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Rhabdomyosarcoma cells show an energy producing anabolic metabolic phenotype compared with primary myocytes.

Fan TW, Kucia M, Jankowski K, Higashi RM, Ratajczak J, Ratajczak MZ, Lane AN - Mol. Cancer (2008)

Bottom Line: Large differences were also evident in de novo biosynthesis of riboses in the free nucleotide pools, as well as entry of glucose carbon into the pyrimidine rings in the free nucleotide pool.The myocytes showed evidence of de novo synthesis of glycogen, which was absent in the Rh30 cells.The data further show that the mitochondria remain functional in Krebs' cycle activity and respiratory electron transfer that enables continued accelerated glycolysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, University of Louisville, KY, USA. twmfan@gmail.com

ABSTRACT

Background: The functional status of a cell is expressed in its metabolic activity. We have applied stable isotope tracing methods to determine the differences in metabolic pathways in proliferating Rhabdomysarcoma cells (Rh30) and human primary myocytes in culture. Uniformly 13C-labeled glucose was used as a source molecule to follow the incorporation of 13C into more than 40 marker metabolites using NMR and GC-MS. These include metabolites that report on the activity of glycolysis, Krebs' cycle, pentose phosphate pathway and pyrimidine biosynthesis.

Results: The Rh30 cells proliferated faster than the myocytes. Major differences in flux through glycolysis were evident from incorporation of label into secreted lactate, which accounts for a substantial fraction of the glucose carbon utilized by the cells. Krebs' cycle activity as determined by 13C isotopomer distributions in glutamate, aspartate, malate and pyrimidine rings was considerably higher in the cancer cells than in the primary myocytes. Large differences were also evident in de novo biosynthesis of riboses in the free nucleotide pools, as well as entry of glucose carbon into the pyrimidine rings in the free nucleotide pool. Specific labeling patterns in these metabolites show the increased importance of anaplerotic reactions in the cancer cells to maintain the high demand for anabolic and energy metabolism compared with the slower growing primary myocytes. Serum-stimulated Rh30 cells showed higher degrees of labeling than serum starved cells, but they retained their characteristic anabolic metabolism profile. The myocytes showed evidence of de novo synthesis of glycogen, which was absent in the Rh30 cells.

Conclusion: The specific 13C isotopomer patterns showed that the major difference between the transformed and the primary cells is the shift from energy and maintenance metabolism in the myocytes toward increased energy and anabolic metabolism for proliferation in the Rh30 cells. The data further show that the mitochondria remain functional in Krebs' cycle activity and respiratory electron transfer that enables continued accelerated glycolysis. This may be a common adaptive strategy in cancer cells.

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Related in: MedlinePlus

Metabolic scheme. Carbon flow from [U-13C]-glucose through glycolysis, Krebs cycle, PPP, pyruvate carboxylation, malate/Asp shuttle, and synthesis of GSH and pyrimidine nucleotides as discussed in the text. Also shown are the expected 13C isotopomers of key metabolites in this network. Excretion of lactate and Ala into serum is part of the alanine and Cori cycles. Solid oval represents the plasma membrane and the dashed oval represents the mitochondrial space. Double-headed arrows: exchange or reversible processes. The label patterns arise from [U-13C]-glucose. Open circles: 12C; filled circles: 13C. Glc = glucose, G6P = glucose-6-phosphate, Rib = nucleotide ribose, DHAP, GAP = dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, PEP = phosphoenolpyruvate, Pyr = pyruvate, Lac = lactate, Cit = citrate, 2 = OG = 2-oxoglutarate, Mal = malate, OAA = oxalacetate, U = uracil base. Critical enzymes are shown in cyan. HK= hexokinase (entry to glycolysis), G6PDH = glucose-6-phosphate dehydrogenase (entry to the oxidative branch of the pentose phosphate pathway), TK, TA transketolase and transaldolase (non-oxidative pentose phosphate pathway), PK = pyruvate kinase, PDH = pyruvate dehydrogenase, PC = pyruvate carboxylase, AAT = aspartate amino transferase, MDH = malate dehydrogenase, ME = malic enzyme. Glutaminolysis is the pathway from Gln to pyruvate via ME, leading to unlabeled malate, Asp, Ala and lactate. Where two patterns are shown in the Krebs' cycle intermediates, this is due to the scrambling at the succinate step (first turn only). For U and OAA, the labeling from PC activity gives rise to a third labeling pattern, shown in red. The isotopomer pattern for U shows the three ring carbons (4,5,6) derived from Asp. The exchange of [13C-1,2,3]-Asp via the malate/Asp shuttle into the cytosol leads to the synthesis of [13C-1,2,3]-malate.
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Figure 1: Metabolic scheme. Carbon flow from [U-13C]-glucose through glycolysis, Krebs cycle, PPP, pyruvate carboxylation, malate/Asp shuttle, and synthesis of GSH and pyrimidine nucleotides as discussed in the text. Also shown are the expected 13C isotopomers of key metabolites in this network. Excretion of lactate and Ala into serum is part of the alanine and Cori cycles. Solid oval represents the plasma membrane and the dashed oval represents the mitochondrial space. Double-headed arrows: exchange or reversible processes. The label patterns arise from [U-13C]-glucose. Open circles: 12C; filled circles: 13C. Glc = glucose, G6P = glucose-6-phosphate, Rib = nucleotide ribose, DHAP, GAP = dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, PEP = phosphoenolpyruvate, Pyr = pyruvate, Lac = lactate, Cit = citrate, 2 = OG = 2-oxoglutarate, Mal = malate, OAA = oxalacetate, U = uracil base. Critical enzymes are shown in cyan. HK= hexokinase (entry to glycolysis), G6PDH = glucose-6-phosphate dehydrogenase (entry to the oxidative branch of the pentose phosphate pathway), TK, TA transketolase and transaldolase (non-oxidative pentose phosphate pathway), PK = pyruvate kinase, PDH = pyruvate dehydrogenase, PC = pyruvate carboxylase, AAT = aspartate amino transferase, MDH = malate dehydrogenase, ME = malic enzyme. Glutaminolysis is the pathway from Gln to pyruvate via ME, leading to unlabeled malate, Asp, Ala and lactate. Where two patterns are shown in the Krebs' cycle intermediates, this is due to the scrambling at the succinate step (first turn only). For U and OAA, the labeling from PC activity gives rise to a third labeling pattern, shown in red. The isotopomer pattern for U shows the three ring carbons (4,5,6) derived from Asp. The exchange of [13C-1,2,3]-Asp via the malate/Asp shuttle into the cytosol leads to the synthesis of [13C-1,2,3]-malate.

