PI3K/Akt signaling regulates the consumption of glucose but not of glutamine. SF188 cells stably expressing Bcl-x_L were treated with AktiVIII at doses ranging from 0–20 μM. Medium was collected and analyzed for glucose, lactate, glutamine, and ammonia. The rates shown were calculated from the difference in metabolite concentration between the medium at the time point shown and fresh medium. The data points presented are the mean ± SD of triplicate samples.

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 (EtOH) for 24 h. The bars shown are normalized to an internal β-actin control and represent the mean ± SD of triplicate samples. Representative data from one of three independent experiments are shown. (B) Oncogenic levels of Myc induce glutamine uptake. MEF MycER treated as in (A) were cultured for 1 min with medium supplemented with [U-¹⁴C₅]glutamine. Uptake of the label was quantified by scintillation counting of the cellular lysate. The data presented are the mean ± SD of triplicate samples. (C) Oncogenic levels of Myc induce flux through glutaminase. MEF MycER treated as in (A) were cultured for 8 h with medium supplemented with L-[γ-¹⁵N]glutamine. Glutaminase activity was determined by measuring the isotopic enrichment of ¹⁵N in NH₄⁺ in the culture medium by GC-MS. The bars shown represent the mean ± SD of triplicate cultures. (D) Oncogenic levels of Myc induce the flux of glutamine into lactate. MEF MycER treated as in (A) were cultured for 6 h in medium supplemented with 4 mM [U-¹³C₅]glutamine. The medium was subsequently removed and analyzed with ¹³C NMR spectroscopy. [2,3-¹³C]lactate is metabolically derived from [U-¹³C₅]glutamine, while [3-¹³C]lactate is metabolically derived from the natural abundance of [1-¹³C]glucose and [6-¹³C]glucose. The data presented are the mean ± SD of triplicate samples. (E) Oncogenic levels of Myc induce the consumption of glutamine from the medium. The glutamine concentration in medium from MEF MycER treated as in (A) was analyzed at the time points shown by the Nova Flex. The data points shown represent the mean ± SD of triplicate samples.

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 then cultured in the absence of glutamine. Cell viability was determined at the time points shown by trypan blue dye exclusion. The data points shown represent the mean ± SD of triplicate samples. (B) Myc-suppressed SF188 cells are resistant to an inhibitor of glutaminolysis. shMYC and shCTRL SF188 cells, described in Fig. 3B, were allowed to plate in the presence of glutamine and then were treated with 500 μM aminooxyacetate (AOA). Cell viability was determined at the time points shown by trypan blue dye exclusion. AOA- treated shCTRL cells were also treated with 7 mM dimethyl α-ketoglutarate (AOA + α-ketoglutarate). The data points shown represent the mean ± SD of triplicate samples.

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 SSA precipitated protein (striped bar) and total glutamine consumed from the medium (gray bar) are presented as the mean ± standard deviation (SD) of four independent experiments. (B) The requirement for glutamine can be satisfied by alpha-ketoglutarate. SF188 cells were allowed to plate in complete medium and then cultured in either glutamine-depleted medium (− glutamine), complete medium (+ glutamine), or glutamine-depleted medium supplemented with 7 mM dimethyl α-ketoglutarate (− glutamine + α-ketoglutarate). Cell viability was determined at the time points shown by trypan blue dye exclusion. The data presented are the mean ± SD of triplicate samples. Representative data from one of three independent experiments are shown.

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 cell line, LN229. (B) Myc is required for glutamine metabolism. SF188 cells were transduced with either lentiviral shRNA against Myc (shMYC) or Luciferase (shCTRL) for 3 days. Glutamine and ammonium levels in the medium were analyzed using the Nova Flex and are presented as the mean ± SD of triplicate samples. Data from one of five independent experiments are shown. Knockdown of Myc protein is depicted in (A). (C) Myc is required for the expression of the proximal enzymes of glutaminolysis. RNA was extracted from shMYC and shCTRL cells and quantified using quantitative RT-PCR (qPCR). The bars shown are normalized to a β-actin control and represent the mean ± SD of triplicate samples. Representative data from one of two independent experiments are shown. EIF1A is included as a negative control. (D) Myc is enriched at the regulatory binding sites of genes involved in glutamine uptake. Sheared chromatin from fixed and lysed SF188 cells was immunoprecipitated using the antibodies indicated. Precipitated DNA fragments were quantified by qPCR. The data presented are the mean ± SD of triplicate samples. The schematic shows the location of Myc-bound E-box elements within the genomic loci of ASCT2 and SN2.

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 8 h. After the culture period, lipids were harvested and ¹⁴C enrichment in phospholipids (PL) was determined by scintillation counting. The bars shown represent the mean ± SD of triplicate samples. Representative data from one of three experiments are shown. (B) Oncogenic levels of Myc induce lactate production. The lactate concentration in the medium from MEF MycER treated with 4-OHT or EtOH for 18 h was quantified by using the Nova Flex Metabolite Analyzer. Each time point is the mean ± SD of triplicate samples. Representative data from one of three experiments are shown. (C) Glutamine's contribution to phospholipid synthesis is maintained in the presence of oncogenic levels of Myc. MEF MycER treated as in Fig. 5A were cultured with medium supplemented with L-[U-¹⁴C]-glutamine for 8 h. After the culture period, lipids were harvested and ¹⁴C enrichment in PL was determined by scintillation counting. The bars shown represent the mean ± SD of triplicate samples. Representative data from one of three independent experiments are shown.