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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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Enhanced glutamine oxidation and activation of fatty acid oxidation in SF188f cells.A. Kinetic OCR response of SF188s and SF188f cells to glutamine (4 mM) (left panel). Insert: calculated glutamine oxidation rate of SF188s and SF188f cells. OCR response (% of baseline) to glutamine and AOA (100 µM) (right panel). SF188s and SF188f cells were plated at 30,000 and 20,000 cells/well, respectively, in XF24 V7 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium. Upon completion of an assay, cells were treated with trypsin and counted for the purpose of normalization. The OCR values were normalized to pmoles/min/104 cells. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4. B and C. OCR response of SF188s and SF188f cells to palmitate-BSA (150 mM) (B) and octanoate (1 mM) (C). SF188s and SF188f cells were plated at 20,000 and 15,000 cells/well, respectively, in XF96 V3 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium supplemented with 5.5 mM glucose and 50 µM carnitine. Fatty acid oxidation was expressed as % OCR plotted using measurement 3 as the baseline. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 6.
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pone-0109916-g008: Enhanced glutamine oxidation and activation of fatty acid oxidation in SF188f cells.A. Kinetic OCR response of SF188s and SF188f cells to glutamine (4 mM) (left panel). Insert: calculated glutamine oxidation rate of SF188s and SF188f cells. OCR response (% of baseline) to glutamine and AOA (100 µM) (right panel). SF188s and SF188f cells were plated at 30,000 and 20,000 cells/well, respectively, in XF24 V7 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium. Upon completion of an assay, cells were treated with trypsin and counted for the purpose of normalization. The OCR values were normalized to pmoles/min/104 cells. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4. B and C. OCR response of SF188s and SF188f cells to palmitate-BSA (150 mM) (B) and octanoate (1 mM) (C). SF188s and SF188f cells were plated at 20,000 and 15,000 cells/well, respectively, in XF96 V3 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium supplemented with 5.5 mM glucose and 50 µM carnitine. Fatty acid oxidation was expressed as % OCR plotted using measurement 3 as the baseline. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 6.

Mentions: Second, we investigated the oxidation of glutamine and fatty acids in the cells. Glutamine oxidation occurred in both, but at a much higher rate in SF188f cells (21±0.3 pmol/min/104 cells) than in SF188s cells (4.6±0.6 pmol/min/104 cells) (Figure 8A). To distinguish the responsible biochemical pathways, we used transaminase inhibitor AOA. As shown in Figure 8A, AOA largely abolished glutamine-stimulated OCR in SF188f cells, but had little effect on SF188s cells. These results suggested that the transaminase pathway in the rapidly dividing SF188f cells was not only activated but became the primary pathway in the oxidation of glutamine. In contrast, the GDH pathway, as inferred from AOA-resistant fraction of glutamine –evoked OCR remained the sole pathway carrying out glutamine oxidation in the slowly-dividing SF188s cells. Similarly, palmitate and octanoate oxidation took place in SF188f cells, but not in the slower-growing SF188s cells (Figure 8B), suggesting SF188f cells had acquired the ability to take up and oxidize both long and medium-chain fatty acids. Collectively, these results indicated that glutamine and fatty acid oxidation was activated as SF188 cells adapted to a nutrient-excessive environment.


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

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

Enhanced glutamine oxidation and activation of fatty acid oxidation in SF188f cells.A. Kinetic OCR response of SF188s and SF188f cells to glutamine (4 mM) (left panel). Insert: calculated glutamine oxidation rate of SF188s and SF188f cells. OCR response (% of baseline) to glutamine and AOA (100 µM) (right panel). SF188s and SF188f cells were plated at 30,000 and 20,000 cells/well, respectively, in XF24 V7 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium. Upon completion of an assay, cells were treated with trypsin and counted for the purpose of normalization. The OCR values were normalized to pmoles/min/104 cells. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4. B and C. OCR response of SF188s and SF188f cells to palmitate-BSA (150 mM) (B) and octanoate (1 mM) (C). SF188s and SF188f cells were plated at 20,000 and 15,000 cells/well, respectively, in XF96 V3 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium supplemented with 5.5 mM glucose and 50 µM carnitine. Fatty acid oxidation was expressed as % OCR plotted using measurement 3 as the baseline. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 6.
© Copyright Policy
Related In: Results  -  Collection

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pone-0109916-g008: Enhanced glutamine oxidation and activation of fatty acid oxidation in SF188f cells.A. Kinetic OCR response of SF188s and SF188f cells to glutamine (4 mM) (left panel). Insert: calculated glutamine oxidation rate of SF188s and SF188f cells. OCR response (% of baseline) to glutamine and AOA (100 µM) (right panel). SF188s and SF188f cells were plated at 30,000 and 20,000 cells/well, respectively, in XF24 V7 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium. Upon completion of an assay, cells were treated with trypsin and counted for the purpose of normalization. The OCR values were normalized to pmoles/min/104 cells. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4. B and C. OCR response of SF188s and SF188f cells to palmitate-BSA (150 mM) (B) and octanoate (1 mM) (C). SF188s and SF188f cells were plated at 20,000 and 15,000 cells/well, respectively, in XF96 V3 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium supplemented with 5.5 mM glucose and 50 µM carnitine. Fatty acid oxidation was expressed as % OCR plotted using measurement 3 as the baseline. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 6.
Mentions: Second, we investigated the oxidation of glutamine and fatty acids in the cells. Glutamine oxidation occurred in both, but at a much higher rate in SF188f cells (21±0.3 pmol/min/104 cells) than in SF188s cells (4.6±0.6 pmol/min/104 cells) (Figure 8A). To distinguish the responsible biochemical pathways, we used transaminase inhibitor AOA. As shown in Figure 8A, AOA largely abolished glutamine-stimulated OCR in SF188f cells, but had little effect on SF188s cells. These results suggested that the transaminase pathway in the rapidly dividing SF188f cells was not only activated but became the primary pathway in the oxidation of glutamine. In contrast, the GDH pathway, as inferred from AOA-resistant fraction of glutamine –evoked OCR remained the sole pathway carrying out glutamine oxidation in the slowly-dividing SF188s cells. Similarly, palmitate and octanoate oxidation took place in SF188f cells, but not in the slower-growing SF188s cells (Figure 8B), suggesting SF188f cells had acquired the ability to take up and oxidize both long and medium-chain fatty acids. Collectively, these results indicated that glutamine and fatty acid oxidation was activated as SF188 cells adapted to a nutrient-excessive environment.

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