Assessing Energy Substrate Oxidation In Vitro with 14CO2 Trapping

Summary

This protocol describes an easy-to-use method to examine substrate oxidation by tracking ¹⁴CO₂ production in vitro.

Abstract

Mitochondria host the machinery for the tricarboxylic acid (TCA) cycle and electron transport chain (ETC), which generate adenosine triphosphate (ATP) to maintain energy homeostasis. Glucose, fatty acids, and amino acids are the major energy substrates fueling mitochondrial respiration in most somatic cells. Evidence shows that different cell types may have a distinct preference for certain substrates. However, substrate utilization by various cells in the skeleton has not been studied in detail. Moreover, as cellular metabolism is attuned to physiological and pathophysiological changes, direct assessments of substrate dependence in skeletal cells may provide important insights into the pathogenesis of bone diseases.

The following protocol is based on the principle of carbon dioxide release from substrate molecules following oxidative phosphorylation. By using substrates containing radioactively labeled carbon atoms (¹⁴C), the method provides a sensitive and easy-to-use assay for the rate of substrate oxidation in cell culture. A case study with primary calvarial preosteoblasts versus bone marrow-derived macrophages (BMMs) demonstrates different utilization of the main substrates between the two cell types.

Introduction

Oxidative phosphorylation (OXPHOS) in eukaryotes is the process by which nutrients are broken down inside mitochondria to release chemical energy in the form of ATP through consumption of oxygen.  Catabolism of various substrates inside mitochondria through the tricarboxylic acid (TCA) cycle generates few ATP molecules directly, but rather stores energy through reduction of the electron carriers nicotinamide adenine dinucleotide (NAD⁺) and flavin adenine dinucleotide (FAD⁺). The reduced carriers are then oxidized by ETC located on the inner membrane of mitochondria to generate a proton concentration gradient across the membrane. The protons eventually flow down their gradient back into the mitochondrial matrix through ATP synthase to produce ATP. OXPHOS is the most efficient means of ATP production from energy substrates and generally preferred in aerobic environments. Previously, aerobic glycolysis-production of lactate from glucose while oxygen is present-was thought to be pathophysiological, often a hallmark of cancer cells. More and more, it is being discovered that some normal cell types use aerobic glycolysis for reasons that have yet to be fully deciphered.

Metabolic flexibility is the capacity for cells or organisms to adapt to changing energy demands and available fuel sources. For example, the energetic demand of skeletal muscle is mainly met by OXPHOS at steady state but by anaerobic glycolysis during high-intensity exercise¹. As the exercise duration increases, glucose and fatty acid oxidation contribute more to overall energy production². However, substrate use is not dependent only on availability, as substrates antagonistically compete during oxidation. Most notably, fatty acid oxidation has been shown to inhibit glucose utilization by skeletal muscle in a phenomenon known as the Randle effect³. A reciprocal effect was demonstrated by subsequent studies^4,5. In addition, many diseases are associated with a change in substrate preference and the development of metabolic inflexibility in cells. For instance, fatty acid oxidation is reduced in the skeletal muscle of type II diabetic patients compared to normal control subjects⁶. The metabolic changes in disease settings are a subject of intense investigation as they may contribute to pathogenesis.

Energy metabolism in skeletal cell types is relatively understudied but has gained attention in recent years⁷. Previous work has shown that aerobic glycolysis is the dominant energy pathway in calvarial osteoblasts, while glucose oxidation through the TCA cycle plays a role in osteoclast formation^8,9. Others have provided evidence for fatty acids as an energy source for osteoblasts¹⁰. Glutamine catabolism has also been shown to support osteoblast differentiation from the progenitors^11,12. However, a comprehensive understanding of substrate utilization by various skeletal cell types is still lacking. In addition, changes in cellular metabolism during cell differentiation or in response to pathological signals are expected to alter fuel substrate utilization. Described below is an easy-to-use protocol for assaying substrate oxidation in vitro.

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Protocol

The use of radioactive materials (RAM) requires prior approval by a designated safety committee at each institution. RAMs used in this protocol have been approved by Environmental Health & Radiation Safety (EHRS) at the University of Pennsylvania. The use of animals requires prior approval by the Institutional Animal Care and Use Committee (IACUC) at the home institution. The following study was approved by IACUC at The Children's Hospital of Philadelphia.

