Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Podsumowanie

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Streszczenie

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

Wprowadzenie

In biological systems, the heat-release or enthalpy change during metabolism is typically monitored either directly (via direct calorimetry) or indirectly via O₂ consumption and/or CO₂ production (via respirometry). Unfortunately, when these techniques are used in isolation, critical information is lost, such as the contribution of anaerobic pathways to cellular metabolism. Calorespirometry is a powerful technique that relies on the concurrent measurement of both heat dissipation and respiration. Pioneering calorespirometric work investigated the anaerobic metabolism in fully oxygenated mammalian cells and demonstrated simultaneous contributions of both aerobic and anaerobic pathways to energy homeostasis despite the transformed cells being in a fully oxygenated environment¹. Calorespirometry has since been applied to a wide variety of biological questions. Some examples include the study of animal energetics at low oxygen levels, the effects of both herbicide and estrogen on the gills of bivalves, the metabolism of terrestrial organisms, and the microbial decomposition of organic soil matter^2,3,4,5,6. Furthermore, calorespirometry has revealed how metabolic preconditioning prior to freezing improves the cryopreservation of mammalian cells⁷. Each approach, both calorimetry and respirometry, has independently increased our knowledge of cellular and organismal bioenergetics. However, fundamental biological questions that can be answered through the use of calorespirometry remain relatively unexplored.

Hess's law states that the total enthalpy change of a reaction is independent of the pathway between the initial and final states. For example, the total enthalpy change for a biochemical pathway is the summation of the change in enthalpies of all reactions within the pathway. Calorimetry offers a real-time approach for measuring cellular heat production, which indiscriminately detects both aerobic and anaerobic pathways. This is based on the foundation that no energy is exchanged in the system except through the walls of the experimental ampule⁸. A change in heat dissipation is equal to the change in enthalpy released from all metabolic reactions in the ampule. Thus, a negative enthalpy correlates to a loss of heat from the system. Exhaustive research over the last four decades has characterized the thermodynamic landscape of both catabolism and anabolism. This is represented by a steady rise in research articles found under the search terms "biological" and "calorimetry" as indexed by the United States National Library of Medicine (NLM) at the National Institutes of Health (PubMed). The search reveals that prior to 1970, a total of 27 publications reference biological calorimetry; meanwhile, in 2016 alone, 546 publications utilized the technique.

Calorimetric methods are well established to determine heat production. However, more flexibility is granted for resolving the respirometric value. The respirometric measurement can consist of O₂, CO₂, or both O₂ and CO₂. Further, the measurement of O₂ or CO₂ can be accomplished by various techniques, including optrodes, Clark-type electrodes, and tunable diode laser absorption spectroscopy^7,9,10,11. While CO₂ production is a valuable metric in many respirometric studies, the medium for cultured cells often utilizes a bicarbonic buffer system for pH control^12,13. To avoid complications of CO₂ measurement in the bicarbonate system, the following protocol for the calorespirometry of cells in culture utilizes O₂ as the sole respirometric parameter.

Concurrent with measuring oxygen flux, certain respirometers (see Table of Materials) are designed for detailed assessments of mitochondrial function. Substrate-uncoupler-inhibitor-titrations (SUIT) protocols are well established and are compatible with experiments designed to measure membrane potential or reactive oxygen species (ROS) formation¹⁴. The presented protocol for calorespirometry of intact cells is compatible with the introduction of chemical uncouplers such as carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and the F₀F₁-ATP synthase inhibitor oligomycin. Through the addition of FCCP, oxygen consumption can be uncoupled from ATP production, which is useful to assess the impact of potential therapeutics on mitochondrial peformance¹⁵. Furthermore, the addition of oligomycin illuminates the extent of leak respiration. Thus, the respirometric measurements performed during calorespirometry are compatible with extensive protocols designed to further elucidate mitochondrial physiology.

