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Rapid analysis of glycolytic and oxidative substrate flux of cancer cells in a microplate.

Pike Winer LS, Wu M - PLoS ONE (2014)

Bottom Line: Using the XF Extracellular Flux analyzer, these methods measure, in real-time, the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of living cells in a microplate as they respond to substrates and metabolic perturbation agents.In proof-of-principle experiments, we analyzed substrate flux and mitochondrial bioenergetics of two human glioblastoma cell lines, SF188s and SF188f, which were derived from the same parental cell line but proliferate at slow and fast rates, respectively.It is plausible that the proton leak of SF188f cells may play a role in allowing continuous glutamine-fueled anaplerotic TCA cycle flux by partially uncoupling the TCA cycle from oxidative phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Seahorse Bioscience Inc., North Billerica, Massachusetts, United States of America.

ABSTRACT
Cancer cells exhibit remarkable alterations in cellular metabolism, particularly in their nutrient substrate preference. We have devised several experimental methods that rapidly analyze the metabolic substrate flux in cancer cells: glycolysis and the oxidation of major fuel substrates glucose, glutamine, and fatty acids. Using the XF Extracellular Flux analyzer, these methods measure, in real-time, the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of living cells in a microplate as they respond to substrates and metabolic perturbation agents. In proof-of-principle experiments, we analyzed substrate flux and mitochondrial bioenergetics of two human glioblastoma cell lines, SF188s and SF188f, which were derived from the same parental cell line but proliferate at slow and fast rates, respectively. These analyses led to three interesting observations: 1) both cell lines respired effectively with substantial endogenous substrate respiration; 2) SF188f cells underwent a significant shift from glycolytic to oxidative metabolism, along with a high rate of glutamine oxidation relative to SF188s cells; and 3) the mitochondrial proton leak-linked respiration of SF188f cells increased significantly compared to SF188s cells. It is plausible that the proton leak of SF188f cells may play a role in allowing continuous glutamine-fueled anaplerotic TCA cycle flux by partially uncoupling the TCA cycle from oxidative phosphorylation. Taken together, these rapid, sensitive and high-throughput substrate flux analysis methods introduce highly valuable approaches for developing a greater understanding of genetic and epigenetic pathways that regulate cellular metabolism, and the development of therapies that target cancer metabolism.

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Analyzing Glycolytic flux.A. Schematic illustration of the glycolytic pathway. NADH produced in the cytosol as glucose is converted to pyruvate and is regenerated by LDH in the cytosol. B. Kinetic ECAR response of SF188s cells to glucose (10 mM) and 2-DG (100 mM) or oxamate (100 mM), respectively. SF188s cells were plated at 30,000 cells/well in XF24 V7 cell culture plates 24–28 hours prior to the assays. The assay medium was substrate-free base medium (as described in Material and Methods) supplemented with 2 mM glutamine. The ECAR value was not normalized. A representative experiment out of at least three is shown here. Each data point represents mean ± SD, n = 4. C. ECAR response of HeLa cells to glucose (10 mM), 2-DG (100 mM) and antimycin (1 µM). Insert: the OCR response in the same experiments showing the Crabtree effect and that glucose did not increase OCR. HeLa cells were plated at 30,000/well in XF24 cell culture plates 24–28 hours prior to the assays. ECAR or OCR values were not normalized. The assay medium was substrate-free base medium (as described in Material and Methods) supplemented with 2 mM glutamine. A representative experiment out of at least three is shown here. Each data point represents mean ± SD, n = 5.
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pone-0109916-g001: Analyzing Glycolytic flux.A. Schematic illustration of the glycolytic pathway. NADH produced in the cytosol as glucose is converted to pyruvate and is regenerated by LDH in the cytosol. B. Kinetic ECAR response of SF188s cells to glucose (10 mM) and 2-DG (100 mM) or oxamate (100 mM), respectively. SF188s cells were plated at 30,000 cells/well in XF24 V7 cell culture plates 24–28 hours prior to the assays. The assay medium was substrate-free base medium (as described in Material and Methods) supplemented with 2 mM glutamine. The ECAR value was not normalized. A representative experiment out of at least three is shown here. Each data point represents mean ± SD, n = 4. C. ECAR response of HeLa cells to glucose (10 mM), 2-DG (100 mM) and antimycin (1 µM). Insert: the OCR response in the same experiments showing the Crabtree effect and that glucose did not increase OCR. HeLa cells were plated at 30,000/well in XF24 cell culture plates 24–28 hours prior to the assays. ECAR or OCR values were not normalized. The assay medium was substrate-free base medium (as described in Material and Methods) supplemented with 2 mM glutamine. A representative experiment out of at least three is shown here. Each data point represents mean ± SD, n = 5.

