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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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The effects of Glu secretion, and GLS inhibition on GBM cells growth and metabolism. (a-b) Cells were incubated for 24h +/− 13C5-Gln. Secretion (positive bars) and consumption (negative bars) rates of Gln and Glu isotopologues are shown. Mean ± S.E.M. n=3 independent experiments. (c-f) Cells were incubated as in (a-b) and the levels of intracellular Gln, Glu, Acetyl CoA, and oleate isotopologues are shown. Mean ± S.E.M. n=3 independent experiments. (g-h) LN18 and SF188 cells were incubated for 24h +/− Gln in media where glucose (g) or alanine (h) where fully replaced by 13C6-glucose or 15N1-alanine respectively. The isotopologues distributions of Glu released in the medium are shown. Mean ± S.E.M. n=3 independent experiments. (i) Scatter plot of Glu secretion observed in the absence of Gln, in relation to the growth inhibition caused by Gln starvation. Mean ± S.E.M. n=3 independent experiments. (j) A schematic representation of the  activity in the context of Glu metabolism. Adapted from Bannai et al.40 (k) LN18 cells were incubated for 24h +/− Gln in media supplemented or not with Glu, α-ketoglutarate dimethylester (dm-αKG), sulfasalazine (SSZ), or cystine, at the indicated concentrations, and the secretion/consumption rates of Glu are shown. (l-o) LN18 cells were incubated as in (k) and the intracellular levels of Glu (l), aspartate (m), citrate (n), and reduced form of glutathione (o) are shown as % of untreated control. (p) LN18 cells were incubated for 72h as described for (k). Cell number is shown as % of untreated control. Mean ± S.E.M. n=4 independent experiments. p values refer to a two-tailed t test for unpaired samples. (q-r) Cells were pre-incubated in medium with 0, 2.5, 5, 10, 15, 30μM BPTES for 3h. At t=0 medium was replaced with one containing 13C5-Gln. The abundance of 13C5-Glu (q) or 13C5-Gln (r) in the medium was monitored over time. In all conditions cells were exposed to 0.3% DMSO. (s) Cells were incubated in medium +/− 2.5μM BPTES for 72h, and counted. DMSO was 0.3% in all conditions. Mean ± S.E.M. n=3 independent experiments. (k, l, m, n, o, q, r) Data derive from one experiment performed once (k, l, m, n, o), or twice (q, r). Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.
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Figure 2: The effects of Glu secretion, and GLS inhibition on GBM cells growth and metabolism. (a-b) Cells were incubated for 24h +/− 13C5-Gln. Secretion (positive bars) and consumption (negative bars) rates of Gln and Glu isotopologues are shown. Mean ± S.E.M. n=3 independent experiments. (c-f) Cells were incubated as in (a-b) and the levels of intracellular Gln, Glu, Acetyl CoA, and oleate isotopologues are shown. Mean ± S.E.M. n=3 independent experiments. (g-h) LN18 and SF188 cells were incubated for 24h +/− Gln in media where glucose (g) or alanine (h) where fully replaced by 13C6-glucose or 15N1-alanine respectively. The isotopologues distributions of Glu released in the medium are shown. Mean ± S.E.M. n=3 independent experiments. (i) Scatter plot of Glu secretion observed in the absence of Gln, in relation to the growth inhibition caused by Gln starvation. Mean ± S.E.M. n=3 independent experiments. (j) A schematic representation of the activity in the context of Glu metabolism. Adapted from Bannai et al.40 (k) LN18 cells were incubated for 24h +/− Gln in media supplemented or not with Glu, α-ketoglutarate dimethylester (dm-αKG), sulfasalazine (SSZ), or cystine, at the indicated concentrations, and the secretion/consumption rates of Glu are shown. (l-o) LN18 cells were incubated as in (k) and the intracellular levels of Glu (l), aspartate (m), citrate (n), and reduced form of glutathione (o) are shown as % of untreated control. (p) LN18 cells were incubated for 72h as described for (k). Cell number is shown as % of untreated control. Mean ± S.E.M. n=4 independent experiments. p values refer to a two-tailed t test for unpaired samples. (q-r) Cells were pre-incubated in medium with 0, 2.5, 5, 10, 15, 30μM BPTES for 3h. At t=0 medium was replaced with one containing 13C5-Gln. The abundance of 13C5-Glu (q) or 13C5-Gln (r) in the medium was monitored over time. In all conditions cells were exposed to 0.3% DMSO. (s) Cells were incubated in medium +/− 2.5μM BPTES for 72h, and counted. DMSO was 0.3% in all conditions. Mean ± S.E.M. n=3 independent experiments. (k, l, m, n, o, q, r) Data derive from one experiment performed once (k, l, m, n, o), or twice (q, r). Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.