The simultaneous measurement of both heat dissipation and oxygen flux allows for the calculation of the calorespirometric (CR) ratio. This ratio is then compared to the Thornton's constant or the theoretical oxycaloric equivalent, which ranges between -430 to -480 kJ mol^-1 depending on the cell line or tissue of interest and the supplemented carbon substrates^1,16. Thus, a more negative CR ratio reveals increased contributions from anaerobic pathways to overall metabolic activity. For example, the CR ratio for routine muscle tissue respiration without the active performance of work ranges from -448 to -468 kJ mol-1 which is within the range of the theoretical oxycaloric equivalent^17,18. Meanwhile, mammalian cancer cells cultured in medium that is high in glucose display enhanced lactic acid fermentation following glycolysis and relatively low mitochondrial engagement¹⁹. This phenotype results in CR ratios in the range of -490 to -800 kJ mol^-1, demonstrating a heightened involvement of the anaerobic pathways in the cellular metabolism as indicated by more negative CR ratios^1,7,16,20.

Both commercial and non-profit cell and tissue distributors currently offer cell lines from over 150 species, with nearly 4,000 cell lines derived from humans. Immortalized cell lines are convenient tools for quickly evaluating the toxicity of potential therapeutics, many of which may directly or indirectly interfere with mitochondrial functions. Using transformed cells during drug screening may be of limited predictive value in part because of the Warburg effect, a hallmark of many cancers. Often, cancers generate ATP from substrate-level phosphorylation and maintain redox balance through the production of lactate without fully engaging the mitochondrion under aerobic conditions¹⁹. Pharmaceutical development is notoriously costly and inefficient, with approximately 8 out of 9 compounds tested in human clinical trials failing to achieve market approval²¹. While potential therapeutics may pass initial screening due to low cytotoxicity in cell lines, it is possible that some of these compounds are mitotoxic. Without a suitable method to detect how these toxins can impair the energy balance in primary cells that do not display the Warburg effect, critical information is often over-looked, bottlenecking therapeutic development in early stages.

Calorespirometry is a practical, noninvasive approach to analyze metabolic activity in a variety of biological samples, including cells and tissues. The core of the presented protocol is compatible with a range of applications. One complication, however, has been identified. Immortalized cells are often cultured in a glucose-free medium supplemented with galactose to increase the contribution of oxidative phosphorylation (OXPHOS) for energy production, in order to sensitize the cells to potential mitotoxins^22,23. This metabolic reprogramming appears to obscure analysis when samples are placed in the stainless steel ampules used by the calorimeter¹⁵. Cells cultured in a glucose medium continue to engage in high metabolic activity for several hours. Meanwhile, cells cultured in galactose medium decrease the heat production within 30 min of their placement in the ampule, making measurements restricted to early experimental time points. This behavior, unfortunately, hinders the opportunity to assess their cellular proliferation. Despite this specific limitation, most applications are compatible with calorespirometric analysis and detailed metabolic information can be obtained through this approach.

Protokół

1. Cell Culture

1. Maintain human hepatocellular carcinoma (HepG2) cells in Dulbecco's Modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and additional substrates (10 mM glucose, 2 mM glutamine, and 1 mM pyruvate) at 37 °C in a cell culture incubator (5% CO₂, 95% air, 100% humidity).
   NOTE: Use the above DMEM formulation when DMEM is referenced in later steps for respirometry and calorimetry.
2. Plate the cells at 1 x 10⁶ cells per 100 mm culture plate and subculture them every 7 days or before reaching 90% confluency and renew the medium every 3 - 4 days.
3. Perform the experiments when cells are 60 - 80% confluent.

2. Preparation of Respirometer and Calorimeter

1. Pipet 2.2 mL of DMEM into the respirometer chamber, insert the stopper fully, and adjust it with the stopper spacer tool to allow for optimal oxygen diffusion from air to medium.
2. Stir continuously at 37 °C until oxygen concentration (blue trace) and oxygen flux (red trace) are stable. Ensure the respirometer's software is programmed to account for the oxygen solubility of the medium used in the experiment.
   NOTE: Different media have different oxygen solubility coefficients (e.g., DMEM = 0.92).
3. After stabilization of the oxygen concentration in DMEM with the chamber open, select a stable portion of the oxygen concentration trace and calibrate it for 100% air saturation.
4. Calibrate for 0% oxygen selecting a stable portion of the oxygen concentration trace when no oxygen is present in the chamber. Perform this step after 20 µL titration of 230 mM sodium dithionite or after all oxygen is consumed by the biological sample at the end of an experiment.
5. After the 100% air calibration of the respirometer, close the stopper and ensure that no bubbles are present in the chamber.
   NOTE: a slight increase in oxygen flux will be detected in the closed chamber as the polarographic oxygen sensor (POS) consumes oxygen in order to measure the oxygen partial pressure.
6. After ~10 min of the stopper being closed, confirm that the respirometer is ready for HepG2 cells by observing a stable oxygen flux.
   NOTE: The calorimeter used here utilizes stainless steel ampules and requires one ampule to be used as a reference.
7. Add 2.5 mL of H₂O (dH₂O) [equal volume as the samples (step 4)] to the reference ampule of the calorimeter and tighten the cap, using pliers if necessary. Clean the sealed ampule with airflow and slowly lower the ampule to position 1 in the reference channel of the calorimeter. Remain at position 1 until biological sample is prepared.

