Determining Basal Energy Expenditure and the Capacity of Thermogenic Adipocytes to Expend Energy in Obese Mice

Summary

This manuscript describes a protocol to measure the basal metabolic rate and the oxidative capacity of thermogenic adipocytes in obese mice.

Abstract

Energy expenditure measurements are necessary to understand how changes in metabolism can lead to obesity. Basal energy expenditure can be determined in mice by measuring whole-body oxygen consumption, CO₂ production, and physical activity using metabolic cages. Thermogenic brown/beige adipocytes (BA) contribute significantly to rodent energy expenditure, particularly at low ambient temperatures. Here, measurements of basal energy expenditure and total BA capacity to expend energy in obese mice are described in two detailed protocols: the first explaining how to set up the assay to measure basal energy expenditure using analysis of covariance (ANCOVA), a necessary analysis given that energy expenditure co-varies with body mass. The second protocol describes how to measure BA energy expenditure capacity in vivo in mice. This procedure involves anesthesia, needed to limit expenditure caused by physical activity, followed by the injection of beta3-adrenergic agonist, CL-316,243, which activates energy expenditure in BA. These two protocols and their limitations are described in sufficient detail to allow a successful first experiment.

Introduction

Metabolism can be defined as the integration of the biochemical reactions responsible for nutrient uptake, storage, transformation, and breakdown that cells use to grow and perform their functions. Metabolic reactions transform the energy contained in nutrients into a form that can be used by cells to synthesize new molecules and execute work. These biochemical reactions are inherently inefficient in transforming this energy into a usable form to sustain life¹. Such inefficiency results in energy dissipation in the form of heat, with this heat production being used to quantify the Standard Metabolic Rate (SMR) of an organism^1<....

Protocol

All experiments were approved by the Institutional Animal Care and Use Committee at the University of California, Los Angeles (UCLA). Mice were administered their diet and water ad libitum in the metabolic cage, housed in a temperature-controlled environment (~21-22 or 30 °C) with a 12h light/dark cycle. 8 week-old female mice fed a high-fat diet or chow diet for 8 weeks were used for this study.

1. Measurement of the Basal Metabolic Rate (BMR)

1. Measure the total body weight of the mouse using a weight scale with accuracy in the range of 0.1 g.
   NOTE: This must be done before housing the mice ....

Results

Figure 4 shows VO₂, VCO₂, Heat production/Energy expenditure (EE), Respiratory Exchange Ratio (RER), and X, Y, Z physical activity values obtained using the metabolic cages of the CLAMS system. The VO₂ and VCO₂ provided by the CLAMS system is the volume of gas (mL) per minute and can be already divided by the body weight or the lean mass values by entering these weight values in the CLAMS software before starting the measurements. However, body wei.......

Discussion

Indirect calorimetry has been used for years to assess whole-body energy expenditure⁴. This protocol described herein provides a straightforward method of measuring the basal metabolic rate and determining BA thermogenic capacity in vivo using metabolic cages.

The indirect calorimetry method described here confirms that dividing energy expenditure values by body weight values can be misleading. For example, it can conclude that energy expenditure is systematica.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CLAMS-Oxymax System	Columbus Instruments	CLAMS-center feeder-ENC	Including enviromental enclosure and Zirconia oxygen sensor
Desktop PC with Oxymax Software	HP/Columbus	N/A	PC needed to be purchased separately
Drierite jug (Calcium Sulfate with Cobalt Chloride Indicator)	Fisher Scientific	23-116681	Needed to dry the gas entering the oxygen sensor, humidity can damage the sensor
NMR for body composition	Echo-MRI	Echo-MRI 100	Measure lean and fat mass in alive mice. It is necessary for ANCOVA analyses.
CL-316-243	Sigma	C5976	Injected to the mice subcutaneously to activate thermogenesis
High fat diet	Research Diets	D12266B	Provided to the mice prior and during measurements
Pentobarbital/Nembutal	Pharmacy at DLAM	N/A	Anesthesia for the mice
Primary standard grade gas (tank and regulator)	Praxair	NI CD5000O6P-K/PRS 2012-2331-590	20.50% Oxygen, 0.50% CO2 balanced with nitrogen used for calibration