1. Preparation of stock solutions for ¹⁴C-labeled substrates

1. Make 4 mM bovine serum albumin (BSA) stock solution by mixing 11 g of BSA and 33.1 mL of Dulbecco's phosphate-buffered saline (DPBS) and shaking it gently at room temperature for ~3 h until BSA is fully dissolved. Warm the BSA stock solution in a 70 °C water bath before use.
2. Air-dry 50 µCi ¹⁴C-oleate (50 µCi/µmol in ethanol) in the vial with the lid off for 8 h in a designated RAM working hood. Add 312.5 µL of H₂O and then 3.5 mg (11.5 µmol) of sodium oleate. Mix thoroughly.
3. Warm the resulting solution to 70 °C in a water bath. Add 0.9375 mL of the prewarmed BSA stock solution to yield 10 mM hot oleate stock solution (containing a 1:11.5 ratio of ¹⁴C-oleate to unlabeled oleate). Aliquot the stock solution and store it at -20 °C for long-term use.
4. Use ¹⁴C-glucose and ¹⁴C-glutamine directly after thawing.

2. Preparation of medium containing ¹⁴C-labeled substrates

NOTE: To ensure reliable concentrations of energy substrates in the media, custom-made media must be used with fresh substrates added shortly before use. Here, a custom-made Minimum Essential Medium (MEMα) without glucose, pyruvate, glutamine, phenol red, or sodium bicarbonate is used. However, an optimal medium should be determined for each cell type.

1. Reconstitute 8.67 g of the medium powder in 1 L of purified water. Add 2.2 g of sodium bicarbonate to achieve a pH of 7.4 and filter the solution using 0.22 µm vacuum filtration.
2. Add fresh ingredients to obtain the complete medium (cMEMα) with final concentrations of 5.5 mM glucose, 2 mM glutamine, 1 mM pyruvate, and 10% fetal bovine serum (FBS). Further supplement cMEMα with 100 mM L-carnitine and 10 µM HEPES to facilitate fatty acid oxidation.
3. Immediately before use, add ¹⁴C-labeled substrates to freshly prepared cMEMα to make hot media. To make the oleate hot medium, add the 10 mM hot oleate stock solution to cMEMα for a final concentration of 100 µM oleate (1:100 dilution, final radioactivity 0.4 µCi/mL).
   NOTE: The 10 mM hot oleate stock containing a 1:11.5 ratio of hot:cold oleate (see step 1.3) is used to achieve a physiological level of 100 µM total oleate in the media while minimizing the amount of radioactive material.
4. Make 10 mM unlabeled oleate stock by adding 24.36 mg of sodium oleate to 2 mL of H₂O in a water bath at 70 °C for ~20 min until completely dissolved before mixing quickly with 6 mL of the 4 mM BSA stock solution prewarmed to 70 °C. Transfer to a 37 °C water bath for 1 h and filter through a 0.22 µm filter. Aliquot and store at -20 °C for long-term use.
5. To make glucose hot medium, add ¹⁴C-glucose to a final concentration of 1.333 µM (1:4,125 dilution, final radioactivity 0.4 µCi/mL). To make glutamine hot medium, add ¹⁴C-glutamine to the final concentration of 2 µM (1:1,000 dilution, final radioactivity 0.4 µCi/mL).
6. Supplement the glucose and glutamine hot medium with a 10 mM stock solution of unlabeled oleate from step 2.4 to achieve the final concentration of 100 µM oleate, the same as that in the oleate hot medium.

3. Preparation of cells

NOTE: Calvarial preosteoblasts and bone marrow macrophages are used as examples here. Users should prepare their cell type of choice according to appropriate protocols. Optimize the concentration of collagenase II to be used for digestion in pilot experiments as the enzymatic activity may vary among different lots.

