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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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Assaying glycolytic flux and glycolytic capacity.A. Kinetic ECAR response of HeLa cells to glucose (10 mM), oligomycin (2 µM), and 2-DG (100 mM). Insert shows OCR response in response to glucose and oligomycin. HeLa cells were plated at 30,000/well in XF24 V7 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium supplemented with 2 mM glutamine. ECAR or OCR values were not normalized. A representative experiment out of at 5 is shown here. Each data point represents mean ± SD, n = 4. B. Calculated glycolytic flux, glycolytic capacity. Glycolytic flux is the difference between the ECARs of measurement 6 and measurement 3. Likewise, glycolytic capacity describes the difference between the ECAR of measurement 9 and that of measurement 3. * p<0.05.
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pone-0109916-g002: Assaying glycolytic flux and glycolytic capacity.A. Kinetic ECAR response of HeLa cells to glucose (10 mM), oligomycin (2 µM), and 2-DG (100 mM). Insert shows OCR response in response to glucose and oligomycin. HeLa cells were plated at 30,000/well in XF24 V7 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium supplemented with 2 mM glutamine. ECAR or OCR values were not normalized. A representative experiment out of at 5 is shown here. Each data point represents mean ± SD, n = 4. B. Calculated glycolytic flux, glycolytic capacity. Glycolytic flux is the difference between the ECARs of measurement 6 and measurement 3. Likewise, glycolytic capacity describes the difference between the ECAR of measurement 9 and that of measurement 3. * p<0.05.

Mentions: To determine both glycolytic flux and glycolytic capacity of the same cell population in one experiment, we measured ECAR while consecutively injecting glucose, oligomycin, and 2-DG. As shown in Figure 2A, adding glucose to HeLa cells, as expected, triggered a glycolytic flux of 19±0.9 mpH/min (EACR at measurement 6 less that at measurement 3) in HeLa cells. The subsequent addition of oligomycin caused a further increase in ECAR to 44±3.8 mpH/min (ECAR at measurement 9 less that at measurement 3), indicating an elevated glucose flux toward lactate and revealing the glycolytic capacity of HeLa cells. The final addition of glycolysis inhibitor 2-DG abolished the overall glycolysis (Figure 2A). The calculated glycolytic flux and glycolytic capacity from the glycolysis experiment are shown in Figure 2B.


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

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

Assaying glycolytic flux and glycolytic capacity.A. Kinetic ECAR response of HeLa cells to glucose (10 mM), oligomycin (2 µM), and 2-DG (100 mM). Insert shows OCR response in response to glucose and oligomycin. HeLa cells were plated at 30,000/well in XF24 V7 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium supplemented with 2 mM glutamine. ECAR or OCR values were not normalized. A representative experiment out of at 5 is shown here. Each data point represents mean ± SD, n = 4. B. Calculated glycolytic flux, glycolytic capacity. Glycolytic flux is the difference between the ECARs of measurement 6 and measurement 3. Likewise, glycolytic capacity describes the difference between the ECAR of measurement 9 and that of measurement 3. * p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0109916-g002: Assaying glycolytic flux and glycolytic capacity.A. Kinetic ECAR response of HeLa cells to glucose (10 mM), oligomycin (2 µM), and 2-DG (100 mM). Insert shows OCR response in response to glucose and oligomycin. HeLa cells were plated at 30,000/well in XF24 V7 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium supplemented with 2 mM glutamine. ECAR or OCR values were not normalized. A representative experiment out of at 5 is shown here. Each data point represents mean ± SD, n = 4. B. Calculated glycolytic flux, glycolytic capacity. Glycolytic flux is the difference between the ECARs of measurement 6 and measurement 3. Likewise, glycolytic capacity describes the difference between the ECAR of measurement 9 and that of measurement 3. * p<0.05.
Mentions: To determine both glycolytic flux and glycolytic capacity of the same cell population in one experiment, we measured ECAR while consecutively injecting glucose, oligomycin, and 2-DG. As shown in Figure 2A, adding glucose to HeLa cells, as expected, triggered a glycolytic flux of 19±0.9 mpH/min (EACR at measurement 6 less that at measurement 3) in HeLa cells. The subsequent addition of oligomycin caused a further increase in ECAR to 44±3.8 mpH/min (ECAR at measurement 9 less that at measurement 3), indicating an elevated glucose flux toward lactate and revealing the glycolytic capacity of HeLa cells. The final addition of glycolysis inhibitor 2-DG abolished the overall glycolysis (Figure 2A). The calculated glycolytic flux and glycolytic capacity from the glycolysis experiment are shown in Figure 2B.

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