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Proc Natl Acad Sci U S A. 2008 Dec 2; 105(48): 18782–18787.
Published online 2008 Nov 24. doi:  10.1073/pnas.0810199105
PMCID: PMC2596212
Cell Biology

Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction


Mammalian cells fuel their growth and proliferation through the catabolism of two main substrates: glucose and glutamine. Most of the remaining metabolites taken up by proliferating cells are not catabolized, but instead are used as building blocks during anabolic macromolecular synthesis. Investigations of phosphoinositol 3-kinase (PI3K) and its downstream effector AKT have confirmed that these oncogenes play a direct role in stimulating glucose uptake and metabolism, rendering the transformed cell addicted to glucose for the maintenance of survival. In contrast, less is known about the regulation of glutamine uptake and metabolism. Here, we report that the transcriptional regulatory properties of the oncogene Myc coordinate the expression of genes necessary for cells to engage in glutamine catabolism that exceeds the cellular requirement for protein and nucleotide biosynthesis. A consequence of this Myc-dependent glutaminolysis is the reprogramming of mitochondrial metabolism to depend on glutamine catabolism to sustain cellular viability and TCA cycle anapleurosis. The ability of Myc-expressing cells to engage in glutaminolysis does not depend on concomitant activation of PI3K or AKT. The stimulation of mitochondrial glutamine metabolism resulted in reduced glucose carbon entering the TCA cycle and a decreased contribution of glucose to the mitochondrial-dependent synthesis of phospholipids. These data suggest that oncogenic levels of Myc induce a transcriptional program that promotes glutaminolysis and triggers cellular addiction to glutamine as a bioenergetic substrate.

Keywords: cancer, mitochondria

Many cancer cell lines depend on a high rate of glucose uptake and metabolism to maintain their viability despite being maintained in an oxygen-replete environment (1). This metabolic phenotype, first observed by Otto Warburg, has been termed aerobic glycolysis (2). Initially, this high rate of glycolysis was believed to result from mutations that impair the ability of cancer cells to carry out oxidative phosphorylation (3). However, such defects appear to be rare in spontaneously arising tumors (4). Recent studies have suggested that activating mutations in phosphoinositol 3-kinase (PI3K) and its downstream effector AKT induce the transformed cell to take up glucose in excess of its bioenergetic needs (5). The resulting high rate of glycolytic metabolism leads to the conversion of mitochondria into synthetic organelles that support glucose-dependent lipid synthesis and nonessential amino acid production (6, 7). Glycolytic pyruvate that accumulates in excess of cellular bioenergetic and synthetic needs is converted to lactate and secreted. A consequence of this metabolic conversion is that cells become addicted to glucose for their ATP production and survival as available lipids and amino acids are redirected from use as bioenergetic substrates and committed to use in anabolic synthesis (5). These data suggest that cancer cell nutrient uptake and metabolism may be under the direct control of the oncogenic signaling pathways that transform the cell. Strategies to exploit the glucose addiction of cells transformed by PI3K mutation for cancer therapy are currently being investigated (4).

In addition to glucose, glutamine can be an essential nutrient for cell growth and viability (8, 9). In vitro addiction to glutamine as a bioenergetic substrate was first observed in HeLa cells but it was not found to be a universal property of cancer cell lines. In cancer patients, some tumors have been reported to consume such an abundance of glutamine that they depress plasma glutamine levels (10, 11). Despite these observations, the high rates of glutamine metabolism and addiction exhibited by some cancer cells are poorly understood. Recently, we reported that glioma cells can exhibit glutamine uptake and metabolism that exceeds the cell's use of glutamine for protein and nucleotide biosynthesis (12). In such cells, the excess glutamine metabolites produced were found to be secreted as either lactate or alanine. This high rate of glutaminolysis was found to be beneficial because it provided the cell a high rate of NADPH production that was used to fuel lipid and nucleotide biosynthesis (see supporting information (SI) Fig. S1 for schematic of glutaminolytic pathway). However, not all tumor cells exhibit glutaminolysis. This suggested that the use of glutamine as a bioenergetic substrate is not induced as an indirect consequence of cell growth, but as a direct consequence of a specific oncogenic event.

