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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.


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Distribution of carbon from glutamine in melanoma cells andmelanocytes emphasizes role of glutamate secretion(A) Yield of carbon from glutamine in total cellular aminoacids (including protein), in secreted metabolites, and estimatedfraction lost as CO2 after culture of cells for 72 h inmedium containing U-13C-glutamine. Abbreviations: Melcytes:melanocytes; Asp-asn: combined cellular aspartate and asparagine;Glu-gln: combined cellular glutamate and glutamine. (B)Metabolites associated with calculated CO2 production(products of glutaminolysis). CO2 production was estimated asthe sum of “missing” 13C in partially labeledend-point metabolites plus CO2 necessarily lost betweenglutamine and the measured metabolites. Metabolites associated with atleast 5% of estimated CO2 production are shown, with Asp-asnrepresenting combined cellular and secreted aspartate and asparagine,and Glu-gln, combined cellular glutamate and glutamine and secretedglutamate. (C) UACC903 cells show net uptake of glutamate,unlike other melanoma cell lines, which secrete glutamate.(D) Fraction of secreted glutamate of TCA-cycle origin(partially 13C-labeled). For UACC903 cells, there was no netsecretion, but there was exchange of labeled glutamate into the medium(Mean ± SEM of N = 3). Source data forparts (A, B) are shown in Supplementary Dataset 2.
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Figure 3: Distribution of carbon from glutamine in melanoma cells andmelanocytes emphasizes role of glutamate secretion(A) Yield of carbon from glutamine in total cellular aminoacids (including protein), in secreted metabolites, and estimatedfraction lost as CO2 after culture of cells for 72 h inmedium containing U-13C-glutamine. Abbreviations: Melcytes:melanocytes; Asp-asn: combined cellular aspartate and asparagine;Glu-gln: combined cellular glutamate and glutamine. (B)Metabolites associated with calculated CO2 production(products of glutaminolysis). CO2 production was estimated asthe sum of “missing” 13C in partially labeledend-point metabolites plus CO2 necessarily lost betweenglutamine and the measured metabolites. Metabolites associated with atleast 5% of estimated CO2 production are shown, with Asp-asnrepresenting combined cellular and secreted aspartate and asparagine,and Glu-gln, combined cellular glutamate and glutamine and secretedglutamate. (C) UACC903 cells show net uptake of glutamate,unlike other melanoma cell lines, which secrete glutamate.(D) Fraction of secreted glutamate of TCA-cycle origin(partially 13C-labeled). For UACC903 cells, there was no netsecretion, but there was exchange of labeled glutamate into the medium(Mean ± SEM of N = 3). Source data forparts (A, B) are shown in Supplementary Dataset 2.

Mentions: To establish more generally how glutamine is used, we quantified the fate ofcarbon derived from glutamine in melanoma cells or melanocytes. Cells werecultured for 72 h with universally-13C-labeled glutamine and themajor metabolites into which 13C accumulated – total cellular(protein-digested) amino acids and secreted metabolites in culture medium - werequantified and their degree of 13C-labeling was determined usingGC-MS. Quantification of 13C in metabolites, relative to the amountof 13C-glutamine taken up by cells, allowed us to calculate thedistribution of carbon from glutamine into cellular and extracellularmetabolites (Figure 3A; see also Supplementary Dataset 2).We also estimated the amount of carbon from glutamine converted toCO2, based on quantities and mass isotopomer distributions of13C-labeled metabolites (Figure 3A,3B; see Methods and Figure 1Bfor details). The combination of cellular amino acids, secreted metabolites andCO2 on average accounted for 90% of glutamine usage by melanomacells or melanocytes (Figure 3A). Secretedglutamate generally represented the largest destination for carbon fromglutamine – as much as 35% of the total in WM793 cells. Exceptions weremelanocytes, where 32% of the carbon from glutamine was assimilated into proteinas glutamate or glutamine but only 12% was secreted as glutamate, and UACC903cells, where extracellular glutamate was a minor product of glutamine (5% oftotal glutamine carbon). Consistent with this, UACC903 cells, unlike other celllines, took up glutamate rather than secreting it (Figure 3C).