3. Preparation of HepG2 Cells for Respirometry and Calorimetry

1. Confirm that the respirometer and calorimeter are prepared and calibrated. Warm DMEM and trypsin to 37 °C in a water bath.
2. Add DMEM to a fresh 100-mm plate and place in the incubator to allow for the equilibration of the medium for both CO₂ and temperature.
   NOTE: This medium will be used for the calorimetry experiment, so the appropriate volume depends on the number of ampules that will be used.
3. Confirm with an inverted light microscope that the cells are at 60 - 80% confluent, then wash the 100-mm plate 2x with 10 mL of room temperature phosphate-buffered saline (PBS).
4. Add 3 mL of the 37-°C trypsin to the 100-mm plate of cells and incubate them for 7 - 10 min at 37 °C in the cell culture incubator. Neutralize the trypsin with 3 mL of 37-°C DMEM and suspend by gently pipetting it ~20 times with a 5 mL pipet.
5. Transfer the 6 mL of resuspended cells in DMEM and trypsin into a sterile 15-mL conical tube and centrifuge it for 5 min at 175 x g. Remove the liquid without disturbing the cell pellet.
6. Resuspend the cell pellet in ~5 mL of DMEM and pipet it thoroughly to ensure the cells are sufficiently dissociated from one another.
7. Remove ~100 µL of the well-mixed sample and perform a thorough cell count utilizing all 8 fields on a hemocytometer to determine the total number of cells. Then calculate the volume for resuspension of the cells in order to generate ~2 x 10⁶ cells per 50 µL.
   NOTE: This will ensure that 2 x 10⁶ cells are added to each chamber of the respirometer with minimal medium displacement.
8. Centrifuge the cells for 5 min at 175 x g. Resuspend the pellet in the calculated volume of DMEM from step 3.7.
9. Perform a cell count to determine the volume required to inject 2 x 10⁶ cells per chamber in the respirometer (this should be ~50 µL). Store the cells for the respirometric measurement on ice until ready.
10. While the concentrated cell sample is on ice, determine the dilution required to produce a concentration of 1 x 10⁵ cells/mL for the calorimetry.
11. Mix the sample well and prepare ~3 mL of the cells at 1 x 10⁵ cells/mL in DMEM for each ampule.
    NOTE: The DMEM used for the sample dilution should be the equilibrated medium prepared in step 3.3.

4. Calorimetry

1. Ensure that calorimeter is connected to the computer and the calorimetric signal in µW is observable and stable.
2. Add 2.5 mL of the cells at 1 x 10⁵ cells/mL (~2.5 x 10⁵ cells) to each ampule, ensuring sufficient air is between the lid of the ampule and the medium to allow for gas diffusion.
3. Immediately tighten the cap, using pliers if necessary. Clean the sealed ampule, using airflow to remove any external debris, and slowly lower the ampule to position 1 of the calorimeter. Record the time the ampule is lowered to position 1.
4. Allow the ampule to sit at position 1 for 15 min.
   NOTE: During this 15 min period, cells are to be injected into the respirometer (step 5). This ensures that the same interval of time is used for both heat production and oxygen consumption when the CR ratio is determined.
5. After 15 min at position 1, slowly lower both the reference and sample ampules to position 2. Record the time the ampules were lowered and measure for at least 30 min.