In the present studies, the oncogenes known to contribute to malignant transformation of glial cells were tested for the ability to induce glutaminolysis. Here, we report that the glutaminolytic phenotype exhibited by tumor cells correlates with a cellular addiction to glutamine metabolism for the maintenance of cell viability. In contrast to glucose, glutamine uptake was not found to be under the direct or indirect control of the PI3K/AKT pathway. Inhibitors of either PI3K or AKT, despite suppressing glucose metabolism in a dose-dependent fashion, had no effect on the glutaminolytic phenotype. In contrast, high level expression of Myc was required to maintain the glutaminolytic phenotype and addiction to glutamine as a bioenergetic substrate. When an inducible Myc transgene was introduced in mouse embryonic fibroblasts (MEF), induction of Myc expression resulted in the induction of glutamine transporters, glutaminase, and lactate dehydrogenase A (LDH-A). Induction of these key regulatory genes involved in glutaminolysis correlated with the Myc-induced increases in glutamine uptake and glutaminase flux. This increase in glutamine uptake was not a compensatory response to increased glutamine incorporation into proteins as a result of Myc-induced protein synthesis, because most of the additional glutamine carbon taken up after Myc induction was secreted as lactate. Myc-induced reprogramming of intermediate metabolism resulted in glutamine addiction, despite the abundant availability of glucose. Glutamine addiction correlated with Myc-induced redirection of glucose carbon away from mitochondria as a result of LDH-A activation. As a result, Myc-transformed cells became dependent on glutamine anapleurosis for the maintenance of mitochondrial integrity and TCA cycle function. Introduction of a Myc-shRNA hairpin reversed the glutamine dependence of Myc-transformed cells. In addition, Myc-transformed cells were sensitive to inhibitors of glutamate conversion to α-ketoglutarate in a Myc-dependent fashion and this sensitivity could be reversed by supplying cells with a cell-penetrant form of the mitochondrial substrate α-ketoglutarate. Taken together, these results suggest that glutamine addiction can be a direct consequence of Myc-induced transformation.


The Human Glioma Line SF188 Depends on Glutamine Catabolism to Maintain Viability.

When SF188 cells were cultured in the presence of 14C-labeled glutamine, <15% of the glutamine the cells took up from the medium was incorporated into newly synthesized protein (Fig. 1A). Despite the fact that only a small fraction of the glutamine was used for anabolic synthesis, SF188 glioma cells were unable to survive in glutamine-deficient medium despite the presence of 25 mM glucose in the medium (Fig. 1B). α-ketoglutarate is the glutamine metabolite that enters the mitochondrial TCA cycle. The replacement of glutamine with a cell-penetrant form of α-ketoglutarate (dimethyl α-ketoglutarate) in the medium suppressed the cell death observed when the cells were cultured in glutamine-deficient medium (Fig. 1B). These data suggest that glutamine addiction does not result from glutamine's role as an amide donor in nucleotide biosynthesis or as the source of nitrogen for the maintenance of nonessential amino acid production because dimethyl α-ketoglutarate is devoid of nitrogen groups and cannot participate in these glutamine-dependent reactions.

Fig. 1.
Glutamine catabolism in the human glioma line SF188. (A) Protein synthesis is a minor fate of glutamine carbon. SF188 cells were cultured in medium supplemented with 0.01% [14C-U5]glutamine relative to unenriched glutamine for 4 h. [14C-U5]glutamine in ...

PI3K/AKT Signaling Regulates the Consumption of Glucose but Not of Glutamine in Glioma Cells.

Previous work has demonstrated that the PI3K/AKT pathway can regulate the expression and surface translocation of a variety of nutrient transporters (5, 13). We therefore investigated whether the PI3K/AKT pathway might also function to up-regulate glutamine uptake and metabolism. To determine whether PI3K/AKT signaling contributes to glutamine metabolism, the effects of the PI3K inhibitor LY294002 or the AKT inhibitor, AKT inhibitor VIII, on glucose and glutamine uptake were studied. SF188 cells with a Bcl-xL transgene were used in this study to prevent apoptosis induced by drug treatment. As presented in Fig. 2, AKT inhibitor VIII suppressed glucose metabolism and lactate production in a dose-dependent fashion. In contrast, neither glutamine metabolism nor ammonia production was inhibited by AKT inhibitor VIII. If anything, there was a compensatory up-regulation of glutamine metabolism in response to increasing doses of the inhibitor. Similar results were observed using the PI3K inhibitor LY294002 (data not shown).