Glutamate and asparagine cataplerosis underlie glutamine addiction in melanoma.

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

Distribution of carbon from glutamine in melanoma cells andmelanocytes emphasizes role of glutamate secretion(A) Yield of carbon from glutamine in total cellular aminoacids (including protein), in secreted metabolites, and estimatedfraction lost as CO2 after culture of cells for 72 h inmedium containing U-13C-glutamine. Abbreviations: Melcytes:melanocytes; Asp-asn: combined cellular aspartate and asparagine;Glu-gln: combined cellular glutamate and glutamine. (B)Metabolites associated with calculated CO2 production(products of glutaminolysis). CO2 production was estimated asthe sum of “missing” 13C in partially labeledend-point metabolites plus CO2 necessarily lost betweenglutamine and the measured metabolites. Metabolites associated with atleast 5% of estimated CO2 production are shown, with Asp-asnrepresenting combined cellular and secreted aspartate and asparagine,and Glu-gln, combined cellular glutamate and glutamine and secretedglutamate. (C) UACC903 cells show net uptake of glutamate,unlike other melanoma cell lines, which secrete glutamate.(D) Fraction of secreted glutamate of TCA-cycle origin(partially 13C-labeled). For UACC903 cells, there was no netsecretion, but there was exchange of labeled glutamate into the medium(Mean ± SEM of N = 3). Source data forparts (A, B) are shown in Supplementary Dataset 2.
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Figure 3: Distribution of carbon from glutamine in melanoma cells andmelanocytes emphasizes role of glutamate secretion(A) Yield of carbon from glutamine in total cellular aminoacids (including protein), in secreted metabolites, and estimatedfraction lost as CO2 after culture of cells for 72 h inmedium containing U-13C-glutamine. Abbreviations: Melcytes:melanocytes; Asp-asn: combined cellular aspartate and asparagine;Glu-gln: combined cellular glutamate and glutamine. (B)Metabolites associated with calculated CO2 production(products of glutaminolysis). CO2 production was estimated asthe sum of “missing” 13C in partially labeledend-point metabolites plus CO2 necessarily lost betweenglutamine and the measured metabolites. Metabolites associated with atleast 5% of estimated CO2 production are shown, with Asp-asnrepresenting combined cellular and secreted aspartate and asparagine,and Glu-gln, combined cellular glutamate and glutamine and secretedglutamate. (C) UACC903 cells show net uptake of glutamate,unlike other melanoma cell lines, which secrete glutamate.(D) Fraction of secreted glutamate of TCA-cycle origin(partially 13C-labeled). For UACC903 cells, there was no netsecretion, but there was exchange of labeled glutamate into the medium(Mean ± SEM of N = 3). Source data forparts (A, B) are shown in Supplementary Dataset 2.
Mentions: To establish more generally how glutamine is used, we quantified the fate ofcarbon derived from glutamine in melanoma cells or melanocytes. Cells werecultured for 72 h with universally-13C-labeled glutamine and themajor metabolites into which 13C accumulated – total cellular(protein-digested) amino acids and secreted metabolites in culture medium - werequantified and their degree of 13C-labeling was determined usingGC-MS. Quantification of 13C in metabolites, relative to the amountof 13C-glutamine taken up by cells, allowed us to calculate thedistribution of carbon from glutamine into cellular and extracellularmetabolites (Figure 3A; see also Supplementary Dataset 2).We also estimated the amount of carbon from glutamine converted toCO2, based on quantities and mass isotopomer distributions of13C-labeled metabolites (Figure 3A,3B; see Methods and Figure 1Bfor details). The combination of cellular amino acids, secreted metabolites andCO2 on average accounted for 90% of glutamine usage by melanomacells or melanocytes (Figure 3A). Secretedglutamate generally represented the largest destination for carbon fromglutamine – as much as 35% of the total in WM793 cells. Exceptions weremelanocytes, where 32% of the carbon from glutamine was assimilated into proteinas glutamate or glutamine but only 12% was secreted as glutamate, and UACC903cells, where extracellular glutamate was a minor product of glutamine (5% oftotal glutamine carbon). Consistent with this, UACC903 cells, unlike other celllines, took up glutamate rather than secreting it (Figure 3C).

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