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

Summary

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.

Abstract

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.

Introduction

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 contr....

Protocol

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 w....

Results

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.

Discussion

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.......

Materials

List of materials used in this article

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