Characterization of Energy Expenditure in Rodents by Indirect Calorimetry

Pēteris Alberts1, Bo G. Johansson2, Robert A. McArthur3

1 Biovitrum, Stockholm, null, 2 Somedic, Hörby, null, 3 McArthur and Associates GmbH, Basel, null
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 9.23D
DOI:  10.1002/0471142301.ns0923ds36
Online Posting Date:  August, 2006
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Abstract

The need for treatment of obesity and obesity-related diseases, such as type 2 diabetes, has been intensified by the epidemic rise of obesity. Recent advances make possible continuous monitoring of metabolically relevant functions in animals to identify novel thermogenic and anorectic compounds. This unit describes non-invasive in vivo calorimetric assessment of energy expenditure using measurements of oxygen consumption and carbon dioxide production, complemented by telemetric monitoring of body core temperature and locomotor activity in mice and rats. Reference compounds are used to illustrate the determination of substance-specific parameters, such as the dose that produces the half-maximal effect (ED50), the maximal effect, as well as the time of onset and duration of compound action. Indirect calorimetry performed at different temperatures provides information on several other well-defined parameters, including resting metabolic rate, basal metabolic rate, lower critical temperature, temperature sensitivity, defended body temperature, and respiratory quotient.

     
 
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Table of Contents

  • Basic Protocol
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

 Basic Protocol
 Materials
  • Normal control mice and rats, for example:
    • Wild type, male C57BL/6J mice, 20- to 22-week-old at the start of the study (e.g., Taconic)
    • Male Balb/c mice (11 weeks old at the start of the study; e.g., B&K Universal)
    • Wild type, male Rj:Wistar (Han) rats, 197 to 242 g body weight (e.g., Elevage Janvier)
  • Mice with characteristics appropriate for studies of obesity and diabetes, for example:
    • Male C57BL/6JBom-Lepob (ob/ob) mice, 12 to 13 weeks old at the start of the study (e.g., Taconic)
    • Male KKAy mice (8 to 12 weeks old at the start of the study; Clea; also available from The Jackson Laboratory)
  • Transgenic mice (e.g., male PTP1B knock-out mice on Balb/c background; originated from Elchebly et al., 1999; McGill University; 11 weeks old at the start of the study)
  • Normal mouse diet (R34; Lactamin) or a high fat diet (32% kcal fat, Research Diets)
  • Isofluran (Baxter)
  • Buprenorphin (Temgesic, Schering-Plough)
  • Bottled synthetic air (21.0% O2 in N2 with <5 ppm water)
  • Calibration gases (18.0% and 25.0% O2 in N2; 200 and 2000 ppm CO2 in synthetic air; AGA Gas)
  • Reference compounds, for example:
    • (±)-CGP-12177A hydrochloride (RBI)
    • (–)-Norepinephrine bitartrate (Sigma-Aldrich)
    • Diazepam (Nordic Drugs)
    • Sibutramine (ChemPacific)
    • d-(+)-Glucose (Sigma-Aldrich)
  • Standard animal cages with nesting material (e.g., aspen L-tubes and M-bricks, Tapvei or Neslets, Ancare)
  • Small surgical scissors
  • Transponder telemetry system (Model PDT-4000 E-Mitter, sterilized, and ER-4000 Energizer Receiver, Mini Mitter)
  • 6-0 silk sutures
  • Sterile disposable skin staplers (9-mm autoclips, MikRon Precision)
  • Indirect calorimetry apparatus (INCA, Somedic; see Fig. 9.23D.1). Major components of the set-up and apparatus as follows:
    • Measuring chamber: a cylindrical (~20-cm high and 24-cm i.d.) acrylic (Perspex) pressure vessel (see step for more details)
    • Flow regulator (Brooks model 8942) maintains a constant airflow through the measuring chamber of 1 liter/min (adjustable within 0.5 to 5 liter/min). The time constant (t1/2) of air exchange of the system is proportional to the chamber volume/airflow × 0.693 (Wilkinson, 2001). Thus, for a volume of 4 liters and airflow of 1 liter/min the time constant is 2.8 min.
    • Mass flow meters (Model AWM 3300V, Micro Switch, Honeywell), to measure in- and outflow to the chamber with their outputs conditioned by an amplifier (4CHAMP, Somedic) before connection to the data collection system
    • Dryer system for removal of water vapor (Silica gel, Safegel 1 to 3 mm with yellow moisture indicator, Merck Prolabo, VWR)
    • CO2 sensor based on the dual channel infrared absorption principle (Model 0633-1240 connected to a 650 Reference instrument, Testo)
    • Relative humidity (RH) sensor (Model HIH-3602-L, Micro Switch, Honeywell)
    • Computer-controlled gas sampler (Somedic)
    • Zirconium O2 analyzer (Series 350, Panametrics)
    • Amplifier (4CHAMP, Somedic)
  • Personal computer with dual serial ports for connection to the carbon dioxide analyzer and transponder systems as well as a built-in data collection card (PCI-1200, National Instruments)
  • Software for the control of the calorimetry apparatus and recording of data (Somedic; written in DasyLab 5.6 for Windows, Datalog)
     FigureFigure 9.23D.1 Schematic flow diagram showing the main components of the calorimeter system (not to scale). The synthetic air and the calibration gases are supplied through stainless steel piping at 16 bar pressure, which is reduced to 1 bar before the calorimetry apparatus. A computer-controlled gas sampler takes samples of chamber in- and outflow as well as calibration gases and supplies the oxygen analyzer with a constant flow of 200 ml/min. A CO2 sensor is placed directly after the output mass flow meter. The output of the O2 sensor (Series 350, Panametrics) is conditioned by an amplifier (4CHAMP, Somedic) before connection to the data collection system. Abbreviations: synthetic air (air), calibration gas (cal.), and relative humidity (RH).