1. Isolation and culture of calvarial preosteoblasts
   1. Sacrifice postnatal day 3-5 (P3-5) pups by decapitation and transfer them to ice-cold DPBS containing Penicillin-Streptomycin (P/S).
   2. Expose the calvaria by removing the skin and soft tissue. Collect the middle region by cutting away the surrounding tissues from back to front in DPBS (Figure 1A). Scratch the inner and outer surfaces of the calvaria gently using tweezers to help release the cells upon subsequent digestion.
   3. Prepare a 2 mg/mL and a 4 mg/mL solution of Collagenase type II in DPBS and filter each solution with a fresh 0.22 µm filter. Digest the cleaned calvaria first with the 2 mg/mL collagenase solution for 15 min and discard the digestion solution. Digest the cleaned samples in 4 mg/mL collagenase solution 3 times for 15 min each time, pooling and saving the digestion solution.
   4. Filter the digestion solution through 70 µm cell strainers and centrifuge the filtrate at 300 × g for 5 min. Resuspend the cell pellet in cMEMα (see step 2.2) containing 10% FBS and P/S.
   5. Count and seed the cells at 4 × 10⁴ cells/cm² in 10 cm plates. Culture the cells in a 37 °C incubator with 5% CO₂ for 3 days.
   6. Dissociate the cells at 37 °C with 0.25% trypsin-EDTA. Count and seed the cells in 24-well cell culture plates with cMEMα containing 10% FBS and P/S at 7.5 × 10⁵/cm². Seed at least 4 wells per cell type per substrate and at least three extra wells for each cell type for cell counting before the assay.
   7. Culture the cells at 37 °C overnight before the substrate oxidation assays.
2. Isolation and culture of BMMs
   NOTE: Culture of BMMs requires macrophage colony-stimulating factor (M-CSF), which can be purchased commercially as a recombinant protein. Conditioned medium from the cell line CMG14-12 engineered to express M-CSF is an economical alternative used here¹³.
   1. Collect femurs and tibias from 8-week-old mice after removing the muscle and connective tissues. Cut and discard both ends of the bones with sharp scissors.
   2. Flush out the bone marrow from one femur and one tibia into a 10 cm Petri dish with a syringe fitted with a 23 G needle containing 15 mL of cMEMα with 10% FBS, P/S, and 10% CMG14-12 conditioned medium (BMM medium). Culture the cells in the Petri dish in a 37 °C incubator with 5% CO₂.
   3. After 3 days, discard the culture media and rinse with DPBS. Dissociate the attached cells at 37 °C with 0.25% trypsin-EDTA for 5 min. Centrifuge and resuspend the cell pellet in the BMM medium.
   4. Count and seed the cells in 24-well cell culture plates at 7.5 × 10⁵/cm² in the same way as described in 3.1.6. Culture at 37 °C overnight in the BMM medium before starting the assays.

4. Substrate oxidation assay with CO₂ trap

NOTE: An appropriate seeding density should be determined for each cell type to achieve 80-90% confluence before the start of the assay. Note that cell density can affect the metabolic state of the cells.

1. Wash the cells in the extra wells twice with DPBS. Dissociate the cells with 0.25% trypsin-EDTA and mixed 20 µL cells resuspended in DPBS with 20 µL of acridine orange/propidium iodide (AO/PI) ready-to-use commercial dye solution. Determine the number of live cells with an automated cell counter and record the number for final calculations.
2. Wash the cells in the assay wells twice with DPBS. Add 500 µL of hot medium to each assay well in the RAM-designated tissue culture hood. Seal the plates with parafilm and incubate the cells in a RAM-designated incubator at 37 °C for 4 h.
3. During incubation, cut filter paper into circular pieces slightly bigger than the area inside the cap of 1.5 ml microcentrifuge tubes and insert the paper snugly into the cap (Figure 1B). Add 200 µL of 1 M perchloric acid to each tube and 20 µL of sodium hydroxide to the filter paper fitted inside the cap.
4. After incubation of the cells, transfer 400 µL of culture medium from each well to the prepared tubes and close the caps immediately. Leave the tubes in a tube rack at room temperature for 1 h.
5. During incubation, set up a scintillation vial for each tube and fill it with 4 mL of scintillation fluid. Transfer each piece of filter paper to a scintillation vial and incubate at room temperature for 30 min.
6. Perform wipe tests on the tissue culture hood, the water bath (used for ¹⁴C-oleate preparation), refrigerator, incubator, sink, ground, and any other working area for potential RAM contamination. Put the paper wipes into scintillation vials containing scintillation fluid.
7. Measure ¹⁴C radioactivity in the scintillation vials with a scintillation counter. Record the reading results. Decontaminate the working environment according to radiation safety guidelines if necessary.

5. Data analysis

NOTE: Assuming that each substrate is fully oxidized to release CO₂, the substrate oxidation rate can be calculated from the trapped CO₂ radioactivity.

1. Calculate the substrate oxidation rate using Eq (1) and the X, Y, and Z values given in Table 1 for different substrates.
   Substrate oxidation rate (µmol/cell/h) =    (1)
   1. Calculate the total disintegrations per min (DPM) for each reaction well. For X, use the DPM value (scintillation counter reading) from 0.4 mL of the reaction medium used for CO₂ trapping out of 0.5 mL of the total reaction medium. Therefore, the total DPM from each reaction well is X × 1.25.
   2. Divide the total DPM by a factor of 2,220,000 to convert to µCi.
   3. Convert the radioactivity in µCi to the number of ¹⁴C-labeled molecules. Divide the µCi value by the specific activity (Y) of each labeled substrate. Use the following Y values (Table 1): ¹⁴C-glucose: 300 mCi/mmol; ¹⁴C-glutamine: 200 mCi/mmol; ¹⁴C-oleate: 50 mCi/mmol.
   4. Calculate the total number of oxidized molecules from the oxidized ¹⁴C-labeled molecules by multiplying the latter with the hot-to-total dilution factor (Z). Use the following Z values (Table 1): Glucose: 4,125; glutamine: 1,000; oleate: 12.5.
   5. Divide the resulting product by cell number (cell#) and 4 (for the reaction time of 4 h) to determine the substrate oxidation rate per cell. Use the cell# determined in the parallel wells at the beginning of the assay.

