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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

Maps of glutamine metabolism(A) Anaplerotic conversion of glutamate to α-ketoglutarate(αKG) is highlighted by green arrows (alternate enzymatic reactions forthis step are contained within the brackets); cataplerotic reactions are shownby red arrows; TCA cycle is within the shaded area. Dashed lines indicatesecretion of metabolites. Genes for key steps are shown: gls1, glutaminase-1;got, glutamate-oxaloacetate transaminase (aspartate aminotransferase); gpt,glutamate-pyruvate transaminase (alanine aminotransferase); glud, glutamatedehydrogenase. Other abbreviations: Pyr, pyruvate; OAA, oxaloacetate; AcCoA,acetyl-coenzyme-A. (B) Using 13C-labeling to quantifyglutaminolysis. Metabolic trafficking from universally 13C-glutamineis indicated by brown arrows, with 13C atoms, including13C lost as CO2, indicated by brown circles, andrecombination with unlabeled (open circles) acetyl-CoA in the TCA cycle. Routesto glutamate, aspartate and lactate are shown, as well as example mass profilesof cellular glutamate and aspartate. The x axis designations of“m+1”, “m+2”, etc., indicatemetabolite mass greater than the unlabeled (no 13C) mass of“m+0”, and together indicate the distribution of13C label. 13C-Glutamine is deamidated to (m+5)glutamate. This is not glutaminolysis, as no energy is produced. But glutamatecan then be converted to α-ketoglutarate and circuit the TCA cycle. Afterone circuit, two carbons are lost as CO2 and replaced by unlabeledacetyl-CoA. This results in m+3 glutamate. Further circuits of the TCAcycle result in exchange of more 13C carbon. Similarly for aspartate,the initial pass through the TCA cycle yields m+4 aspartate (one carbonlost as CO2), and another circuit through the cycle yields m+2aspartate (three carbons lost as CO2). Glutaminolysis can bequantified in terms of CO2 production by comparing the 13Ccontent and quantities of metabolites after 13C-glutamine labelingwith the amount of 13C-glutamine taken up by cells.
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Figure 1: Maps of glutamine metabolism(A) Anaplerotic conversion of glutamate to α-ketoglutarate(αKG) is highlighted by green arrows (alternate enzymatic reactions forthis step are contained within the brackets); cataplerotic reactions are shownby red arrows; TCA cycle is within the shaded area. Dashed lines indicatesecretion of metabolites. Genes for key steps are shown: gls1, glutaminase-1;got, glutamate-oxaloacetate transaminase (aspartate aminotransferase); gpt,glutamate-pyruvate transaminase (alanine aminotransferase); glud, glutamatedehydrogenase. Other abbreviations: Pyr, pyruvate; OAA, oxaloacetate; AcCoA,acetyl-coenzyme-A. (B) Using 13C-labeling to quantifyglutaminolysis. Metabolic trafficking from universally 13C-glutamineis indicated by brown arrows, with 13C atoms, including13C lost as CO2, indicated by brown circles, andrecombination with unlabeled (open circles) acetyl-CoA in the TCA cycle. Routesto glutamate, aspartate and lactate are shown, as well as example mass profilesof cellular glutamate and aspartate. The x axis designations of“m+1”, “m+2”, etc., indicatemetabolite mass greater than the unlabeled (no 13C) mass of“m+0”, and together indicate the distribution of13C label. 13C-Glutamine is deamidated to (m+5)glutamate. This is not glutaminolysis, as no energy is produced. But glutamatecan then be converted to α-ketoglutarate and circuit the TCA cycle. Afterone circuit, two carbons are lost as CO2 and replaced by unlabeledacetyl-CoA. This results in m+3 glutamate. Further circuits of the TCAcycle result in exchange of more 13C carbon. Similarly for aspartate,the initial pass through the TCA cycle yields m+4 aspartate (one carbonlost as CO2), and another circuit through the cycle yields m+2aspartate (three carbons lost as CO2). Glutaminolysis can bequantified in terms of CO2 production by comparing the 13Ccontent and quantities of metabolites after 13C-glutamine labelingwith the amount of 13C-glutamine taken up by cells.