Fig. 2.
PI3K/Akt signaling regulates the consumption of glucose but not of glutamine. SF188 cells stably expressing Bcl-xL were treated with AktiVIII at doses ranging from 0–20 μM. Medium was collected and analyzed for glucose, lactate, glutamine, ...

Myc Can Regulate Glutaminolysis.

Another oncogene associated with a poor prognosis in glial tumors is Myc (14). SF188 cells were originally isolated from a patient whose tumor displayed amplification of Myc (15). Western blot analysis of the SF188 cells used in these studies revealed Myc protein expression in excess of that observed in proliferating fibroblasts or a tumor cell line lacking amplified Myc (Fig. 3A). To determine whether Myc is required for the maintenance of oxidative glutamine metabolism in SF188 cells, the effect of suppressing Myc using shRNA was investigated. SF188 cells were transduced with a lentivirus containing shRNA against MYC (shMYC) or a lentivirus containing a control shRNA (shCTRL) and the rate of glutamine consumption and ammonia production was examined. shMYC cells had an ≈80% reduction in their Myc level (Fig. 3A). This level of Myc reduction lead to a statistically significant reduction in glutamine consumption (P < 0.01) and ammonia production (P < 0.05) (Fig. 3B).

Fig. 3.
Myc activates the transcription of genes involved in glutamine uptake and metabolism. (A) Myc protein is over-expressed in SF188 cells. Western blot reveals over-expression of c-Myc in SF188 glioblastoma cells compared with MEF and another glioblastoma ...

Myc Activates the Transcription of Genes Required for Glutamine Uptake and Metabolism.

Myc's transforming properties depend on its ability to bind to DNA and modify gene transcription (16). By using quantitative RT-PCR (qPCR), we observed that shMYC cells expressed significantly lower levels of the high affinity glutamine importers ASCT2 and SN2 (P < 0.01) without expressing significantly lower levels of the control transcript EIF1A (Fig. 3C). Furthermore, when Myc antibodies were used to perform chromatin immuno-precipitation (ChIP), Myc was found to selectively bind to the promoter regions of both ASCT2 and SN2 (Fig. 3D). This selectivity was comparable to that of the established Myc target CYCLIN D2 (Fig. 3D). Thus, Myc appears to bind to the promoter elements of glutamine transporters and this binding is associated with enhanced levels of glutamine transporter mRNA.

Myc Activates Glutaminolysis in MEF.

The above data demonstrate that Myc transcription contributes to the high level of glutaminolysis exhibited by SF188 glioma cells. We next wanted to determine whether the induction of Myc transcription was sufficient to induce increased glutamine metabolism. To address this question, immortalized MEF that stably express a 4-hydroxy tamoxifen-inducible MycER construct (17) were analyzed to study the effects of Myc activation on glutamine metabolism. Treatment of cells with 4-hydroxy tamoxifen for 24 h resulted in increased levels of the transcripts for not only the glutamine transporter ASCT2, but also for glutaminase (P < 0.005), the enzyme that deamidates glutamine to glutamate, resulting in its intracellular capture, and LDH-A (P < 0.005), which converts glutamine-derived pyruvate into lactate (Fig. 4A). These increases in mRNA levels correlated with enhanced functional activity. Treatment with 4-hydroxy tamoxifen resulted in statistically significant increases in glutamine uptake (P < 0.05) (Fig. 4B), glutaminase flux (P < 0.005) (Fig. 4C), and production of glutamine-derived lactate (P < 0.05) (Fig. 4D) in the MEF expressing MycER. As a result, the rate at which glutamine was consumed from the medium was significantly greater (Fig. 4E). Furthermore, the glutamine uptake in vehicle-treated cells began to reach an asymptote by 6 h, whereas the glutamine metabolized by the 4-hydroxy tamoxifen treated cells increased linearly over the culture period (Fig. 4E). Thus, the ability of Myc-induced cells to metabolize glutamine does not appear to be saturatable over this period as would be predicted for glutaminolysis, which ends not in the cellular accumulation of glutamine-derived metabolites over time, but in the secretion of glutamine-derived lactate into the medium. Despite increasing glutamine consumption, Myc induction did not increase the proliferative expansion of normal MEF under the same conditions. After 24 h of 4-hydroxy tamoxifen or vehicle treatment of cells that were initially plated at 1 × 105 cells per well the day before induction, the control cells increased to 5.2 ± 0.5 × 105 cells per well (mean ± SD) whereas the Myc-induced cells had only increased to 3.5 ± 0.5 × 105 cells per well (mean ± SD).

