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Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma.

Tardito S, Oudin A, Ahmed SU, Fack F, Keunen O, Zheng L, Miletic H, Sakariassen PØ, Weinstock A, Wagner A, Lindsay SL, Hock AK, Barnett SC, Ruppin E, Mørkve SH, Lund-Johansen M, Chalmers AJ, Bjerkvig R, Niclou SP, Gottlieb E - Nat. Cell Biol. (2015)

Bottom Line: However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation.Moreover, Gln-starved cells are not rescued by TCA cycle replenishment.In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons.

View Article: PubMed Central - PubMed

Affiliation: Cancer Metabolism Research Unit, Cancer Research UK, Beatson Institute, Switchback Road, Glasgow G61 1BD, UK.

ABSTRACT
L-Glutamine (Gln) functions physiologically to balance the carbon and nitrogen requirements of tissues. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by glutamine synthetase (GS; cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons. Finally, the Gln required for the growth of GBM tumours is contributed only marginally by the circulation, and is mainly either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes.

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Astrocytes feed with Gln GBM cells. (a, b) Astrocytes were incubated in SMEM for 6 days with 0, 0.1, 0.3, 0.65, 1, 2, 4mM Gln (a) or for the indicated times with 0 and 0.65mM Gln (b). (c) Astrocytes derived from two independent extractions, and cell lines, were incubated for 3 days +/− Gln and protein expression assessed. Unprocessed scans of western blots are shown in Supplementary Figure 8. (d-j) Astrocytes were incubated in SMEM for 24h in the presence of 5.56mM Glucose (13C6 or 13C0), 0.65mM Gln (13C5 or 13C0), 0.8mM 15NH4+, and 1mM MSO as indicated. Secretion/consumption rates (positive/negative bars respectively) are shown for Gln (d) and Glu (e). Intracellular levels of Gln (f) and Glu (g) isotopologues are reported as % of control (total of isotopologues in Gln fed conditions). The intracellular isotopologues of Gln (h), Glu (i), AMP (j) are shown as % of control (total isotopologues in the presence of Gln and 15NH4. (k) Astrocytes were incubated for 6 days +/− 0.65mM Gln, and 1mM MSO and counted. (l) Astrocytes were grown to confluence in multi-well plates. iRFP4 cells were seeded in wells +/− astrocytes, and +/− Gln. The fluorescence of iRFP4 cells in representative wells is shown. The experiment was performed twice with comparable results. (m) Astrocytes were grown to confluence in multi-well plates. iRFP4 cells seeded in transwell inserts were co-cultured +/− astrocytes, +/−Gln, and +/− Erwinase (Erw) as indicated. Fluorescence of iRFP4 cells in representative inserts is shown. At day 5 astrocytes were stained with sulphorodamine-B and the fluorescence of representative wells is shown. (n) Quantification of the iRFP4 fluorescence as described for (m). (a, b, d, e, f, g, h, i, j, k, n) Data derive from one experiment performed twice. Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.
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Figure 8: Astrocytes feed with Gln GBM cells. (a, b) Astrocytes were incubated in SMEM for 6 days with 0, 0.1, 0.3, 0.65, 1, 2, 4mM Gln (a) or for the indicated times with 0 and 0.65mM Gln (b). (c) Astrocytes derived from two independent extractions, and cell lines, were incubated for 3 days +/− Gln and protein expression assessed. Unprocessed scans of western blots are shown in Supplementary Figure 8. (d-j) Astrocytes were incubated in SMEM for 24h in the presence of 5.56mM Glucose (13C6 or 13C0), 0.65mM Gln (13C5 or 13C0), 0.8mM 15NH4+, and 1mM MSO as indicated. Secretion/consumption rates (positive/negative bars respectively) are shown for Gln (d) and Glu (e). Intracellular levels of Gln (f) and Glu (g) isotopologues are reported as % of control (total of isotopologues in Gln fed conditions). The intracellular isotopologues of Gln (h), Glu (i), AMP (j) are shown as % of control (total isotopologues in the presence of Gln and 15NH4. (k) Astrocytes were incubated for 6 days +/− 0.65mM Gln, and 1mM MSO and counted. (l) Astrocytes were grown to confluence in multi-well plates. iRFP4 cells were seeded in wells +/− astrocytes, and +/− Gln. The fluorescence of iRFP4 cells in representative wells is shown. The experiment was performed twice with comparable results. (m) Astrocytes were grown to confluence in multi-well plates. iRFP4 cells seeded in transwell inserts were co-cultured +/− astrocytes, +/−Gln, and +/− Erwinase (Erw) as indicated. Fluorescence of iRFP4 cells in representative inserts is shown. At day 5 astrocytes were stained with sulphorodamine-B and the fluorescence of representative wells is shown. (n) Quantification of the iRFP4 fluorescence as described for (m). (a, b, d, e, f, g, h, i, j, k, n) Data derive from one experiment performed twice. Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.

