Measuring fatty acid oxidation in muscle cells

A sensitive non-radiometric real-time assay of FAO

For fatty acid metabolism analyses, extracellular flux assays (XF) enable higher throughput, allow use of reduced sample sizes, eliminate the need for radioactive compounds and provide kinetic resolution rates that can be typically obtained within several minutes.

Altered fatty acid metabolism by skeletal muscle can lead to insulin resistance and the accumulation of lipid in non-adipose tissue. This accumulation plays a critical role in the pathogenesis of chronic diseases including diabetes and heart failure.

To study the mechanisms underlying these processes investigators measured fatty acid utilization in muscle cells as it relates to obesity and weight loss, and in response to known and novel potential drugs affecting fatty acid oxidation (FAO). Key to many of these studies is the detection of altered FAO, commonly obtained by quantifying ¹⁴CO₂, a radioactive end product of the oxidation reaction [1]. While this and similar assays allow effective, specific measurements, they are time consuming, require the use of radiolabeled fatty acids such as palmitate or oleate, and offer only limited kinetic resolution as rates are often measured over an hour or longer.

In a previously published manuscript [2], we compared results obtained using the XF assay directly measuring oxygen consumption rate (OCR) to those obtained with a radiometric assay measuring an FAO reaction product, 3HOH. The extracellular flux assay (XF) performed in the XF Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA) detects changes in oxidative respiration, as measured by the OCR resulting from exogenous addition of fatty acids to muscle cells in culture. Based on OCR detection in real-time, we show that XF assays can provide estimates of metabolic fluxes with accuracies comparable to those obtained using radiometric assays.

We used L6 myoblasts untreated or treated with the biguanide metformin (Glucophage), a marketed drug for Type 2 diabetes that acts by upregulating AMPK, as the model system for this assay. Figure 1 shows the similarities and differences between an XF and a radiometric assay performed in parallel by determining cellular responses to exogenous palmitate.

Figure 1a

Figure 1b

Cellular OCR increases from basal rate upon addition of palmitate and specificity for FAO is determined by addition of the CPT1 inhibitor, etomoxir. The XF assay in 1A follows similar kinetics as 3HOH accumulation from radiolabeled palmitate shown in 1B. Some Cells were pretreated for 2 hours with 1 mM metformin (met) to activate the AMP-kinase, which stimulates FAO.

In Figure 1, the XF and radiometric assays both clearly showed a rise in oxidative respiration due to exogenous addition of fatty acid in the form of palmitate. Pretreatment for 2 hours with metformin enhanced palmitate usage as expected. However, a fundamental difference is that the XF assay measures the rate of oxygen consumption in nearly real-time (Figure 1A). For the radiometric assay, aliquots were taken at the indicated time points but the accumulated 3HOH was measured separately, hours later, after processing (Figure 1B).

Because the 3HOH arises only from beta-oxidation of the labeled palmitate, the radiometric assay only reports the utilization of that specific substrate. However, OCR in the XF assay measures total oxidative metabolism from not only fatty acids but glucose and amino acids as well. To determine the fraction of OCR specific to palmitate utilization, a specific inhibitor of FAO such as etomoxir, a carnitine palmitoyl transferase 1 inhibitor can be injected from the drug injection ports on the XF Analyzer as shown in Figure 1A. This allows the XF assay to uniquely discriminate the fraction of OCR associated with fatty acid utilization versus other substrates.

By using the drug injection ports on the XF Analyzer to inject glucose or palmitate into wells containing either metformin treated or untreated C2C12 myotubes, a shift of substrate preference away from glucose oxidation to palmitate can be measured in real-time (manuscript in preparation). This preferential shift is related to the ability of metformin to inhibit mitochondrial respiration at complex I [3].

Discussion

Extracellular metabolite fluxes can be measured quickly, accurately and conveniently using XF assays to detect and quantify oxygen consumption rate (OCR), a measure of mitochondrial respiration, and extracellular acidification rate (ECAR), an indicator of glycolysis. In this Note, instead of following the fate of radiolabeled fatty acid tracers, OCR is used to measure FAO in response to differentially treated muscle cell populations, in real-time.