Fig. 4.
Myc activates glutaminolysis in MEF. (A) Oncogenic levels of Myc induce the expression of genes involved in glutaminolysis. qPCR analysis of target genes from total RNA isolated from MEF MycER treated with 200 nM 4-hydroxytamoxifen (4-OHT) or vehicle ...

Myc Diverts Glucose Away from Mitochondrial Metabolism.

Previous studies have suggested that proliferating nontransformed cells maintain de novo phospholipid biosynthesis from glucose (7). However, when MEF stably expressing MycER were treated with 4-hydroxy tamoxifen, the use of glucose as a precursor for phospholipid synthesis was suppressed (Fig. 5A). Concomitantly, an increased amount of the glucose-derived carbon was secreted from the cell as lactate (Fig. 5B). In contrast, the contribution of glutamine to phospholipid synthesis in Myc-induced cells was maintained and even increased despite increased secretion of glutamine-derived lactate (Figs. 4D and and55C). Together, these data demonstrate that Myc-induction leads to the diversion of glucose-derived pyruvate away from mitochondria, its conversion to lactate, and secretion from the cell. As a result, Myc induction enhances cellular dependence on glutamine to maintain phospholipid synthesis and TCA cycle anapleurosis.

Fig. 5.
Myc diverts glucose away from mitochondrial metabolism in MEF. (A) Oncogenic levels of Myc suppress the contribution of glucose to phospholipid synthesis. MEF MycER treated as in Fig. 4A were cultured with medium supplemented with D-[U-14C]-glucose for ...

The Glutamine Addiction Exhibited by SF188 Glioma Cells Is Myc-Dependent.

The above data suggest that Myc is both necessary and potentially sufficient for the glutaminolytic metabolism exhibited by SF188 cells. To confirm that Myc is also involved in the glutamine addiction observed by these cells, SF188 cells were transduced with either a lentivirus containing a MYC-shRNA (shMYC) or a control shRNA (shCTRL). The resulting cells were incubated in glutamine-depleted or complete medium. Cells transduced with MYC-shRNA had a statistically significant increase (P < 0.01) in their resistance to glutamine starvation relative to cells transduced with a control shRNA (Fig. 6A).

Fig. 6.
The glutamine addiction exhibited by SF188 glioma cells is Myc-dependent. (A) Myc-suppressed SF188 cells are resistant to glutamine starvation. shMYC and shCTRL SF188 cells, described in Fig. 3B, were allowed to plate in the presence of glutamine and ...

As a further confirmation that this glutamine addiction is Myc-dependent, the cells were treated with aminooxyacetate (AOA), a chemical inhibitor of glutamate-dependent transaminases that convert glutamate into α-ketoglutarate in the glutaminolytic pathway (18). AOA selectively induced the death of the cells transduced with control shRNA without affecting the viability of cells transduced with MYC-shRNA (P < 0.01). Although AOA is a well-characterized inhibitor of the transaminases (19), a chemical inhibitor can have nonspecific effects on the viability of cells. To confirm the specificity of AOA's effects, the ability of dimethyl α-ketoglutarate to reverse the AOA-induced toxicity to SF188 cells was examined. Addition of 7 mM dimethyl α-ketoglutarate completely suppressed the death induced by AOA treatment of SF188 parental and control transduced cells (P < 0.01) (Fig. 6B and data not shown).


