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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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Determining fatty acid oxidation.A. Schematic illustration of biochemical pathway of fatty acid oxidation. B. Kinetic OCR response of SF188f cells to palmitate (150 µM). C. Kinetic OCR response in SF188f cells to octanoate (1 mM). SF188f cells were plated at 15,000 cells/well in XF96 V3 cell culture plates 24–28 hours prior to the assays. Assay medium was the substrate-free base medium supplemented with 5.5 mM glucose and 50 µM carnitine. The OCR value was not normalized. A representative experiment out of four is shown here. Each data point represents mean ± SD, n = 6.
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pone-0109916-g005: Determining fatty acid oxidation.A. Schematic illustration of biochemical pathway of fatty acid oxidation. B. Kinetic OCR response of SF188f cells to palmitate (150 µM). C. Kinetic OCR response in SF188f cells to octanoate (1 mM). SF188f cells were plated at 15,000 cells/well in XF96 V3 cell culture plates 24–28 hours prior to the assays. Assay medium was the substrate-free base medium supplemented with 5.5 mM glucose and 50 µM carnitine. The OCR value was not normalized. A representative experiment out of four is shown here. Each data point represents mean ± SD, n = 6.

Mentions: Fatty acids are an important energy source for meeting a high energy demand and maintaining cellular functions in many cells, such as skeletal and cardio muscle cells. Fatty acid oxidation in cancer cells has also been reported but its relevance remains to be fully understood [30]–[34]. Exogenous fatty acids are taken up by cells, converted to fatty acyl CoA in the cytosol, then converted to acetyl CoA in the mitochondria via β–oxidation and ultimately broken down to CO2 and H2O (Figure 5A). The process of biochemical conversion of fatty acids to CO2 and H2O consuming oxygen is referred to here as fatty acid oxidation (FAO).


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

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

Determining fatty acid oxidation.A. Schematic illustration of biochemical pathway of fatty acid oxidation. B. Kinetic OCR response of SF188f cells to palmitate (150 µM). C. Kinetic OCR response in SF188f cells to octanoate (1 mM). SF188f cells were plated at 15,000 cells/well in XF96 V3 cell culture plates 24–28 hours prior to the assays. Assay medium was the substrate-free base medium supplemented with 5.5 mM glucose and 50 µM carnitine. The OCR value was not normalized. A representative experiment out of four is shown here. Each data point represents mean ± SD, n = 6.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0109916-g005: Determining fatty acid oxidation.A. Schematic illustration of biochemical pathway of fatty acid oxidation. B. Kinetic OCR response of SF188f cells to palmitate (150 µM). C. Kinetic OCR response in SF188f cells to octanoate (1 mM). SF188f cells were plated at 15,000 cells/well in XF96 V3 cell culture plates 24–28 hours prior to the assays. Assay medium was the substrate-free base medium supplemented with 5.5 mM glucose and 50 µM carnitine. The OCR value was not normalized. A representative experiment out of four is shown here. Each data point represents mean ± SD, n = 6.
Mentions: Fatty acids are an important energy source for meeting a high energy demand and maintaining cellular functions in many cells, such as skeletal and cardio muscle cells. Fatty acid oxidation in cancer cells has also been reported but its relevance remains to be fully understood [30]–[34]. Exogenous fatty acids are taken up by cells, converted to fatty acyl CoA in the cytosol, then converted to acetyl CoA in the mitochondria via β–oxidation and ultimately broken down to CO2 and H2O (Figure 5A). The process of biochemical conversion of fatty acids to CO2 and H2O consuming oxygen is referred to here as fatty acid oxidation (FAO).

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