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Glutamate and asparagine cataplerosis underlie glutamine addiction in melanoma.

Ratnikov B, Aza-Blanc P, Ronai ZA, Smith JW, Osterman AL, Scott DA - Oncotarget (2015)

Bottom Line: Glutamine dependence is a prominent feature of cancer metabolism, and here we show that melanoma cells, irrespective of their oncogenic background, depend on glutamine for growth.In the absence of glutamine, TCA cycle metabolites were liable to depletion through aminotransferase-mediated α-ketoglutarate-to-glutamate conversion and glutamate secretion.Melanocytes use more glutamine for protein synthesis rather than secreting it as glutamate and are less prone to loss of glutamate and TCA cycle metabolites when starved of glutamine.

View Article: PubMed Central - PubMed

Affiliation: Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Glutamine dependence is a prominent feature of cancer metabolism, and here we show that melanoma cells, irrespective of their oncogenic background, depend on glutamine for growth. A quantitative audit of how carbon from glutamine is used showed that TCA-cycle-derived glutamate is, in most melanoma cells, the major glutamine-derived cataplerotic output and product of glutaminolysis. In the absence of glutamine, TCA cycle metabolites were liable to depletion through aminotransferase-mediated α-ketoglutarate-to-glutamate conversion and glutamate secretion. Aspartate was an essential cataplerotic output, as melanoma cells demonstrated a limited capacity to salvage external aspartate. Also, the absence of asparagine increased the glutamine requirement, pointing to vulnerability in the aspartate-asparagine biosynthetic pathway within melanoma metabolism. In contrast to melanoma cells, melanocytes could grow in the absence of glutamine. Melanocytes use more glutamine for protein synthesis rather than secreting it as glutamate and are less prone to loss of glutamate and TCA cycle metabolites when starved of glutamine.

No MeSH data available.


Related in: MedlinePlus

Testing the metabolic functions of aminotransferases and glutamatedehydrogenase (GLUD1) in Lu1205 cells(A) Comparative enrichment of α-ketoglutarate orglutamate from glucose or glutamine in Lu1205 cells (left panel) ormelanocytes (right panel) ± 0.5 mM AOA. (B–D)Effects of combined AOA treatment and GLUD1 knockdownon metabolism and growth. (B) Labeling of TCA cyclemetabolites fumarate and citrate from 13C-glutamine.(C) Quantities of cellular free glutamate andglutamine. (D) Mass distribution of secreted glutamate(corrected for natural labeling and 13C-glutamine impurity).(Mean ± SEM of N = 3 for all, except mean± range of N = 2 for D).**p < 0.05, ***p <0.01 by Student's unpaired t-test.
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Figure 4: Testing the metabolic functions of aminotransferases and glutamatedehydrogenase (GLUD1) in Lu1205 cells(A) Comparative enrichment of α-ketoglutarate orglutamate from glucose or glutamine in Lu1205 cells (left panel) ormelanocytes (right panel) ± 0.5 mM AOA. (B–D)Effects of combined AOA treatment and GLUD1 knockdownon metabolism and growth. (B) Labeling of TCA cyclemetabolites fumarate and citrate from 13C-glutamine.(C) Quantities of cellular free glutamate andglutamine. (D) Mass distribution of secreted glutamate(corrected for natural labeling and 13C-glutamine impurity).(Mean ± SEM of N = 3 for all, except mean± range of N = 2 for D).**p < 0.05, ***p <0.01 by Student's unpaired t-test.

Mentions: To assay the roles of aminotransferases in controlling flux into and out of theTCA cycle, we labeled Lu1205 cells or melanocytes with either13C-glucose or 13C-glutamine, and compared the effects ofAOA treatment on the 13C-enrichment of glutamate orα-ketoglutarate. In untreated Lu1205 cells (Figure 4A, left panel), cellular α-ketoglutarate andglutamate were both approximately 30% derived from glucose and 50% derived fromglutamine, demonstrating equilibration in the interconversion of thesemetabolites. In AOA-treated cells, 13C input from glutamine (viaglutamate) to α-ketoglutarate was maintained or increased, indicatingactive glutamate to α-ketoglutarate conversion. However, 13Cinput from glucose (via α-ketoglutarate) to glutamate was much reduced,indicating that the α-ketoglutarate to glutamate reaction was inhibited(see Figure 1A). Therefore, these cellsconvert α-ketoglutarate to glutamate largely using AOA-sensitiveaminotransferases, but they retain the capacity to convert glutamate toα-ketoglutarate even when aminotransferases are inhibited (apparently dueto the contribution of oxidative deamination by GLUD1 as discussed below).