The factors that regulate glutamine uptake and metabolism during cell growth and transformation have remained poorly understood. In this manuscript, we provide evidence that oncogenic levels of Myc reprogram intermediate metabolism, leading to glutamine addiction for the maintenance of mitochondrial TCA cycle integrity. Previous work has demonstrated that LDH-A induction by Myc is required for Myc-transformation (20). This results in diversion of glucose-derived pyruvate into lactate. Despite this, Myc-transformed cells display an increased mitochondrial mass and increased rate of O2 consumption (21). Furthermore, Morrish et al. (22) have reported that Myc-over-expressing cells are exquisitely sensitive to inhibition of the mitochondrial electron transport chain. To explain this apparent paradox, they suggested that mitochondrial respiration might be maintained by catabolizing alternative bioenergetic substrates. In this article, we report that the alternative substrate is glutamine. Myc-transformation leads to conversion from glucose to glutamine as the oxidizable substrate used to maintain TCA cycle activity and cell viability. Myc binds to the promoters and induces the expression of several key regulatory genes involved in glutaminolytic metabolism. Our studies suggest that supraphysiological levels of Myc associated with oncogenic transformation are both necessary and sufficient for the induction of glutaminolysis to levels that result in glutamine addiction.

Yuneva et al. (23) have reported that some, but not all, Myc transformants were dependent on glutamine. They also demonstrated that over-expression of Bcl-2 suppressed the death of Myc-transformants deprived of glutamine. Cell types and cell lines vary greatly in their level of expression in Bcl-2 family members and this may account for the differences observed between cell lines. Consistent with this, when SF188 cells were transfected with Bcl-xL, they underwent cell cycle arrest but did not die when deprived of glutamine (data not shown). Like Yuneva et al. (23), we also found that cell-penetrant TCA cycle intermediates could suppress Myc-induced apoptosis. Together, these results suggest that glutamine addiction does not result from the use of glutamine as an amine donor, but rather because glutamine metabolism is essential to maintain mitochondrial integrity and function in Myc-transformed cells.

Whether levels of Myc expression induced in response to mitogenic stimulation also play a critical role in glutamine uptake and metabolism in the growth of nontransformed cells remains to be determined. At lower levels of expression, Myc-induced increases in protein synthesis and cell growth will also stimulate the incorporation of glutamine into newly synthesized proteins (24, 25). There are undoubtedly additional signaling pathways that contribute to the regulation of glutamine uptake. Cells lacking oncogenic Myc levels, while not glutamine dependent, still take up sufficient glutamine to fuel both nucleotide and protein biosynthesis for cell growth and proliferation. Perhaps that is what is most surprising about the current results. Little of the glutamine uptake stimulated by Myc is used for macromolecular synthesis. Previous 13C-NMR studies found that during glutaminolysis, >60% of glutamine-derived carbon is released from the cell as either lactate or CO2 (12). Although the TCA cycle was also replenished by glutamine, only 5% of glutamine fluxing through the TCA cycle was incorporated into fatty acids. Here, we show that only 15% of the glutamine carbon taken up by the cell is incorporated in protein. Nevertheless, the additional stimulation of glutaminolysis by oncogenic levels of Myc results in cellular addiction to glutamine.

The ability of Myc to induce glutaminolysis does have a potentially beneficial effect for the transformed cell. Glutaminolysis results in the robust production of NADPH, thus providing an energy source for a wide variety of synthetic reactions required for cell growth (12). It has long been believed that the major source of NADPH production during cell growth occurs through the oxidative arm of the pentose phosphate shunt (26). However, recent evidence suggests that transformed cells exhibiting aerobic glycolysis derive the majority of their ribose biosynthesis through the nonoxidative arm of the pentose phosphate shunt (27). Under these conditions, G6PD cannot be used to produce a supply of NADPH to support macromolecular synthesis of fatty acids or nucleotides. Without a compensatory mechanism to generate NADPH, de novo nucleotide synthesis using ribose produced in the nonoxidative arm of the pentose phosphate shunt would rapidly lead to intracellular depletion of NADPH. The ability of Myc to stimulate NADPH production through glutamine-dependent degradation provides the transformed cell with a mechanism to produce the quantities of NADPH needed to meet the demands of cell proliferation.

The data presented here also demonstrate that glutamine uptake is controlled independently of glucose uptake. Although the PI3K/AKT pathway plays a major role in regulating glucose uptake, it does not appear to be required for the uptake and catabolism of glutamine in Myc-transformed cells. Thus, the two main bioenergetic substrates used by proliferating cells appear to be under independent control by oncogenic signaling pathways. The remarkable ability of Myc and Akt to cooperate in transforming cells may result in part from their ability to complement each other in stimulating the uptake of these two critical nutrients.