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Results

In this example, the CO₂ trapping method is used to compare substrate oxidation by primary calvarial preosteoblasts versus BMMs, which are frequently used for in vitro osteoblast or osteoclast differentiation, respectively. After the primary cells are passaged and cultured in cMEMα overnight, they typically reach 80-90% of confluence and exhibit their characteristic morphology. The calvarial preosteoblasts are notably larger than BMMs (Figure 2). Each cell type is su...

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Discussion

The protocol provides an easy-to-use method to determine the oxidation rate of major energy substrates. It is a simpler alternative to other protocols that use flasks containing a central well and capped with rubber stoppers^14,15,16. Although the example study here is performed with cell culture, the method can be easily adapted for tissue explants or tissue homogenates containing intact mitochondria, as previously described

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Materials

Name	Company	Catalog Number	Comments
0.22 µm filters	Sigma-Aldrich	SLGVM33RS	Used to filter BSA solution
0.25% Trypsin-EDTA	Gibco	25200056	Dissociate cells from cell culture plates
1.5 mL Eppendorf tubes	PR1MA	PR MCT17 RB	Used for reaction incubation
10 cm plates	TPP	93100	Used for cell culture
10 mL syringe	BD	302995	Used to flush marrow from long bones
10% FBS	Atlanta biologicals	S11550	For Cell culture medium preparation
14C-Glucose	PerkinElmer	NEC042X050UC	Used to make hot media
14C-glutamine	PerkinElmer	NEC451050UC	Used to make hot media
14C-oleate	PerkinElmer	NEC317050UC	Used to make hot media
23 G needle	BD	305120	Used to flush marrow from long bones
24-well plates	TPP	92024	Used for cell culture
70 μm cell strainers	MIDSCI	70CELL	Used to filter supernatant during cavarial digestion
Acridine Orange/Propidium Iodide (AO/PI) dye	Nexcelom Biosciences	CS2-0106	Stains live cells to determine seed density
Bovine Serum Ablumin	Proliant Biologicals	68700	Used for fatty acid conjugation
Cellometer Auto 2000	Nexcelom Biosciences		Determine the number of viable cells
Centrifuge	Thermo Fisher	Legend Micro 21R	Used to pellet cells
Collagenase type II	Worthington	LS004176	Dissociate cells from tissue
Custom MEM alpha	GIBCO	SKU: ME 18459P1	Used to create custom hot media
Dulbecco's Phosphate-Buffered Saline	Gibco	10010023	Used to dissolve and dilute reagents, and wash culture dishes
Filter Paper	Millipore-Sigma	WHA1001090	Traps CO2 with sodium hydroxide
Glucose	Sigma-Aldrich	g7528	Used to make custom media
HEPES	Gibco	15630080	Traps CO2 during cell culture
L-carnitine	Sigma-Aldrich	C0283	Supplemented for fatty acid oxidation
L-Glutamine	Sigma-Aldrich	g3126	Used to make custom media
MEM alpha	Thermo	A10490	Cell culture medium
Parafilm	Pecheney Plastic Packaging	PM998	Used to seal cell culture dishes
Penicillin-Streptomycin	Thermo Fisher	15140122	Prevents contamination in cell culture
Perchloric Acid	Sigma-Aldrich	244252	Releases CO2 during metabolic assay
Pyruvate	Sigma-Aldrich	p5280	Used to make custom media
Scintillation Counter	Beckman Coulter	LS6500	Determines radioactivity from the filter paper
Scintillation Fluid	MP Biomedicals	882453	Absorb the energy emitted by RAMs and re-emit it as flashes of light
Scintillation Vial	Fisher Scientific	03-337-1	Reaction containers for scintillation fluid
Sodium carbonate	Sigma-Aldrich	S5761	Balance buffer for medium
Sodium Hydroxide	Sigma-Aldrich	58045	Traps CO2 during metaboilc assay
Sodium oleate	SANTA CRUZ	SC-215879	BSA conjugated fatty acid preparation
Vaccum filtration 1000	TPP	99950	Filter cMEMα