Mentions: We have previously shown that glycolysis accounts for ∼80% of total ECAR in a number of cancer cells as determined through two methods: a) removing glucose from the assay medium and b) adding glycolytic pathway inhibitors such as hexokinase inhibitor 2-DG and lactate dehydrogenase (LDH) inhibitor oxamate [22]. The remaining 20% of the ECAR can be attributed to other metabolic processes, such as the TCA cycle CO2 evolution. In order to measure glycolysis using ECAR more accurately and easily, we took the following approach. Glucose is added to cells that are incubated in a glucose-free medium, but supplemented with glutamine (see Materials and Methods). The ECAR increase following the addition of glucose establishes the glycolysis rate. A subsequent addition of a glycolysis inhibitor eliminates the glucose-induced ECAR increase. Any acidification due to other metabolic processes such as the TCA cycle CO2 release (from any substrate but glucose) is detected as ECAR prior to glucose addition. The OCR response to glucose, monitored concurrently with ECAR, serves as an indicator of whether glucose is also catabolized through mitochondrial respiration (Figure 1A).


Rapid analysis of glycolytic and oxidative substrate flux of cancer cells in a microplate.

Pike Winer LS, Wu M - PLoS ONE (2014)

Analyzing Glycolytic flux.A. Schematic illustration of the glycolytic pathway. NADH produced in the cytosol as glucose is converted to pyruvate and is regenerated by LDH in the cytosol. B. Kinetic ECAR response of SF188s cells to glucose (10 mM) and 2-DG (100 mM) or oxamate (100 mM), respectively. SF188s cells were plated at 30,000 cells/well in XF24 V7 cell culture plates 24–28 hours prior to the assays. The assay medium was substrate-free base medium (as described in Material and Methods) supplemented with 2 mM glutamine. The ECAR value was not normalized. A representative experiment out of at least three is shown here. Each data point represents mean ± SD, n = 4. C. ECAR response of HeLa cells to glucose (10 mM), 2-DG (100 mM) and antimycin (1 µM). Insert: the OCR response in the same experiments showing the Crabtree effect and that glucose did not increase OCR. HeLa cells were plated at 30,000/well in XF24 cell culture plates 24–28 hours prior to the assays. ECAR or OCR values were not normalized. The assay medium was substrate-free base medium (as described in Material and Methods) supplemented with 2 mM glutamine. A representative experiment out of at least three is shown here. Each data point represents mean ± SD, n = 5.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4215881&req=5

