The most prevalent human diseases in industrialized countries include obesity, type 2 diabetes, cancer, neurodegenerative, and cardiovascular diseases. A common feature of these ailments is dysregulation of cellular energy metabolism. Mitochondria generate 90% of cellular ATP via oxidative phosphorylation and are central to intermediary metabolism, ROS generation, and apoptosis. However, there is a lack of rapid and sensitive assays to measure metabolism in vitro, especially for drug discovery.
To address this, Seahorse Bioscience (www.seahorsebio.com) introduced the XF24 Extracellular Flux Analyzer. This instrument noninvasively quantifies physiological changes in cellular energetics by measuring the two major energy yielding pathways—mitochondrial respiration and glycolysis—in a microplate format. With the analyzer, compounds affecting mitochondrial function and Fatty Acid Oxidation (FAO) can be detected and their EC50 values and kinetics determined.
Extracellular Flux (XF) assays provide comparable performance to biochemical and radioactive methods without the preparation and use of complicated labels or radioactive materials. The XF24 can also be used to detect physiological signaling of receptors, such as GPCRs, ion channels, and receptor tyrosine kinases.
The Seahorse XF24 measures the rate of change of oxygen (Oxygen Consumption Rate, OCR) and proton concentrations (Extracellular Acidification Rate, ECAR) in the media immediately surrounding living cells in a 24-well microplate (Figure 1A). Therefore, a measurement of the analyte fluxes in the media can be used to determine rates of cellular metabolism. Because XF measurements are non-destructive, cells can be profiled over a period of minutes, hours, or days.
The XF24 can simultaneously determine both the oxidative and glycolytic components of cellular bioenergetics in response to metabolic pathway inhibitors (Figure 1B). Untreated A549 cells show a normal basal level of oxidative and glycolytic metabolism. When exposed for 10 minutes to 100 mM of the glycolysis inhibitor 2-deoxyglucose, the cells shift to an almost exclusive oxidative metabolism. Conversely, when incubated for 30 minutes in 1µM of the Complex I inhibitor, rotenone, the cells shift to glycolytic energy production. Lastly, incubation with both 2-DG and rotenone results in a precipitous drop in activity for both energy yielding pathways.
Assessing Mitochondrial Function
In the mitochondria, molecular oxygen is the final electron acceptor for the electrochemical gradient that provides the energy for coupled ATP synthesis. Thus, quantifying oxygen consumption is a direct physiological measurement of mitochondrial oxidative phosphorylation.
Overnight, cultures of HepG2 cells were exposed to increasing concentrations of FCCP (Figure 2), an uncoupler of oxidative phosphorylation, that was injected automatically during the experiment. Plotting the OCR and ECAR responses together produces a bioenergetic chart, or “Power Grid”, indicative of both mitochondrial respiration and glycolysis, respectively. By injecting, in sequence, three dose series of FCCP, the minimum and maximal responses were determined.
The impact of two agonists on FAO, Metformin and an Acetyl CoA carboxylase (ACC) inhibitor, were determined in the murine skeletal muscle cell line C2C12 (Figure 3A). C2C12 myocytes were differentiated for 6 days in Seahorse 24-well microplates and then preincubated for 18 hours with either 10 µM ACC inhibitor or 1 mM Metformin.
By using the XF24 Analyzer, three baseline measurements of OCR and ECAR were made to establish the basal metabolic rates and pathways. Then palmitate complexed to fatty acid-free BSA was injected, and readings were taken to detect a shift toward aerobic respiration, indicative of FAO. Overnight treatment with 10 µM ACC inhibitor or 1 mM Metformin caused 4.6-fold and 3.2-fold increases in OCR, respectively (Figure 3A).
XF and Radiometric Assays
Radiometric FAO assays measure the quantity of radiolabeled CO2 produced by the metabolism of fatty acids, such as Palmitate or Oleate containing 14C, or the quantity of radioactive water produced by the metabolism of these substrates labeled with 3H. To compare the data quality and relative sensitivities of XF and radiometric assays, the induction and inhibition of FAO in parallel C2C12 myoctes cultures was measured (Figure 3B). FAO was induced by adding exogenous palmitate and inhibited by subsequently adding the CPT-1 inhibitor, Etoxomir.
Both XF24 and Radiometric assays observed a significant and comparable inhibition of FAO in the presence of Etomoxir. However, in contrast to traditional radiometric assays, XF assays do not require radioactive materials, strong acids and alkalines, long assay times, or scintillation counting steps. Also, cells may be measured multiple times to profile kinetics of FAO induction and can be re-used after testing.
Agonists of FAO may have clinical potential to reduce co-morbidity factors associated with metabolic syndrome. C2C12 myocytes pretreated overnight with increasing doses of an ACC inhibitor caused a dose-dependent increase in FAO as measured by an escalating increase in OCR (Figure 3C). To confirm that OCR was indicative of the oxidation of fatty acids as compared to glucose or amino acids, Etomoxir was added at the end, resulting in a greater than 90% reduction in OCR.
This FAO assay has been performed in both primary and immortalized cells derived from muscle, liver, adipose, and heart.
Detection of GPCR Signaling
Since 1990, experiments with the microphysiometer have established that many cellular receptors, in response to ligand or agonist, cause the release of protons into the media from the cells expressing them. These protons acidify the media causing an increase in ECAR. Although the literature demonstrates this principle almost exclusively in cells engineered to overexpress receptors of interest, the XF24 has the ability to detect GPCR signaling in native receptors as well (Figure 3D).
It turns out that the signaling of many GPCRs, ion channels, and receptor tyrosine kinases are detectable by proton flux, and this is especially valuable for those receptors that do not signal via calcium or cAMP and hence are not detectable by other common technologies.