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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 glutamine oxidation and demonstrating transaminase pathway activity.A. Schematic illustration of biochemical pathway for glutamine oxidation in the mitochondria. B. Kinetic OCR response in SF188f cells to glutamine (4 mM). SF188f cells were plated at 20,000 cells/well in XF24 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium. The OCR value was not normalized. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4. C. OCR response (% of baseline) in SF188f cells to glutamine (4 mM) and AOA (100 µM). Glutamine-induced OCR reached 60% over the baseline (OCR at measurement 6 divided by that at measurement 3) while AOA addition reduced it to 20% (OCR at Measurement 9 divided by measurement 3). SF188f cells were plated at 20,000 cells/well in XF24 V7 cell culture plates 24–28 hours before the assays. The % OCR was plotted using measurement 3 as the baseline. The assay medium was the substrate-free base medium. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4.
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pone-0109916-g004: Assaying glutamine oxidation and demonstrating transaminase pathway activity.A. Schematic illustration of biochemical pathway for glutamine oxidation in the mitochondria. B. Kinetic OCR response in SF188f cells to glutamine (4 mM). SF188f cells were plated at 20,000 cells/well in XF24 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium. The OCR value was not normalized. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4. C. OCR response (% of baseline) in SF188f cells to glutamine (4 mM) and AOA (100 µM). Glutamine-induced OCR reached 60% over the baseline (OCR at measurement 6 divided by that at measurement 3) while AOA addition reduced it to 20% (OCR at Measurement 9 divided by measurement 3). SF188f cells were plated at 20,000 cells/well in XF24 V7 cell culture plates 24–28 hours before the assays. The % OCR was plotted using measurement 3 as the baseline. The assay medium was the substrate-free base medium. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4.

Mentions: As illustrated in Figure 4A, glutamine enters the mitochondria and is converted to CO2 and H2O in an oxygen-consuming process, which we refer to here as glutamine oxidation. Glutamine can also be partially oxidized to malate via the TCA cycle, which then exits the mitochondria and is converted to pyruvate by malic enzyme in the cytosol, a process known as glutaminolysis. For simplicity, we refer to both processes as glutamine oxidation.


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

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

Assaying glutamine oxidation and demonstrating transaminase pathway activity.A. Schematic illustration of biochemical pathway for glutamine oxidation in the mitochondria. B. Kinetic OCR response in SF188f cells to glutamine (4 mM). SF188f cells were plated at 20,000 cells/well in XF24 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium. The OCR value was not normalized. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4. C. OCR response (% of baseline) in SF188f cells to glutamine (4 mM) and AOA (100 µM). Glutamine-induced OCR reached 60% over the baseline (OCR at measurement 6 divided by that at measurement 3) while AOA addition reduced it to 20% (OCR at Measurement 9 divided by measurement 3). SF188f cells were plated at 20,000 cells/well in XF24 V7 cell culture plates 24–28 hours before the assays. The % OCR was plotted using measurement 3 as the baseline. The assay medium was the substrate-free base medium. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4215881&req=5

pone-0109916-g004: Assaying glutamine oxidation and demonstrating transaminase pathway activity.A. Schematic illustration of biochemical pathway for glutamine oxidation in the mitochondria. B. Kinetic OCR response in SF188f cells to glutamine (4 mM). SF188f cells were plated at 20,000 cells/well in XF24 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium. The OCR value was not normalized. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4. C. OCR response (% of baseline) in SF188f cells to glutamine (4 mM) and AOA (100 µM). Glutamine-induced OCR reached 60% over the baseline (OCR at measurement 6 divided by that at measurement 3) while AOA addition reduced it to 20% (OCR at Measurement 9 divided by measurement 3). SF188f cells were plated at 20,000 cells/well in XF24 V7 cell culture plates 24–28 hours before the assays. The % OCR was plotted using measurement 3 as the baseline. The assay medium was the substrate-free base medium. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4.
Mentions: As illustrated in Figure 4A, glutamine enters the mitochondria and is converted to CO2 and H2O in an oxygen-consuming process, which we refer to here as glutamine oxidation. Glutamine can also be partially oxidized to malate via the TCA cycle, which then exits the mitochondria and is converted to pyruvate by malic enzyme in the cytosol, a process known as glutaminolysis. For simplicity, we refer to both processes as glutamine 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