Mentions: To assess this potential interaction, rat primary cortical astrocytes were cultured and their Gln requirement and metabolism investigated. Similar to GBM cells, the minimal Gln concentration required for maximal astrocyte growth was ~0.65 mM (Fig. 8a). Nonetheless, astrocyte proliferation was barely affected by Gln deprivation (Fig. 8b). As observed in the human TMA (Fig. 6a), GS protein levels in astrocytes and in the highest expressing GBM cells were comparable (Fig. 8c). However, only astrocytes demonstrated no net Gln consumption but rather, rapid Glu uptake (Fig. 8d-e), in line with the expression of Excitatory Amino Acids Transporters (EAAT) in this cell type34. Under Gln starvation, Glu consumption was unaffected and paralleled by an equimolar net Gln efflux (Fig. 8d-e). The absence of Gln in the medium reduced intracellular Gln, but not Glu (Fig. 8f-g). Moreover, 13C6-glucose tracing showed that only 30-40% of both intracellular Glu and Gln (Fig. 8f-g) were glucose-derived. Astrocytes maintained ~30% of the control level of intracellular Gln under Gln starvation (Fig. 8f and 8h), fitting with high GS expression. Gln maintenance depended on GS activity, as seen from both 15N1-ammonia tracing and GS inhibition by MSO (Fig. 8h). Moreover, GS inhibition dramatically elevated the intracellular amounts of its substrate, Glu (Fig. 8i), without changing the steady state levels of the Gln-product, AMP (Fig. 8j). However, combined Gln withdrawal and GS inhibition significantly reduced the labelled fraction of AMP derived from de novo synthesis (15N2 and 15N3; Fig. 8j), and hindered proliferation (Fig. 8k).


Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma.

Tardito S, Oudin A, Ahmed SU, Fack F, Keunen O, Zheng L, Miletic H, Sakariassen PØ, Weinstock A, Wagner A, Lindsay SL, Hock AK, Barnett SC, Ruppin E, Mørkve SH, Lund-Johansen M, Chalmers AJ, Bjerkvig R, Niclou SP, Gottlieb E - Nat. Cell Biol. (2015)

