Limits...
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.

Show MeSH

Related in: MedlinePlus

Schematic illustration of cellular metabolism pathways along with assays of glucose, glutamine, and fatty acid oxidation, glycolytic flux and mitochondrial bioenergetics.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4215881&req=5

pone-0109916-g010: Schematic illustration of cellular metabolism pathways along with assays of glucose, glutamine, and fatty acid oxidation, glycolytic flux and mitochondrial bioenergetics.

Mentions: By measuring OCR and ECAR responses of living cells to the addition of substrates along with metabolic perturbation agents, the methods presented here have cleared a path for easy and systematic analyses of metabolic substrate flux in wide range of cancer cell lines (Figure 10). These microplate-based substrate flux methods use only small amounts of biological materials, making it possible to analyze large numbers of natural, genetically altered, or compound-treated cancer or non-cancer cell samples in a short period of time. The approach is highly valuable, particularly when integrated with genomic, proteomic, and metabolomics technologies with the purpose of understanding of genetic, epigenetic, and signaling pathways that regulate cancer cell metabolism and cancer cells' metabolic vulnerabilities. Given the growing appreciation of metabolic shifts and nutrient substrate switches in many other diseases, including cardiovascular, neurological and immune ailments, these new methods are also very useful for investigating pathobiology in a wide variety of disease models.


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

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

Schematic illustration of cellular metabolism pathways along with assays of glucose, glutamine, and fatty acid oxidation, glycolytic flux and mitochondrial bioenergetics.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0109916-g010: Schematic illustration of cellular metabolism pathways along with assays of glucose, glutamine, and fatty acid oxidation, glycolytic flux and mitochondrial bioenergetics.
Mentions: By measuring OCR and ECAR responses of living cells to the addition of substrates along with metabolic perturbation agents, the methods presented here have cleared a path for easy and systematic analyses of metabolic substrate flux in wide range of cancer cell lines (Figure 10). These microplate-based substrate flux methods use only small amounts of biological materials, making it possible to analyze large numbers of natural, genetically altered, or compound-treated cancer or non-cancer cell samples in a short period of time. The approach is highly valuable, particularly when integrated with genomic, proteomic, and metabolomics technologies with the purpose of understanding of genetic, epigenetic, and signaling pathways that regulate cancer cell metabolism and cancer cells' metabolic vulnerabilities. Given the growing appreciation of metabolic shifts and nutrient substrate switches in many other diseases, including cardiovascular, neurological and immune ailments, these new methods are also very useful for investigating pathobiology in a wide variety of disease models.

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