Mentions: A glutamine requirement for the proliferation of many cell types is well established[4, 5]and certain tumor or oncogene-modified cell lines undergo apoptosis when deprived ofglutamine [6–8]. The metabolism of glutamine begins by its conversion to glutamate byglutaminase, or other amidases which have various functions in biosynthetic pathways[9]. Deamination of glutamate yieldsα-ketoglutarate, an intermediate in the TCA cycle, and thereby glutamine acts asan anaplerotic substrate [10], contributing tothe maintenance of pools of carboxylic acids in the TCA cycle and sustaining cellularoxidative phosphorylation (Figure 1A). This use ofglutamine as an energy substrate is known as glutaminolysis, by analogy to glycolysis[11]. Additionally, the carbon contributed tothe TCA cycle by glutamine can be used in biosynthetic reactions [12] (Figure 1A), and theprocesses of glutaminolysis and biosynthesis can run in parallel. Anaplerosis andcataplerosis (green and red arrows respectively in Figure 1A) must be balanced to maintain the TCA cycle in equilibrium [13]. Studies on tumor cells using13C-glutamine have confirmed the use of glutamine as an anaplerotic substrate[3, 14–18], and have outlined someof the cataplerotic roles of carbon derived from glutamine, including use for fatty acidsynthesis under hypoxia [3, 19–21] and exportfrom the mitochondrion as aspartate and generation of NADPH (which maintains cellularredox state) via malic enzyme activity [16]. Itis also well known that tumor or normal cells fed with glutamine will secrete glutamate[22, 23], and the secretion of some partially-13C glutamate from cellslabeled with universally-13C-glutamine, which indicates TCA cycle-origin(Figure 1B), has been reported [15]. Glutamate derived from the TCA cyclecontributes to cataplerosis and therefore provides a potential route for loss of TCAcycle metabolites, a process which has been observed in glutamine-starved cellsundergoing apoptosis [6].


Glutamate and asparagine cataplerosis underlie glutamine addiction in melanoma.

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

Maps of glutamine metabolism(A) Anaplerotic conversion of glutamate to α-ketoglutarate(αKG) is highlighted by green arrows (alternate enzymatic reactions forthis step are contained within the brackets); cataplerotic reactions are shownby red arrows; TCA cycle is within the shaded area. Dashed lines indicatesecretion of metabolites. Genes for key steps are shown: gls1, glutaminase-1;got, glutamate-oxaloacetate transaminase (aspartate aminotransferase); gpt,glutamate-pyruvate transaminase (alanine aminotransferase); glud, glutamatedehydrogenase. Other abbreviations: Pyr, pyruvate; OAA, oxaloacetate; AcCoA,acetyl-coenzyme-A. (B) Using 13C-labeling to quantifyglutaminolysis. Metabolic trafficking from universally 13C-glutamineis indicated by brown arrows, with 13C atoms, including13C lost as CO2, indicated by brown circles, andrecombination with unlabeled (open circles) acetyl-CoA in the TCA cycle. Routesto glutamate, aspartate and lactate are shown, as well as example mass profilesof cellular glutamate and aspartate. The x axis designations of“m+1”, “m+2”, etc., indicatemetabolite mass greater than the unlabeled (no 13C) mass of“m+0”, and together indicate the distribution of13C label. 13C-Glutamine is deamidated to (m+5)glutamate. This is not glutaminolysis, as no energy is produced. But glutamatecan then be converted to α-ketoglutarate and circuit the TCA cycle. Afterone circuit, two carbons are lost as CO2 and replaced by unlabeledacetyl-CoA. This results in m+3 glutamate. Further circuits of the TCAcycle result in exchange of more 13C carbon. Similarly for aspartate,the initial pass through the TCA cycle yields m+4 aspartate (one carbonlost as CO2), and another circuit through the cycle yields m+2aspartate (three carbons lost as CO2). Glutaminolysis can bequantified in terms of CO2 production by comparing the 13Ccontent and quantities of metabolites after 13C-glutamine labelingwith the amount of 13C-glutamine taken up by cells.
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Related In: Results  -  Collection