Mentions: To investigate cellular metabolic alterations upon Gln starvation, the exchange rate of metabolites between cells and medium was analysed by HPLC-MS. Gln was the second most consumed nutrient by all cell lines (Supplementary Fig. 2 and Supplementary Table 2). However, no clear relationship emerged between Gln consumption and Gln dependency (Fig. 2a and Supplementary Fig.3a). In contrast, all cell lines showed a net secretion of Glu despite its presence in the medium (Fig. 2b). Tracing 13C5-labeled Gln revealed that 38 ± 8% to 60 ± 19% (GUVW and U87 cells, respectively) of the Gln consumed was deamidated and secreted as 13C5-Glu (Fig. 2a-b). Unexpectedly, even Gln-starved cells discharged Glu (Fig. 2b, empty bars). Here, intracellular Gln was almost exhausted (Fig. 2c) and Glu concentration fell by more than 50% (Fig. 2d) yet, neither acetyl-CoA (Fig. 2e) nor oleate (Fig. 2f) levels were reduced, re-affirming that Gln does not sustain fatty acids biosynthesis under normoxic condition16-20. Consistently, in all cell lines, less than 15% of the citrate was derived from reductive carboxylation (13C5-Citrate; Supplementary Fig. 3b), and labelled acetyl-CoA and oleate were barely detectable (Fig. 2e-f).


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)

The effects of Glu secretion, and GLS inhibition on GBM cells growth and metabolism. (a-b) Cells were incubated for 24h +/− 13C5-Gln. Secretion (positive bars) and consumption (negative bars) rates of Gln and Glu isotopologues are shown. Mean ± S.E.M. n=3 independent experiments. (c-f) Cells were incubated as in (a-b) and the levels of intracellular Gln, Glu, Acetyl CoA, and oleate isotopologues are shown. Mean ± S.E.M. n=3 independent experiments. (g-h) LN18 and SF188 cells were incubated for 24h +/− Gln in media where glucose (g) or alanine (h) where fully replaced by 13C6-glucose or 15N1-alanine respectively. The isotopologues distributions of Glu released in the medium are shown. Mean ± S.E.M. n=3 independent experiments. (i) Scatter plot of Glu secretion observed in the absence of Gln, in relation to the growth inhibition caused by Gln starvation. Mean ± S.E.M. n=3 independent experiments. (j) A schematic representation of the  activity in the context of Glu metabolism. Adapted from Bannai et al.40 (k) LN18 cells were incubated for 24h +/− Gln in media supplemented or not with Glu, α-ketoglutarate dimethylester (dm-αKG), sulfasalazine (SSZ), or cystine, at the indicated concentrations, and the secretion/consumption rates of Glu are shown. (l-o) LN18 cells were incubated as in (k) and the intracellular levels of Glu (l), aspartate (m), citrate (n), and reduced form of glutathione (o) are shown as % of untreated control. (p) LN18 cells were incubated for 72h as described for (k). Cell number is shown as % of untreated control. Mean ± S.E.M. n=4 independent experiments. p values refer to a two-tailed t test for unpaired samples. (q-r) Cells were pre-incubated in medium with 0, 2.5, 5, 10, 15, 30μM BPTES for 3h. At t=0 medium was replaced with one containing 13C5-Gln. The abundance of 13C5-Glu (q) or 13C5-Gln (r) in the medium was monitored over time. In all conditions cells were exposed to 0.3% DMSO. (s) Cells were incubated in medium +/− 2.5μM BPTES for 72h, and counted. DMSO was 0.3% in all conditions. Mean ± S.E.M. n=3 independent experiments. (k, l, m, n, o, q, r) Data derive from one experiment performed once (k, l, m, n, o), or twice (q, r). Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4663685&req=5