In conclusion, the results presented here provide evidence that Myc transformation is associated with induction of a level of glutamine metabolism that results in glutamine addiction. Such addiction may ultimately be exploited through the use of inhibitors of the enzymes involved in the glutaminolytic pathway. The ability of the transaminase inhibitor AOA to induce the death of Myc-transformed cells but not isogenic cells in which Myc is suppressed by a MYC-shRNA provides evidence that such an intervention would have a selectively toxic effect on Myc-transformed cells. Myc-activation/amplification is one of the most common oncogenic events observed in a wide variety of cancers and is known to drive the progression of human lymphomas (28, 29), neuroblastoma (30), and small cell lung cancer (31). Despite this, therapeutics that inhibit the transcriptional properties of Myc have so far eluded drug discovery efforts. The identification of Myc's role in glutaminolysis may provide a number of enzymatic targets through which to selectively impair the growth and survival of Myc-transformed tumor cells. Finally, the present results demonstrate that nutrient uptake in mammalian cells is under distinct and specific regulation as a result of the properties of known oncogenes. Previous results have suggested that an activating mutation in PI3K or AKT facilitate the uptake and metabolism of glucose in an mTOR-dependent manner (13). In addition, induction of HIF-1 in response to hypoxia or mitochondrial ROS can also reprogram the intracellular fate of glucose (6). The present studies suggest that oncogenic Myc activation selectively induces addiction to glutamine, the other major catabolic substrate used by mammalian cells to maintain bioenergetics during cell growth and proliferation. Whether other essential nutrients are under similar control by these or other oncogenic signaling pathways remains to be determined.


Cell Culture and Media.

SF188 cells (UC Brain Tumor Research Center, SF, CA) and SV40-immortalized MEF stably transfected with MycER (a gift from Drs. AT Tikhonenko and R.A. Amaravadi of University of Pennsylvania, Philadelphia, PA) were cultured in DMEM (Invitrogen), 10% FBS (Gemini Biosystems), 100 units/ml Penicillin, 100 μg/ml Streptomycin, 25 mM glucose, and 6 mM L-glutamine at 37°C in a 5% CO2 incubator. To avoid depleting oxygen, all experiments were carried out under subconfluent conditions. For glutamine starvation experiments, DMEM without glutamine (Invitrogen) was supplemented with 10% dialyzed FBS (Gemini Biosystems). For metabolic tracing experiments, DMEM without glutamine and with 10% dialyzed FBS was supplemented with either L-glutamine that was unenriched or with L-[U-13C5]glutamine (Isotec), [U-14C5]glutamine (GE Amersham), and [γ-15N]glutamine (Cambridge Isotope Laboratories). DMEM without glucose (Sigma) was supplemented with [U-14C6]glucose (Sigma). To activate MycER, cells were incubated with 200 nM 4-hydroxytamoxifen for 24 h. Lentiviral transduction, qPCR, immunoblotting, and metabolite analysis were performed as previously described (6, 12, 33, 34).

Glutamine Uptake.

Cells were incubated with 4 mM glutamine and 1 μM [U-14C5]glutamine in 2 ml of medium in a 6-well plate for 1 min at 37°C in a 5% CO2 incubator. After incubation, the medium was aspirated, and cells were washed three times with unlabeled medium, after which cells were lysed with 200 μl of a 0.2%SDS/0.2N NaOH solution, incubated for 1 h, neutralized with 10 μl of 2N HCl, and analyzed with a beta scintillation counter (PerkinElmer Life Sciences) (32).

NMR Analysis.

Cells were grown to 80% confluency, after which the culture medium was removed and replaced with medium that contained 4 mM [U-13C5]glutamine (and no unenriched glutamine) for 6 h. The resulting medium was then analyzed in a 20-mm NMR tube with a 9.4 Tesla spectrometer at 100.66 MHz. A 90° excitation pulse was applied every 20 seconds (fully relaxed), with broad-band decoupling used only during 13C data acquisition (no NOE enhancement). Spectra were acquired with 16384 points, a bandwidth of 25,000 Hz and 3,000 excitations. Free induction decays were apodized with exponential multiplication (3 Hz line broadening). Peak intensities in Fourier transformed spectra were determined with Nuts NMR (Acorn NMR, Livermore, CA). Carbon-3 of lactate derived from glutamine produced a doublet (21.5 and 20.3 ppm) due to splitting from 13C at carbon-2, whereas lactate derived from the natural abundance 13C of glucose, produced a singlet at 20.9 ppm. All peaks were well resolved from each other.

