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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 glucose oxidation.A. Schematic illustration of biochemical pathway for glucose oxidation. The NADH produced in the cytosol as glucose is converted to pyruvate is imported into the mitochondria via the malate-aspartate shuttle and regenerated via the ETC to maintain continuous glucose oxidation. B. Kinetic OCR response of PC-3 cells to glucose (10 mM); C. OCR response to glucose (10 mM), oligomycin (1 µM) and FCCP (0.3 µM). PC-3 cells were plated at 25,000/well in XF24 V7 culture plates. The assay medium was the substrate-free base medium. The OCR values were not normalized. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4.
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pone-0109916-g003: Assaying glucose oxidation.A. Schematic illustration of biochemical pathway for glucose oxidation. The NADH produced in the cytosol as glucose is converted to pyruvate is imported into the mitochondria via the malate-aspartate shuttle and regenerated via the ETC to maintain continuous glucose oxidation. B. Kinetic OCR response of PC-3 cells to glucose (10 mM); C. OCR response to glucose (10 mM), oligomycin (1 µM) and FCCP (0.3 µM). PC-3 cells were plated at 25,000/well in XF24 V7 culture plates. The assay medium was the substrate-free base medium. The OCR values were not normalized. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4.

Mentions: Glucose-derived pyruvate can also enter the mitochondria, where it is converted to acetyl CoA by pyruvate dehydrogenase and enters the TCA cycle via citrate synthase (or as oxaloacetate via pyruvate carboxylase [29]. The acetyl moiety is eventually oxidized to CO2 and H2O (Figure 3A). The oxygen-consuming process of glucose oxidation first to pyruvate and then to CO2 and H2O is referred to here as glucose oxidation.


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

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

Assaying glucose oxidation.A. Schematic illustration of biochemical pathway for glucose oxidation. The NADH produced in the cytosol as glucose is converted to pyruvate is imported into the mitochondria via the malate-aspartate shuttle and regenerated via the ETC to maintain continuous glucose oxidation. B. Kinetic OCR response of PC-3 cells to glucose (10 mM); C. OCR response to glucose (10 mM), oligomycin (1 µM) and FCCP (0.3 µM). PC-3 cells were plated at 25,000/well in XF24 V7 culture plates. The assay medium was the substrate-free base medium. The OCR values were not normalized. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0109916-g003: Assaying glucose oxidation.A. Schematic illustration of biochemical pathway for glucose oxidation. The NADH produced in the cytosol as glucose is converted to pyruvate is imported into the mitochondria via the malate-aspartate shuttle and regenerated via the ETC to maintain continuous glucose oxidation. B. Kinetic OCR response of PC-3 cells to glucose (10 mM); C. OCR response to glucose (10 mM), oligomycin (1 µM) and FCCP (0.3 µM). PC-3 cells were plated at 25,000/well in XF24 V7 culture plates. The assay medium was the substrate-free base medium. The OCR values were not normalized. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4.
Mentions: Glucose-derived pyruvate can also enter the mitochondria, where it is converted to acetyl CoA by pyruvate dehydrogenase and enters the TCA cycle via citrate synthase (or as oxaloacetate via pyruvate carboxylase [29]. The acetyl moiety is eventually oxidized to CO2 and H2O (Figure 3A). The oxygen-consuming process of glucose oxidation first to pyruvate and then to CO2 and H2O is referred to here as glucose oxidation.

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