Mentions: Skeletal muscle uses both glycolysis and oxidative phosphorylation for providing energy for contraction. Under conditions of hard exercise, glycogen stores are rapidly depleted, and oxidative phosphorylation of fuels such as fatty acids cannot keep up with demand. The muscle resorts to a greatly increased rate of glycolysis (> 100 fold [18]) and the production of lactate. Lactate, and to some extent Ala, is exported from the myocytes into the blood, when they are transported the liver for resynthesis into glucose via gluconeogenesis (cf. Figure 1). These comprise the Cori and alanine cycles [19,20]. Thus, myocytes have the capacity to make and secrete large amounts of lactate. The secretion of lactate via the monocarboxylate transporter is often accompanied by a proton symport [21,22]. Moreover, most cells can actively export protons via various exchangers, such as the H+/Na+ antiporter [23], Many cancer cells pump prodigious quantities of H+ into the extracellular environment, which may offer survival advantages, particularly in the light of their accelerated glycolysis [24,25].


Rhabdomyosarcoma cells show an energy producing anabolic metabolic phenotype compared with primary myocytes.

Fan TW, Kucia M, Jankowski K, Higashi RM, Ratajczak J, Ratajczak MZ, Lane AN - Mol. Cancer (2008)

Metabolic scheme. Carbon flow from [U-13C]-glucose through glycolysis, Krebs cycle, PPP, pyruvate carboxylation, malate/Asp shuttle, and synthesis of GSH and pyrimidine nucleotides as discussed in the text. Also shown are the expected 13C isotopomers of key metabolites in this network. Excretion of lactate and Ala into serum is part of the alanine and Cori cycles. Solid oval represents the plasma membrane and the dashed oval represents the mitochondrial space. Double-headed arrows: exchange or reversible processes. The label patterns arise from [U-13C]-glucose. Open circles: 12C; filled circles: 13C. Glc = glucose, G6P = glucose-6-phosphate, Rib = nucleotide ribose, DHAP, GAP = dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, PEP = phosphoenolpyruvate, Pyr = pyruvate, Lac = lactate, Cit = citrate, 2 = OG = 2-oxoglutarate, Mal = malate, OAA = oxalacetate, U = uracil base. Critical enzymes are shown in cyan. HK= hexokinase (entry to glycolysis), G6PDH = glucose-6-phosphate dehydrogenase (entry to the oxidative branch of the pentose phosphate pathway), TK, TA transketolase and transaldolase (non-oxidative pentose phosphate pathway), PK = pyruvate kinase, PDH = pyruvate dehydrogenase, PC = pyruvate carboxylase, AAT = aspartate amino transferase, MDH = malate dehydrogenase, ME = malic enzyme. Glutaminolysis is the pathway from Gln to pyruvate via ME, leading to unlabeled malate, Asp, Ala and lactate. Where two patterns are shown in the Krebs' cycle intermediates, this is due to the scrambling at the succinate step (first turn only). For U and OAA, the labeling from PC activity gives rise to a third labeling pattern, shown in red. The isotopomer pattern for U shows the three ring carbons (4,5,6) derived from Asp. The exchange of [13C-1,2,3]-Asp via the malate/Asp shuttle into the cytosol leads to the synthesis of [13C-1,2,3]-malate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2577687&req=5

