow cells were cultured in the presence or absence of glucose for 4 d. For all indicated panels, P < 0.05 (*), P < 0.005 (**), and P < 0.0005 (***).

Figure 2.

Metabolite pools are depleted in the absence of glucose. (A) IL-3-dependent cells were withdrawn from glucose for 24 h, after which 15 mM ¹³C₆-glucose was added into the culture medium. Cells were harvested and LC-MS/MS metabolite analysis was performed on duplicate samples 1 h after glucose addition. Select metabolites in glycolysis (hexose-phosphate), the pentose phosphate pathway (ribose-phosphate), the TCA cycle (citrate), and the hexosamine pathway (UDP-GlcNAc), with background subtracted, are shown. Data represent total (labeled [▪] + unlabeled [□]) metabolite pools (for additional metabolites, see Supplemental Tables S1, S2). (B) IL-3-dependent cells were withdrawn from glucose for 24 h, after which 15 mM glucose was added into the culture medium. Cell size was measured over the next 24 h. Result is representative of two independent experiments.

Figure 3.

IL-3Rα surface expression is dependent on glucose availability. (A) Western blot analysis from IL-3-dependent cells cultured in the presence or absence of glucose for 48 h. (B) Cells were cultured for 48 h in replete media (with both glucose and IL-3) or in media lacking either glucose or IL-3. Surface expression of IL-3Rα and IL-3Rβ_c was examined by FACS analysis. (C) Cells were cultured for 48 h in glucose-free media supplemented with 0, 1, 4, or 25 mM glucose, and IL-3Rα surface expression was analyzed by FACS. (ic) Isotype control. Data represent mean ± SD of triplicates. (***) P < 0.0005.

Figure 4.

Metabolite flux through the hexosamine pathway restores IL-3Rα surface expression and signaling in the absence of glucose. (A) The hexosamine biosynthetic pathway is gated by the rate-limiting enzyme GFAT (glutamine fructose-6-phosphate amidotransferase), which transfers an amide group from glutamine to fructose-6-phosphate to produce glucosamine-6-phosphate. Glucosamine-6-P is acetylated by Gnpnat (glucosamine phosphate N-acetyltransferase) to generate GlcNAc-6-P. The final product of the pathway is UDP-GlcNAc, which is used for both N-linked glycosylation in the ER and Golgi and for O-GlcNAc protein modification in the nucleus and cytoplasm. The metabolite GlcNAc can enter the hexosamine pathway after phosphorylation by the salvage pathway enzyme NAGK. (B) IL-3-dependent cells were cultured in IL-3-containing medium in the presence or absence of glucose or in glucose-free medium supplemented with 15 mM GlcNAc for 48 h. IL-3 was present in all conditions. FACS analysis of surface expression of IL-3Rα or surface binding of L-PHA was performed. Results are representative of at least three independent experiments. (C) Cells were treated for 2 d in the presence or absence of glucose, GlcNAc, and IL-3, as indicated. Cell surface proteins were biotinylated, isolated over NeutrAvidin column, and analyzed by Western blot and Coomassie stain. (D) Cells were withdrawn from glucose for 24 h and then treated with GlcNAc in the presence or absence of Jak inhibitor for an additional 24 h. IL-3 was present in all conditions. Cells were harvested and signaling was analyzed by Western blot. (E) IL-3-dependent cells were starved of glucose for 24 h (in the presence of IL-3), and then 15 mM ¹³C₆-glucose, 15 mM ¹³C₆-GlcNAc, or equal volume water control was added to cells. Cells were harvested at 1, 24, and 72 h after nutrient addition. LC-MS/MS analysis of labeled metabolites was performed. The experiment was done with duplicates for each condition. Representative labeled pools of metabolites from the hexosamine pathway (GlcNAc-P and UDP-GlcNAc), glycolysis (Hexose-P), pentose phosphate pathway (Ribose-P), and the TCA cycle (citrate) are shown at each time point. For additional metabolites in each pathway, see Supplemental Table S2.

Figure 5.