Lipid Biochemistry.

To determine the rate of lipid synthesis from glucose or glutamine, 1–4 × 105 MEF were plated in 6-well plates and cultured with DMEM supplemented with either 2.2 μM D-[U-14C6] glucose (GE Amersham) or 0.54 μM L-[U-14C5]glutamine (GE Amersham), both supplemented to 0.01% of their respective unenriched nutrients. After 8 h, cells were trypsinized, washed in PBS, and lysed in 0.4 ml of 0.5% Triton X-100. Total lipids were extracted and phospholipids were collected using TLC (33). Total incorporated label in the phospholipid fraction was analyzed with a scintillation counter (PerkinElmer Life Sciences).

ChIP Analysis.

SF188 cells were plated on 15-cm dishes and were fixed in 1% formaldehyde. Chromatin was sheared to an average size of 500–1,000 bp by sonication (30 times with 30-s pulses, on a Diagenode Bioruptor). Lysates corresponding to 5–10 × 106 cells were rotated at 4°C overnight with 2 μg of polyclonal antibodies specific for c-MYC (sc-764, Santa Cruz Biotechnology) or normal rabbit IgG. Precipitated DNA fragments were quantified using PCR.

Akt Inhibitor.

SF188 cells stably expressing Bcl-xL were plated at a density of 4 × 105 cells in a 6-well plate format, and incubated in DMEM with 10% FCS for 36 h before treatment with AKT inhibitor VIII (Calbiochem) at 0, 0.1, 2, 5, and 10 μM concentrations in triplicate. After 8 h, medium samples were collected for metabolite analysis, and viable cells were counted using trypan blue exclusion.

Contribution of Glutamine to Protein Synthesis.

SF188 cells were cultured in medium supplemented with 0.01% [U-14C5]glutamine relative to unenriched glutamine for 4 h. Cells were washed three times with PBS, extracted using 0.5% Triton-X, acidified using 10% (vol/vol) of 35% (wt/v) sulfosalicylic acid, and then spun down at 13,000 rpm for 15 min at 4°C. The pellet was then resolubilized in NaOH at 37°C. 14C incorporated into the protein product were quantified using a scintillation counter (PerkinElmer Life Sciences). Efficiency of protein recovery was controlled for by calculating the recovery of 14C BSA (Sigma) added after lysis. Recovery of intracellular [U-14C5]glutamine in its free form was controlled for by calculating the recovery of [U-14C5]glutamine added just before SSA precipitation. The recovered counts were sensitive to 5 μg/ml cycloheximide. The glutamine consumption rate was then calculated using the GLN2 glutamine assay kit (Sigma). The data presented are the mean ± SD from four independent experiments.

Gas Chromatography–Mass Spectrometry.

Cells were plated at 1.2 × 106 per 6-cm dish. At 80% confluency, they were fed with 1.5 ml of DMEM containing 4 mM l-[γ-15N]glutamine (Cambridge Isotope Laboratories). Every 2 h, medium was collected to determine the concentration of NH3 using the Nova Biomedical Flex Analyzer. An aliquot was used to determine isotopic enrichment in NH3 with published methods (35).

AOA Inhibitor Experiments.

SF188 cells were treated with AOA (Sigma) at doses ranging from 500 nM – 500 μM and viability was assessed 24 h post-treatment (18). Five hundred μM was the lowest dose that killed a significant fraction of the cells. SF188 cells with Myc or control shRNA were replated 3 days post viral transduction. After allowing to plate overnight, cells were treated with 500 μM for 24 h, and then viability was assessed using trypan blue dye exclusion.

Supplementary Material

Supporting Information:


We thank members of the Thompson laboratory for thoughtful discussions and Tullia Lindsten and Tamar Schwartz for their review of the manuscript. This work was supported by grants from the National Cancer Institute; the National Institutes of Health, including K08 DK072565 (to R.J.D.) and HD26979 and NS054900 (to M.Y.); the Abramson Family Cancer Research Institute (C.B.T.); a Medical Scientist Training grant and a Cancer Research Institute grant (to D.R.W.). S.B.M. was supported by grants from the National Institutes of Health and Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health.


The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0810199105/DCSupplemental.


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