Figure 2: The effects of Glu secretion, and GLS inhibition on GBM cells growth and metabolism. (a-b) Cells were incubated for 24h +/− 13C5-Gln. Secretion (positive bars) and consumption (negative bars) rates of Gln and Glu isotopologues are shown. Mean ± S.E.M. n=3 independent experiments. (c-f) Cells were incubated as in (a-b) and the levels of intracellular Gln, Glu, Acetyl CoA, and oleate isotopologues are shown. Mean ± S.E.M. n=3 independent experiments. (g-h) LN18 and SF188 cells were incubated for 24h +/− Gln in media where glucose (g) or alanine (h) where fully replaced by 13C6-glucose or 15N1-alanine respectively. The isotopologues distributions of Glu released in the medium are shown. Mean ± S.E.M. n=3 independent experiments. (i) Scatter plot of Glu secretion observed in the absence of Gln, in relation to the growth inhibition caused by Gln starvation. Mean ± S.E.M. n=3 independent experiments. (j) A schematic representation of the activity in the context of Glu metabolism. Adapted from Bannai et al.40 (k) LN18 cells were incubated for 24h +/− Gln in media supplemented or not with Glu, α-ketoglutarate dimethylester (dm-αKG), sulfasalazine (SSZ), or cystine, at the indicated concentrations, and the secretion/consumption rates of Glu are shown. (l-o) LN18 cells were incubated as in (k) and the intracellular levels of Glu (l), aspartate (m), citrate (n), and reduced form of glutathione (o) are shown as % of untreated control. (p) LN18 cells were incubated for 72h as described for (k). Cell number is shown as % of untreated control. Mean ± S.E.M. n=4 independent experiments. p values refer to a two-tailed t test for unpaired samples. (q-r) Cells were pre-incubated in medium with 0, 2.5, 5, 10, 15, 30μM BPTES for 3h. At t=0 medium was replaced with one containing 13C5-Gln. The abundance of 13C5-Glu (q) or 13C5-Gln (r) in the medium was monitored over time. In all conditions cells were exposed to 0.3% DMSO. (s) Cells were incubated in medium +/− 2.5μM BPTES for 72h, and counted. DMSO was 0.3% in all conditions. Mean ± S.E.M. n=3 independent experiments. (k, l, m, n, o, q, r) Data derive from one experiment performed once (k, l, m, n, o), or twice (q, r). Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.
Mentions: To investigate cellular metabolic alterations upon Gln starvation, the exchange rate of metabolites between cells and medium was analysed by HPLC-MS. Gln was the second most consumed nutrient by all cell lines (Supplementary Fig. 2 and Supplementary Table 2). However, no clear relationship emerged between Gln consumption and Gln dependency (Fig. 2a and Supplementary Fig.3a). In contrast, all cell lines showed a net secretion of Glu despite its presence in the medium (Fig. 2b). Tracing 13C5-labeled Gln revealed that 38 ± 8% to 60 ± 19% (GUVW and U87 cells, respectively) of the Gln consumed was deamidated and secreted as 13C5-Glu (Fig. 2a-b). Unexpectedly, even Gln-starved cells discharged Glu (Fig. 2b, empty bars). Here, intracellular Gln was almost exhausted (Fig. 2c) and Glu concentration fell by more than 50% (Fig. 2d) yet, neither acetyl-CoA (Fig. 2e) nor oleate (Fig. 2f) levels were reduced, re-affirming that Gln does not sustain fatty acids biosynthesis under normoxic condition16-20. Consistently, in all cell lines, less than 15% of the citrate was derived from reductive carboxylation (13C5-Citrate; Supplementary Fig. 3b), and labelled acetyl-CoA and oleate were barely detectable (Fig. 2e-f).

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