Activation of the hexosamine pathway with GlcNAc is sufficient to promote growth in the absence of glucose. (A) IL-3-dependent cells were cultured in the presence or absence of glucose plus 0, 7.5, or 15 mM GlcNAc, as indicated. IL-3 was present in all conditions. Cell size after 4 d was measured in femtoliters (fL) (mean ± SD of triplicates). (B) Cells were starved of glucose for 11 d, with 15 mM GlcNAc added at day 0, day 3, or day 6, indicated by the arrows. Media were changed (including fresh IL-3) every 3 d. Cell size was measured on indicated days (mean ± SD of triplicates). (C) Cells were starved of glucose or IL-3 ±15 mM GlcNAc for 4 d, and cell size was measured by Coulter Counter (mean ± SD of triplicates). (D) Cells were withdrawn from glucose for 24 h, and then GlcNAc was added to cells for an additional 48 h in the presence of 0.5 μM Jaki (JAK inhibitor I), 10 μM Ly (LY294002, PI-3K inhibitor), 10 μM Akti (Akt inhibitor VIII), 100 nM Rapa (Rapamycin, mTOR inhibitor), and 30 μM PD (PD 98059; MAPKK inhibitor). (UT) Untreated. IL-3 was present in all conditions. Cell size was measured in femtoliters (fL) (mean ± SD of triplicate wells). (E) Cells were starved of glucose in the presence or absence of 15 mM GlcNAc and 1 μg/mL cycloheximide for 5 d. (UT) Untreated. IL-3 was present in all conditions. Protein content was measured by BCA protein assay and normalized to total cell number (mean ± SD of triplicate wells). Result is representative of two independent experiments. (F) Cell size in glucose-starved cells was measured (mean ± SD of triplicates) after 3 d of treatment in the presence or absence of GlcNAc and 2 μg/mL cycloheximide. (UT) Untreated. IL-3 was present in all conditions. Results are representative of three independent experiments. (G) Cells were cultured ±glucose (11 mM) and ±GlcNAc (15 mM) for 5 d. IL-3 was present in all conditions. Cell number was assessed by Coulter counter (mean ± SD of triplicates). (H) Cells were starved of glucose for 24 h, after which either 15 mM glucose or 15 mM GlcNAc was added to cells in the presence of 10 μM BrdU. BrdU incorporation was measured by FACS every 6 h for 24 h. Result is representative of two independent experiments. For all indicated panels, P < 0.05 (*), P < 0.005 (**), and P < 0.0005 (***).

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 (***).

Figure 7.

Increased glutamine consumption promotes growth through multiple mechanisms. (A) IL-3-dependent cells were withdrawn from glucose in the presence or absence of 7.5 or 15 mM GlcNAc for 3 d. IL-3 was present in all conditions. Cells were then incubated with ¹⁴C₅-glutamine and production of ¹⁴CO₂ was measured after 8 h (mean ± SD of triplicates). Results are representative of two independent experiments. (B) Oxygen consumption was measured (mean ± SD of quadruplicate samples) after 2 d of glucose withdrawal in the presence of IL-3 ±7.5 or 15 mM GlcNAc. Results are representative of three independent experiments. (C) IL-3-dependent cells were starved of glucose for 24 h, and then 15 mM ¹³C₆-glucose, 15 mM ¹³C₆-GlcNAc, or equal volume water control was added to cells. Cells were harvested at 1, 24, and 72 h after nutrient addition. LC-MS/MS analysis of labeled and unlabeled metabolites was performed. The experiment was done with duplicates for each condition. The ratio of unlabeled metabolite pools in GlcNAc-treated versus glucose-withdrawn control cells was determined at each time point for metabolites in glycolytic and pentose phosphate pathways and the TCA cycle. Pools of TCA cycle metabolites, but not glycolytic or pentose phosphate metabolites, increased in response to GlcNAc treatment. See also Supplemental Tables S1 and S2. (D) Cells were starved of glucose overnight and then glucose or GlcNAc was added to cells, along with ¹⁴C₅-glutamine, for 24 h. Cells were harvested, lipids were extracted, and incorporation of ¹⁴C into lipids was measured by scintillation counting (mean ± SD of triplicates). Results are representative of two independent experiments. (E) Cells were starved of glucose for 24 h, and then either 15 mM glucose or 15 mM GlcNAc was added to cells. Uptake of ¹⁴C-leucine was measured at indicated time points (mean ± SD of triplicate wells). (F) Cells were withdrawn from glucose for 24 h in IL-3-containing medium ±60 μM DON. GlcNAc was added to cells in each condition for an additional 24 h and uptake of ¹⁴C-leucine was assessed (mean ±SD of triplicates). For all indicated panels, P < 0.005 (**) and P < 0.0005 (***).

Figure 8.

Glutamine is required for continued signaling downstream from the IL-3R. (A) Cells were starved of glucose for 3 d in medium containing either 4 mM glutamine or 1 mM dimethyl-αKG ±15 mM GlcNAc and ±60 μM DON. Signaling was analyzed by Western blot. Cell size in femtoliters (fL) was assessed prior to lysis of cells. Results are representative of three independent experiments. (B) Cells were withdrawn from glucose ±0.4 mM leucine and ±15 mM GlcNAc for 2 d as indicated, and signaling was assessed by Western blot. Results are representative of two independent experiments. (C) Surface expression of the IL-3Rα was determined by flow cytometry in cells treated with 60 μM DON.