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Show All Figures
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Figure 1: Maps of glutamine metabolism(A) Anaplerotic conversion of glutamate to α-ketoglutarate(αKG) is highlighted by green arrows (alternate enzymatic reactions forthis step are contained within the brackets); cataplerotic reactions are shownby red arrows; TCA cycle is within the shaded area. Dashed lines indicatesecretion of metabolites. Genes for key steps are shown: gls1, glutaminase-1;got, glutamate-oxaloacetate transaminase (aspartate aminotransferase); gpt,glutamate-pyruvate transaminase (alanine aminotransferase); glud, glutamatedehydrogenase. Other abbreviations: Pyr, pyruvate; OAA, oxaloacetate; AcCoA,acetyl-coenzyme-A. (B) Using 13C-labeling to quantifyglutaminolysis. Metabolic trafficking from universally 13C-glutamineis indicated by brown arrows, with 13C atoms, including13C lost as CO2, indicated by brown circles, andrecombination with unlabeled (open circles) acetyl-CoA in the TCA cycle. Routesto glutamate, aspartate and lactate are shown, as well as example mass profilesof cellular glutamate and aspartate. The x axis designations of“m+1”, “m+2”, etc., indicatemetabolite mass greater than the unlabeled (no 13C) mass of“m+0”, and together indicate the distribution of13C label. 13C-Glutamine is deamidated to (m+5)glutamate. This is not glutaminolysis, as no energy is produced. But glutamatecan then be converted to α-ketoglutarate and circuit the TCA cycle. Afterone circuit, two carbons are lost as CO2 and replaced by unlabeledacetyl-CoA. This results in m+3 glutamate. Further circuits of the TCAcycle result in exchange of more 13C carbon. Similarly for aspartate,the initial pass through the TCA cycle yields m+4 aspartate (one carbonlost as CO2), and another circuit through the cycle yields m+2aspartate (three carbons lost as CO2). Glutaminolysis can bequantified in terms of CO2 production by comparing the 13Ccontent and quantities of metabolites after 13C-glutamine labelingwith the amount of 13C-glutamine taken up by cells.
Mentions: A glutamine requirement for the proliferation of many cell types is well established[4, 5]and certain tumor or oncogene-modified cell lines undergo apoptosis when deprived ofglutamine [6–8]. The metabolism of glutamine begins by its conversion to glutamate byglutaminase, or other amidases which have various functions in biosynthetic pathways[9]. Deamination of glutamate yieldsα-ketoglutarate, an intermediate in the TCA cycle, and thereby glutamine acts asan anaplerotic substrate [10], contributing tothe maintenance of pools of carboxylic acids in the TCA cycle and sustaining cellularoxidative phosphorylation (Figure 1A). This use ofglutamine as an energy substrate is known as glutaminolysis, by analogy to glycolysis[11]. Additionally, the carbon contributed tothe TCA cycle by glutamine can be used in biosynthetic reactions [12] (Figure 1A), and theprocesses of glutaminolysis and biosynthesis can run in parallel. Anaplerosis andcataplerosis (green and red arrows respectively in Figure 1A) must be balanced to maintain the TCA cycle in equilibrium [13]. Studies on tumor cells using13C-glutamine have confirmed the use of glutamine as an anaplerotic substrate[3, 14–18], and have outlined someof the cataplerotic roles of carbon derived from glutamine, including use for fatty acidsynthesis under hypoxia [3, 19–21] and exportfrom the mitochondrion as aspartate and generation of NADPH (which maintains cellularredox state) via malic enzyme activity [16]. Itis also well known that tumor or normal cells fed with glutamine will secrete glutamate[22, 23], and the secretion of some partially-13C glutamate from cellslabeled with universally-13C-glutamine, which indicates TCA cycle-origin(Figure 1B), has been reported [15]. Glutamate derived from the TCA cyclecontributes to cataplerosis and therefore provides a potential route for loss of TCAcycle metabolites, a process which has been observed in glutamine-starved cellsundergoing apoptosis [6].

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