NOTE: To adapt the system to larger animals, e.g., when changing from mouse to rat, the higher absolute oxygen consumption and the need for greater animal space requires that a number of parameters be increased. In the case of the rat, the following has been found to be suitable:
  • increase the volume of the measuring chamber to 13 liters,
  • increase the chamber airflow rate to 2 liter/min,
  • increase the volume of the air dryer to 250 ml, and
  • renew the air dryer after every experiment.
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Figures

  •  FigureFigure 9.23D.1 Schematic flow diagram showing the main components of the calorimeter system (not to scale). The synthetic air and the calibration gases are supplied through stainless steel piping at 16 bar pressure, which is reduced to 1 bar before the calorimetry apparatus. A computer-controlled gas sampler takes samples of chamber in- and outflow as well as calibration gases and supplies the oxygen analyzer with a constant flow of 200 ml/min. A CO2 sensor is placed directly after the output mass flow meter. The output of the O2 sensor (Series 350, Panametrics) is conditioned by an amplifier (4CHAMP, Somedic) before connection to the data collection system. Abbreviations: synthetic air (air), calibration gas (cal.), and relative humidity (RH).
    View Image
  •  FigureFigure 9.23D.2 Schematic plot of the environmental temperature dependence of the metabolic rate (MR). Using Equations 2 and 3 and the relationship between environmental temperature and metabolic rate, the following parameters are calculated: basal metabolic rate (BMR), temperature sensitivity (TS), lower critical temperature (LCT), and defended body temperature (DBT). The thermoneutral zone and the upper critical temperature are defined but not calculated. Above the upper critical temperature, the MR increased as a result of panting to maintain the body temperature.
    View Image
  •  FigureFigure 9.23D.3 A typical continuous monitoring of O2 consumption (V¢O2 or MR; ml/min/kg0.75), respiratory quotient (RQ; %), total locomotor activity (activity counts/min; left axis), and body core temperature (°C; right axis) in the KKAy mouse at an environmental temperature of 15°C.
    View Image
  •  FigureFigure 9.23D.4 The environmental temperature-dependence of metabolic rate (MR) in male mice. (A) C57BL/6J, (B) ob/ob, (C) Balb/c, (D) KKAy on normal food, (E) KKAy on high-fat diet (32%) for 2 to 10 weeks, and (F) PTP1B knockout mice. The graphs are drawn using the fitted constants from Equations 2 and 3 (Table 9.23D.2).
    View Image
  •  FigureFigure 9.23D.5 (A) Enhancing effect on metabolic rate (MR; at 30°C) in male C57BL/6J mice of norepinephrine (s.c.), or (±)-CGP-12177A (s.c.). The MR is expressed relative to the lowest dose with no or minimal effect as AUC liter/kg0.75 (%). The lowest O2 consumption values were 0.60 ± 0.089 liter/kg0.75(n = 5) and 0.90 ± 0.089 liter/kg0.75(n = 5), respectively. The lines in the dose-response curves are drawn using the fitted constants from Equation 15 (Table 9.23D.3). (B) Effect of environmental temperature on respiratory quotient (RQ; unitless ratio) in KKAy mice on normal food or high-fat diet (32%); the same animals as in Figure 9.23D.4. Statistical differences (p) are: * p < 0.05, ** p < 0.01, *** p < 0.001. (C) Effect of environmental temperature on respiratory quotient (RQ; unitless ratio) in C57BL/6J and ob/ob mice; the same animals as in Figure 9.23D.4. (D) Effect of norepinephrine on respiratory quotient (RQ) in C57BL/6J and ob/ob mice; the same animals as in (A). The ob/ob mice do not tolerate the 3 mg/kg dose.
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  •  FigureFigure 9.23D.6 The effects of sibutramine on metabolic rate (MR) and respiratory quotient (RQ; unitless quotient) in the Wistar rat. (A) The effect of intraperitoneal (i.p.) vehicle or sibutramine (20 mg/kg) on O2 consumption (V¢O2 or MR) in the oral glucose tolerance test (OGTT). Glucose (5 g/kg) or vehicle (distilled water) was given orally (p.o.). (B) The RQ in the same experiment. (C) The effect of i.p. vehicle or sibutramine (5, 10 or 20 mg/kg) dissolved in physiological saline on the RQ. (C. Froger, V. Castagne, and P. Lacroix, unpub. observ.). All tests were conducted at 30°C with n = 8 rats/group.
    View Image

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Literature Cited

Literature Cited
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 Key References
    Even et al., 1994. See above.

An excellent description of the theory of indirect calorimetry.

    Ferrannini, 1988. See above.

A review of indirect calorimetry.

    Frayn, 1996. See above.

An introduction to metabolism, RQ, and metabolic parameters.

    International Union of Physiological Sciences (IUPS Thermal Commission), 2001. See above.

Internationally accepted terminology.

    Kenakin, 1997. See above.

An excellent description of the theoretical aspects of pharmacology.

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