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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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Markedly reprogrammed mitochondrial bioenergetic machinery of SF188s and SF188f cells.A. Kinetic OCR response of SF188s and SF188f cells to oligomycin (1 and 2 µM respectively) to determine ATP coupled respiration, FCCP (0.3 µM) to establish maximal respiratory capacity, and myxothiazol (1 µM) and rotenone (1 µM) cocktail) to define mitochondrial respiration (left). Calculated ATP-coupled respiration, proton leak-linked respiration, maximal mitochondrial respiratory capacity (right). 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 supplemented with 25 mM glucose, 6 mM glutamine and 1 mM pyruvate. Upon completion of an assay, cells were treated with trypsin and counted for the purpose of normalization. OCR values were normalized to pmoles/104 cells. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 3. *p<0.05. B. Kinetic OCR response (% of baseline, baseline = measurement 3) of SF188f (right) and SF188s cell (left) with distribution of ATP-coupled respiration (% of oligomycin-sensitive at measurement 4) and proton leak-linked respiration (oligomycin-resistant at measurement 4 but myxothiazol-sensitive mitochondrial OCR at measurement 12) and non-mitochondria respiration (myxothiazol-resistant OCR at measurement 12).
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pone-0109916-g009: Markedly reprogrammed mitochondrial bioenergetic machinery of SF188s and SF188f cells.A. Kinetic OCR response of SF188s and SF188f cells to oligomycin (1 and 2 µM respectively) to determine ATP coupled respiration, FCCP (0.3 µM) to establish maximal respiratory capacity, and myxothiazol (1 µM) and rotenone (1 µM) cocktail) to define mitochondrial respiration (left). Calculated ATP-coupled respiration, proton leak-linked respiration, maximal mitochondrial respiratory capacity (right). 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 supplemented with 25 mM glucose, 6 mM glutamine and 1 mM pyruvate. Upon completion of an assay, cells were treated with trypsin and counted for the purpose of normalization. OCR values were normalized to pmoles/104 cells. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 3. *p<0.05. B. Kinetic OCR response (% of baseline, baseline = measurement 3) of SF188f (right) and SF188s cell (left) with distribution of ATP-coupled respiration (% of oligomycin-sensitive at measurement 4) and proton leak-linked respiration (oligomycin-resistant at measurement 4 but myxothiazol-sensitive mitochondrial OCR at measurement 12) and non-mitochondria respiration (myxothiazol-resistant OCR at measurement 12).

Mentions: Having established differential oxidation of glutamine and fatty acids in SF188s and SF188f cells, we proceeded to investigate the mitochondrial bioenergetics machinery [36], [37] by consecutively adding oligomycin, FCCP, and complex III inhibitor myxothiazol in the presence of saturating amounts of a full set of energy substrates (25 mM glucose, 6 mM glutamine and 1 mM pyruvate for both cell lines). As shown in Figure 9, the mitochondrial bioenergetic state of SF188f differed strikingly from that of SF188s. First of all, in addition to a higher basal OCR, the mitochondrial respiratory capacity (determined by FCCP-stimulated OCR, Figure 9A)) was much higher while the fraction of basal OCR contributing to ATP-coupled respiration (revealed by oligomycin-sensitive OCR) in SF188f cells was greatly attenuated (Figure 9B). Intriguingly, proton leak (the difference between oligomycin-resistant but myxothiazol-sensitive OCR) in SF188f was markedly increased to 48% of the baseline OCR compared with 17% in SF188s cells (Figure 9B). These results suggested that basal respiration in SF188f cells was largely uncoupled from phosphorylation of ADP to ATP.


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

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

Markedly reprogrammed mitochondrial bioenergetic machinery of SF188s and SF188f cells.A. Kinetic OCR response of SF188s and SF188f cells to oligomycin (1 and 2 µM respectively) to determine ATP coupled respiration, FCCP (0.3 µM) to establish maximal respiratory capacity, and myxothiazol (1 µM) and rotenone (1 µM) cocktail) to define mitochondrial respiration (left). Calculated ATP-coupled respiration, proton leak-linked respiration, maximal mitochondrial respiratory capacity (right). 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 supplemented with 25 mM glucose, 6 mM glutamine and 1 mM pyruvate. Upon completion of an assay, cells were treated with trypsin and counted for the purpose of normalization. OCR values were normalized to pmoles/104 cells. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 3. *p<0.05. B. Kinetic OCR response (% of baseline, baseline = measurement 3) of SF188f (right) and SF188s cell (left) with distribution of ATP-coupled respiration (% of oligomycin-sensitive at measurement 4) and proton leak-linked respiration (oligomycin-resistant at measurement 4 but myxothiazol-sensitive mitochondrial OCR at measurement 12) and non-mitochondria respiration (myxothiazol-resistant OCR at measurement 12).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4215881&req=5

pone-0109916-g009: Markedly reprogrammed mitochondrial bioenergetic machinery of SF188s and SF188f cells.A. Kinetic OCR response of SF188s and SF188f cells to oligomycin (1 and 2 µM respectively) to determine ATP coupled respiration, FCCP (0.3 µM) to establish maximal respiratory capacity, and myxothiazol (1 µM) and rotenone (1 µM) cocktail) to define mitochondrial respiration (left). Calculated ATP-coupled respiration, proton leak-linked respiration, maximal mitochondrial respiratory capacity (right). 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 supplemented with 25 mM glucose, 6 mM glutamine and 1 mM pyruvate. Upon completion of an assay, cells were treated with trypsin and counted for the purpose of normalization. OCR values were normalized to pmoles/104 cells. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 3. *p<0.05. B. Kinetic OCR response (% of baseline, baseline = measurement 3) of SF188f (right) and SF188s cell (left) with distribution of ATP-coupled respiration (% of oligomycin-sensitive at measurement 4) and proton leak-linked respiration (oligomycin-resistant at measurement 4 but myxothiazol-sensitive mitochondrial OCR at measurement 12) and non-mitochondria respiration (myxothiazol-resistant OCR at measurement 12).
Mentions: Having established differential oxidation of glutamine and fatty acids in SF188s and SF188f cells, we proceeded to investigate the mitochondrial bioenergetics machinery [36], [37] by consecutively adding oligomycin, FCCP, and complex III inhibitor myxothiazol in the presence of saturating amounts of a full set of energy substrates (25 mM glucose, 6 mM glutamine and 1 mM pyruvate for both cell lines). As shown in Figure 9, the mitochondrial bioenergetic state of SF188f differed strikingly from that of SF188s. First of all, in addition to a higher basal OCR, the mitochondrial respiratory capacity (determined by FCCP-stimulated OCR, Figure 9A)) was much higher while the fraction of basal OCR contributing to ATP-coupled respiration (revealed by oligomycin-sensitive OCR) in SF188f cells was greatly attenuated (Figure 9B). Intriguingly, proton leak (the difference between oligomycin-resistant but myxothiazol-sensitive OCR) in SF188f was markedly increased to 48% of the baseline OCR compared with 17% in SF188s cells (Figure 9B). These results suggested that basal respiration in SF188f cells was largely uncoupled from phosphorylation of ADP to ATP.

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