pone-0109916-g001: Analyzing Glycolytic flux.A. Schematic illustration of the glycolytic pathway. NADH produced in the cytosol as glucose is converted to pyruvate and is regenerated by LDH in the cytosol. B. Kinetic ECAR response of SF188s cells to glucose (10 mM) and 2-DG (100 mM) or oxamate (100 mM), respectively. SF188s cells were plated at 30,000 cells/well in XF24 V7 cell culture plates 24–28 hours prior to the assays. The assay medium was substrate-free base medium (as described in Material and Methods) supplemented with 2 mM glutamine. The ECAR value was not normalized. A representative experiment out of at least three is shown here. Each data point represents mean ± SD, n = 4. C. ECAR response of HeLa cells to glucose (10 mM), 2-DG (100 mM) and antimycin (1 µM). Insert: the OCR response in the same experiments showing the Crabtree effect and that glucose did not increase OCR. HeLa cells were plated at 30,000/well in XF24 cell culture plates 24–28 hours prior to the assays. ECAR or OCR values were not normalized. The assay medium was substrate-free base medium (as described in Material and Methods) supplemented with 2 mM glutamine. A representative experiment out of at least three is shown here. Each data point represents mean ± SD, n = 5.
Mentions: We have previously shown that glycolysis accounts for ∼80% of total ECAR in a number of cancer cells as determined through two methods: a) removing glucose from the assay medium and b) adding glycolytic pathway inhibitors such as hexokinase inhibitor 2-DG and lactate dehydrogenase (LDH) inhibitor oxamate [22]. The remaining 20% of the ECAR can be attributed to other metabolic processes, such as the TCA cycle CO2 evolution. In order to measure glycolysis using ECAR more accurately and easily, we took the following approach. Glucose is added to cells that are incubated in a glucose-free medium, but supplemented with glutamine (see Materials and Methods). The ECAR increase following the addition of glucose establishes the glycolysis rate. A subsequent addition of a glycolysis inhibitor eliminates the glucose-induced ECAR increase. Any acidification due to other metabolic processes such as the TCA cycle CO2 release (from any substrate but glucose) is detected as ECAR prior to glucose addition. The OCR response to glucose, monitored concurrently with ECAR, serves as an indicator of whether glucose is also catabolized through mitochondrial respiration (Figure 1A).

Bottom Line: Using the XF Extracellular Flux analyzer, these methods measure, in real-time, the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of living cells in a microplate as they respond to substrates and metabolic perturbation agents.In proof-of-principle experiments, we analyzed substrate flux and mitochondrial bioenergetics of two human glioblastoma cell lines, SF188s and SF188f, which were derived from the same parental cell line but proliferate at slow and fast rates, respectively.It is plausible that the proton leak of SF188f cells may play a role in allowing continuous glutamine-fueled anaplerotic TCA cycle flux by partially uncoupling the TCA cycle from oxidative phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Seahorse Bioscience Inc., North Billerica, Massachusetts, United States of America.

ABSTRACT
Cancer cells exhibit remarkable alterations in cellular metabolism, particularly in their nutrient substrate preference. We have devised several experimental methods that rapidly analyze the metabolic substrate flux in cancer cells: glycolysis and the oxidation of major fuel substrates glucose, glutamine, and fatty acids. Using the XF Extracellular Flux analyzer, these methods measure, in real-time, the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of living cells in a microplate as they respond to substrates and metabolic perturbation agents. In proof-of-principle experiments, we analyzed substrate flux and mitochondrial bioenergetics of two human glioblastoma cell lines, SF188s and SF188f, which were derived from the same parental cell line but proliferate at slow and fast rates, respectively. These analyses led to three interesting observations: 1) both cell lines respired effectively with substantial endogenous substrate respiration; 2) SF188f cells underwent a significant shift from glycolytic to oxidative metabolism, along with a high rate of glutamine oxidation relative to SF188s cells; and 3) the mitochondrial proton leak-linked respiration of SF188f cells increased significantly compared to SF188s cells. It is plausible that the proton leak of SF188f cells may play a role in allowing continuous glutamine-fueled anaplerotic TCA cycle flux by partially uncoupling the TCA cycle from oxidative phosphorylation. Taken together, these rapid, sensitive and high-throughput substrate flux analysis methods introduce highly valuable approaches for developing a greater understanding of genetic and epigenetic pathways that regulate cellular metabolism, and the development of therapies that target cancer metabolism.

Show MeSH
Related in: MedlinePlus