Astrocytes feed with Gln GBM cells. (a, b) Astrocytes were incubated in SMEM for 6 days with 0, 0.1, 0.3, 0.65, 1, 2, 4mM Gln (a) or for the indicated times with 0 and 0.65mM Gln (b). (c) Astrocytes derived from two independent extractions, and cell lines, were incubated for 3 days +/− Gln and protein expression assessed. Unprocessed scans of western blots are shown in Supplementary Figure 8. (d-j) Astrocytes were incubated in SMEM for 24h in the presence of 5.56mM Glucose (13C6 or 13C0), 0.65mM Gln (13C5 or 13C0), 0.8mM 15NH4+, and 1mM MSO as indicated. Secretion/consumption rates (positive/negative bars respectively) are shown for Gln (d) and Glu (e). Intracellular levels of Gln (f) and Glu (g) isotopologues are reported as % of control (total of isotopologues in Gln fed conditions). The intracellular isotopologues of Gln (h), Glu (i), AMP (j) are shown as % of control (total isotopologues in the presence of Gln and 15NH4. (k) Astrocytes were incubated for 6 days +/− 0.65mM Gln, and 1mM MSO and counted. (l) Astrocytes were grown to confluence in multi-well plates. iRFP4 cells were seeded in wells +/− astrocytes, and +/− Gln. The fluorescence of iRFP4 cells in representative wells is shown. The experiment was performed twice with comparable results. (m) Astrocytes were grown to confluence in multi-well plates. iRFP4 cells seeded in transwell inserts were co-cultured +/− astrocytes, +/−Gln, and +/− Erwinase (Erw) as indicated. Fluorescence of iRFP4 cells in representative inserts is shown. At day 5 astrocytes were stained with sulphorodamine-B and the fluorescence of representative wells is shown. (n) Quantification of the iRFP4 fluorescence as described for (m). (a, b, d, e, f, g, h, i, j, k, n) Data derive from one experiment performed twice. Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.
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Figure 8: Astrocytes feed with Gln GBM cells. (a, b) Astrocytes were incubated in SMEM for 6 days with 0, 0.1, 0.3, 0.65, 1, 2, 4mM Gln (a) or for the indicated times with 0 and 0.65mM Gln (b). (c) Astrocytes derived from two independent extractions, and cell lines, were incubated for 3 days +/− Gln and protein expression assessed. Unprocessed scans of western blots are shown in Supplementary Figure 8. (d-j) Astrocytes were incubated in SMEM for 24h in the presence of 5.56mM Glucose (13C6 or 13C0), 0.65mM Gln (13C5 or 13C0), 0.8mM 15NH4+, and 1mM MSO as indicated. Secretion/consumption rates (positive/negative bars respectively) are shown for Gln (d) and Glu (e). Intracellular levels of Gln (f) and Glu (g) isotopologues are reported as % of control (total of isotopologues in Gln fed conditions). The intracellular isotopologues of Gln (h), Glu (i), AMP (j) are shown as % of control (total isotopologues in the presence of Gln and 15NH4. (k) Astrocytes were incubated for 6 days +/− 0.65mM Gln, and 1mM MSO and counted. (l) Astrocytes were grown to confluence in multi-well plates. iRFP4 cells were seeded in wells +/− astrocytes, and +/− Gln. The fluorescence of iRFP4 cells in representative wells is shown. The experiment was performed twice with comparable results. (m) Astrocytes were grown to confluence in multi-well plates. iRFP4 cells seeded in transwell inserts were co-cultured +/− astrocytes, +/−Gln, and +/− Erwinase (Erw) as indicated. Fluorescence of iRFP4 cells in representative inserts is shown. At day 5 astrocytes were stained with sulphorodamine-B and the fluorescence of representative wells is shown. (n) Quantification of the iRFP4 fluorescence as described for (m). (a, b, d, e, f, g, h, i, j, k, n) Data derive from one experiment performed twice. Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.
Mentions: To assess this potential interaction, rat primary cortical astrocytes were cultured and their Gln requirement and metabolism investigated. Similar to GBM cells, the minimal Gln concentration required for maximal astrocyte growth was ~0.65 mM (Fig. 8a). Nonetheless, astrocyte proliferation was barely affected by Gln deprivation (Fig. 8b). As observed in the human TMA (Fig. 6a), GS protein levels in astrocytes and in the highest expressing GBM cells were comparable (Fig. 8c). However, only astrocytes demonstrated no net Gln consumption but rather, rapid Glu uptake (Fig. 8d-e), in line with the expression of Excitatory Amino Acids Transporters (EAAT) in this cell type34. Under Gln starvation, Glu consumption was unaffected and paralleled by an equimolar net Gln efflux (Fig. 8d-e). The absence of Gln in the medium reduced intracellular Gln, but not Glu (Fig. 8f-g). Moreover, 13C6-glucose tracing showed that only 30-40% of both intracellular Glu and Gln (Fig. 8f-g) were glucose-derived. Astrocytes maintained ~30% of the control level of intracellular Gln under Gln starvation (Fig. 8f and 8h), fitting with high GS expression. Gln maintenance depended on GS activity, as seen from both 15N1-ammonia tracing and GS inhibition by MSO (Fig. 8h). Moreover, GS inhibition dramatically elevated the intracellular amounts of its substrate, Glu (Fig. 8i), without changing the steady state levels of the Gln-product, AMP (Fig. 8j). However, combined Gln withdrawal and GS inhibition significantly reduced the labelled fraction of AMP derived from de novo synthesis (15N2 and 15N3; Fig. 8j), and hindered proliferation (Fig. 8k).

Bottom Line: However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation.Moreover, Gln-starved cells are not rescued by TCA cycle replenishment.In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons.

View Article: PubMed Central - PubMed

Affiliation: Cancer Metabolism Research Unit, Cancer Research UK, Beatson Institute, Switchback Road, Glasgow G61 1BD, UK.

ABSTRACT
L-Glutamine (Gln) functions physiologically to balance the carbon and nitrogen requirements of tissues. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by glutamine synthetase (GS; cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons. Finally, the Gln required for the growth of GBM tumours is contributed only marginally by the circulation, and is mainly either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes.

Show MeSH
Related in: MedlinePlus