Glutamate and asparagine cataplerosis underlie glutamine addiction in melanoma.

Ratnikov B, Aza-Blanc P, Ronai ZA, Smith JW, Osterman AL, Scott DA - Oncotarget (2015)

Testing the metabolic functions of aminotransferases and glutamatedehydrogenase (GLUD1) in Lu1205 cells(A) Comparative enrichment of α-ketoglutarate orglutamate from glucose or glutamine in Lu1205 cells (left panel) ormelanocytes (right panel) ± 0.5 mM AOA. (B–D)Effects of combined AOA treatment and GLUD1 knockdownon metabolism and growth. (B) Labeling of TCA cyclemetabolites fumarate and citrate from 13C-glutamine.(C) Quantities of cellular free glutamate andglutamine. (D) Mass distribution of secreted glutamate(corrected for natural labeling and 13C-glutamine impurity).(Mean ± SEM of N = 3 for all, except mean± range of N = 2 for D).**p < 0.05, ***p <0.01 by Student's unpaired t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480687&req=5

Figure 4: Testing the metabolic functions of aminotransferases and glutamatedehydrogenase (GLUD1) in Lu1205 cells(A) Comparative enrichment of α-ketoglutarate orglutamate from glucose or glutamine in Lu1205 cells (left panel) ormelanocytes (right panel) ± 0.5 mM AOA. (B–D)Effects of combined AOA treatment and GLUD1 knockdownon metabolism and growth. (B) Labeling of TCA cyclemetabolites fumarate and citrate from 13C-glutamine.(C) Quantities of cellular free glutamate andglutamine. (D) Mass distribution of secreted glutamate(corrected for natural labeling and 13C-glutamine impurity).(Mean ± SEM of N = 3 for all, except mean± range of N = 2 for D).**p < 0.05, ***p <0.01 by Student's unpaired t-test.
Mentions: To assay the roles of aminotransferases in controlling flux into and out of theTCA cycle, we labeled Lu1205 cells or melanocytes with either13C-glucose or 13C-glutamine, and compared the effects ofAOA treatment on the 13C-enrichment of glutamate orα-ketoglutarate. In untreated Lu1205 cells (Figure 4A, left panel), cellular α-ketoglutarate andglutamate were both approximately 30% derived from glucose and 50% derived fromglutamine, demonstrating equilibration in the interconversion of thesemetabolites. In AOA-treated cells, 13C input from glutamine (viaglutamate) to α-ketoglutarate was maintained or increased, indicatingactive glutamate to α-ketoglutarate conversion. However, 13Cinput from glucose (via α-ketoglutarate) to glutamate was much reduced,indicating that the α-ketoglutarate to glutamate reaction was inhibited(see Figure 1A). Therefore, these cellsconvert α-ketoglutarate to glutamate largely using AOA-sensitiveaminotransferases, but they retain the capacity to convert glutamate toα-ketoglutarate even when aminotransferases are inhibited (apparently dueto the contribution of oxidative deamination by GLUD1 as discussed below).

Bottom Line: Glutamine dependence is a prominent feature of cancer metabolism, and here we show that melanoma cells, irrespective of their oncogenic background, depend on glutamine for growth.In the absence of glutamine, TCA cycle metabolites were liable to depletion through aminotransferase-mediated α-ketoglutarate-to-glutamate conversion and glutamate secretion.Melanocytes use more glutamine for protein synthesis rather than secreting it as glutamate and are less prone to loss of glutamate and TCA cycle metabolites when starved of glutamine.

View Article: PubMed Central - PubMed

Affiliation: Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Glutamine dependence is a prominent feature of cancer metabolism, and here we show that melanoma cells, irrespective of their oncogenic background, depend on glutamine for growth. A quantitative audit of how carbon from glutamine is used showed that TCA-cycle-derived glutamate is, in most melanoma cells, the major glutamine-derived cataplerotic output and product of glutaminolysis. In the absence of glutamine, TCA cycle metabolites were liable to depletion through aminotransferase-mediated α-ketoglutarate-to-glutamate conversion and glutamate secretion. Aspartate was an essential cataplerotic output, as melanoma cells demonstrated a limited capacity to salvage external aspartate. Also, the absence of asparagine increased the glutamine requirement, pointing to vulnerability in the aspartate-asparagine biosynthetic pathway within melanoma metabolism. In contrast to melanoma cells, melanocytes could grow in the absence of glutamine. Melanocytes use more glutamine for protein synthesis rather than secreting it as glutamate and are less prone to loss of glutamate and TCA cycle metabolites when starved of glutamine.

No MeSH data available.


Related in: MedlinePlus