Recent applications of this assay include a study by Crunckhorn et al that showed that obesity and saturated fatty acids decrease PGC-1, and mitochondrial gene expression and function via p38 MAPK-dependent transcriptional pathways. This study also showed that palmitate reduced the expression of tricarboxylic acid cycle (TCA) and oxidative phosphorylation mitochondrial genes and reduced oxygen consumption in C2C12 myotubes [4].

In another study, Weinberg et al explored the Warburg hypothesis for tumor cell growth that suggests defects in mitochondrial oxidative phosphorylation forces the tumor to rely on high levels of aerobic glycolysis to provide the energy and biosynthetic intermediates to fuel rapid growth. Using a Krasmediated oncogenic model, the authors were able to demonstrate that TCA cycling via amino acids and fatty acid oxidation was critical for oncogene-induced tumorigenicity [5].

In a study to determine the effects of SRT1720 on energy metabolism, investigators measured oxygen consumption and determined AMPK activation in C2C12 myotubes treated with the sirtuin over 24 hours without activating AMPK kinase in the myotubes [6].

And finally, using oleate as the fatty acid fuel source in situ, investigators were able to demonstrate the effect of SIRT1 on AMPK control of mitochondrial respiration by showing that AICAR-induced increases in mitochondrial O2 consumption were severely blunted by knocking down SIRT1. This was the first direct evidence that AMPK regulation of mitochondrial and lipid metabolism genes through modulation of PGC-1a is heavily influenced by SIRT1 [7].

Materials and Methods

Cells: L6 myoblasts were seeded at 20,000 cells per well in an XF24 well culture microplates and incubated overnight in a 37°C/10% CO₂ incubator. (Approximately 40% fewer cells would be seeded in an XF96 cell culture microplate.) Concentrated stocks of sodium palmitate (2 mM) were conjugated with a 0.34 mM (2.267 g/dL) ultra fatty acid free bovine serum albumin (BSA). Ultra fatty acid free BSA was purchased from Roche Diagnostics (Indianapolis, IN). Seahorse has developed an optimized protocol for preparing the palmitate-BSA complex (available on the website.)

The assay medium for FAO is low-buffered KHB buffer consisting of 110 mM NaCl, 4.7 mM KCl, 2 mM MgSO4 1.2 mM Na2HPO4, 2.5 mM glucose adjusted to pH7.4 supplemented with 0.5mM carnitine, 100nM insulin unless specified. Glucose concentrations may affect the propensity of cells to uptake and utilize palmitate. For induction of FAO, palmitate was injected to a final concentration of 200 μM.

XF Analysis

XF analyses were performed in the XF Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA), a fully integrated, multi-well instrument that measures the uptake and excretion of metabolic end products in real-time for all assays. Both OCR and ECAR were measured using XF assay kits. The disposable assay kits contain either an XF24 or XF96 XF cell culture plate and a solid state sensor cartridge, embedded with either 24 or 96 dual-florescent biosensors (O₂ or H⁺). Each sensor cartridge is also equipped with two or four drug delivery chambers per well for injecting testing agents into wells during an assay. OCR is reported in the unit of pmoles/minute and ECAR in mpH/minute.

To prepare the assay, as illustrated in Figure 2, the L6 culture media was removed from the XF culture plates containing the L6 treated and untreated cells. The wells were then washed and resuspended with assay medium. In parallel, the disposable sensor cartridge injection ports were preloaded with either palmitic acid complexed to BSA or the BSA vehicle alone. Combining the culture plate and sensor cartridge and inserting it into the instrument starts the XF experimental run. Three basal rates were measured prior to automated injection of palmitic acid (0.2 mM) in BSA vehicle or BSA vehicle alone. After treatment for 58 min, the carnitine palmitoyl transferase-1 inhibitor, Etomoxir (ETO, 50 μM), was added. Some cells were pretreated for 2 h with 1 mM metformin (met) to activate the AMP-kinase, which stimulates FAO.

References

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