Figure 1: Metabolic scheme. Carbon flow from [U-13C]-glucose through glycolysis, Krebs cycle, PPP, pyruvate carboxylation, malate/Asp shuttle, and synthesis of GSH and pyrimidine nucleotides as discussed in the text. Also shown are the expected 13C isotopomers of key metabolites in this network. Excretion of lactate and Ala into serum is part of the alanine and Cori cycles. Solid oval represents the plasma membrane and the dashed oval represents the mitochondrial space. Double-headed arrows: exchange or reversible processes. The label patterns arise from [U-13C]-glucose. Open circles: 12C; filled circles: 13C. Glc = glucose, G6P = glucose-6-phosphate, Rib = nucleotide ribose, DHAP, GAP = dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, PEP = phosphoenolpyruvate, Pyr = pyruvate, Lac = lactate, Cit = citrate, 2 = OG = 2-oxoglutarate, Mal = malate, OAA = oxalacetate, U = uracil base. Critical enzymes are shown in cyan. HK= hexokinase (entry to glycolysis), G6PDH = glucose-6-phosphate dehydrogenase (entry to the oxidative branch of the pentose phosphate pathway), TK, TA transketolase and transaldolase (non-oxidative pentose phosphate pathway), PK = pyruvate kinase, PDH = pyruvate dehydrogenase, PC = pyruvate carboxylase, AAT = aspartate amino transferase, MDH = malate dehydrogenase, ME = malic enzyme. Glutaminolysis is the pathway from Gln to pyruvate via ME, leading to unlabeled malate, Asp, Ala and lactate. Where two patterns are shown in the Krebs' cycle intermediates, this is due to the scrambling at the succinate step (first turn only). For U and OAA, the labeling from PC activity gives rise to a third labeling pattern, shown in red. The isotopomer pattern for U shows the three ring carbons (4,5,6) derived from Asp. The exchange of [13C-1,2,3]-Asp via the malate/Asp shuttle into the cytosol leads to the synthesis of [13C-1,2,3]-malate.
Mentions: Skeletal muscle uses both glycolysis and oxidative phosphorylation for providing energy for contraction. Under conditions of hard exercise, glycogen stores are rapidly depleted, and oxidative phosphorylation of fuels such as fatty acids cannot keep up with demand. The muscle resorts to a greatly increased rate of glycolysis (> 100 fold [18]) and the production of lactate. Lactate, and to some extent Ala, is exported from the myocytes into the blood, when they are transported the liver for resynthesis into glucose via gluconeogenesis (cf. Figure 1). These comprise the Cori and alanine cycles [19,20]. Thus, myocytes have the capacity to make and secrete large amounts of lactate. The secretion of lactate via the monocarboxylate transporter is often accompanied by a proton symport [21,22]. Moreover, most cells can actively export protons via various exchangers, such as the H+/Na+ antiporter [23], Many cancer cells pump prodigious quantities of H+ into the extracellular environment, which may offer survival advantages, particularly in the light of their accelerated glycolysis [24,25].

Bottom Line: Large differences were also evident in de novo biosynthesis of riboses in the free nucleotide pools, as well as entry of glucose carbon into the pyrimidine rings in the free nucleotide pool.The myocytes showed evidence of de novo synthesis of glycogen, which was absent in the Rh30 cells.The data further show that the mitochondria remain functional in Krebs' cycle activity and respiratory electron transfer that enables continued accelerated glycolysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, University of Louisville, KY, USA. twmfan@gmail.com

ABSTRACT

Background: The functional status of a cell is expressed in its metabolic activity. We have applied stable isotope tracing methods to determine the differences in metabolic pathways in proliferating Rhabdomysarcoma cells (Rh30) and human primary myocytes in culture. Uniformly 13C-labeled glucose was used as a source molecule to follow the incorporation of 13C into more than 40 marker metabolites using NMR and GC-MS. These include metabolites that report on the activity of glycolysis, Krebs' cycle, pentose phosphate pathway and pyrimidine biosynthesis.

Results: The Rh30 cells proliferated faster than the myocytes. Major differences in flux through glycolysis were evident from incorporation of label into secreted lactate, which accounts for a substantial fraction of the glucose carbon utilized by the cells. Krebs' cycle activity as determined by 13C isotopomer distributions in glutamate, aspartate, malate and pyrimidine rings was considerably higher in the cancer cells than in the primary myocytes. Large differences were also evident in de novo biosynthesis of riboses in the free nucleotide pools, as well as entry of glucose carbon into the pyrimidine rings in the free nucleotide pool. Specific labeling patterns in these metabolites show the increased importance of anaplerotic reactions in the cancer cells to maintain the high demand for anabolic and energy metabolism compared with the slower growing primary myocytes. Serum-stimulated Rh30 cells showed higher degrees of labeling than serum starved cells, but they retained their characteristic anabolic metabolism profile. The myocytes showed evidence of de novo synthesis of glycogen, which was absent in the Rh30 cells.

Conclusion: The specific 13C isotopomer patterns showed that the major difference between the transformed and the primary cells is the shift from energy and maintenance metabolism in the myocytes toward increased energy and anabolic metabolism for proliferation in the Rh30 cells. The data further show that the mitochondria remain functional in Krebs' cycle activity and respiratory electron transfer that enables continued accelerated glycolysis. This may be a common adaptive strategy in cancer cells.

Show MeSH
Related in: MedlinePlus