5. Respirometry

1. During the 15-min interval in which the ampule is at position 1 in the calorimeter, begin the respirometric studies.
2. Thoroughly mix the cell sample by pipetting to ensure a homogenous solution and inject <50 µL of the cell sample into each chamber of the respirometer for a final concentration of ~2 x 10⁶ cells/chamber. Record the time the cells are injected into the respirometer.
3. After the injection, the HepG2 cells are continuously stirred at 37 °C. Wait at least 20 - 30 min for both a stabilization of the oxygen flux and a steady decrease in the oxygen concentration.
4. Select the O₂ flux per V tab on the right of the screen and highlight a stable portion of oxygen flux. Select marks, then statistics and record data for O₂ flux per V. This recording will be used in calculation of the CR ratio.
   NOTE: Steps 5.5 and 5.6 are optional. Leak respiration and maximal uncoupled respiration will be revealed by titrations of oligomycin and FCCP.
5. Inject 1 µL of 4mg/mL oligomycin into each chamber and allow ~ 10 min for oxygen flux stabilization. Record stable leak respiration rate by following steps performed in 5.4 by highlighting a stable portion of the oxygen flux trace after oligomycin addition.
6. Titrate 2-µL injections of 0.2 mM FCCP into each chamber, allowing for oxygen flux stabilization after each injection. Continue the titrations until the maximal flux is reached, as indicated by a slight decline in the subsequent FCCP titration. Record the stable respiration rate during the maximal flux.

6. Calculation of Calorespirometric Ratio

1. Perform a final cell count of the samples prepared at ~ 1 x 10⁵ cells/mL utilizing all 8 fields of a hemocytometer to determine the total number of cells added to the ampule (2.5 mL).
2. Determine a time to record measurements from both calorimeter and respirometer at which stable signals are present at identical times. Reference the time recorded when the ampule was lowered to position 2 and the time of injection of cells into the respirometer.
3. Record the heat production by placing the cursor over the trace displaying heat output for the respected ampule at the time determined in step and express as µW/million cells. Collect oxygen consumption data and ensure that it is expressed as pmol O₂ x s^-1 x million cells.
4. To calculate the CR ratio, first convert µW to kJ/s, and then divide kJ/s over mol of O₂/s to obtain the CR ratio expressed as kJ/mol O₂. NOTE: The CR ratio is presented in kJ/mol O₂.
   (see Supplementary FIle 1)

Wyniki

The reproducibility of calorespirometric measurements depends on a proper and consistent sample preparation. Samples prepared from cell culture should not be used if the plates are overgrown, as cell counts can become inaccurate due to clumping. Further, reduced heat flows due to limited substrate diffusion in the clumped cells may occur. Therefore, when using adherent cells, it is critical to select a plate with the confluency between 60 - 80% and to change the medium 24 h prior to the experiment.

Dyskusje

The objective of calorespirometry is to quantitatively evaluate the contributions of aerobic and anaerobic pathways to metabolic activity and obtain a composite view of cellular energy flux. This is accomplished by a simultaneous measurement of heat dissipation and oxygen flux followed by a comparison of the calculated CR ratio with the theoretical oxycaloric equivalent. Several critical steps must be considered for reproducible and reliable data. The maintenance of a sterile, healthy cell culture is critical. Contaminat...

Materiały

Name	Company	Catalog Number	Comments
HepG2 Cells	American Type Culture Collection	HB-8065	Cells used for calorespirometry
O2k-Respirometer	Oroboros Instruments	10022-02	Respirometer
LKB 2277 thermal activity monitor (TAM)	Thermometric AB		Thermometric was purchased by TA Instruments
Sodium Pyruvate (100 mM)	Thermofisher Scientific	11360070	100x solution added to DMEM medium
Fetal Bovine Serum - Premiuim Select	Atlanta Biologicals	S11550	Added to 10% in DMEM medium
Trypsn-EDTA (0.25%)	Thermofisher Scientific	25200072	Cell dissociation reagent
Oligomycin from Streptomyces diastatochromogenes	Sigma Aldrich	O4876	Mitochondrial Inhibitor
Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone	Sigma Aldrich	C2920	Mitochondrial Uncoupler
Corning 100 mm TC-Treated Culture Dish	Corning Corporation	430167	Tissue culture dish
Glucose, powder	Thermofisher Scientific	15023021	Glucose for DMEM medium
Galactose, powder	Fischer Scientific	BP656500	Galactose for DMEM medium
L-Glutamine (200 mM)	Thermofisher Scientific	25030081	Glutamine for DMEM medium
DMEM, no glucose	Thermofisher Scientific	11966025	Cell culture medium