Name: determining basal energy expenditure and the capacity of thermogenic adipocytes to expend energy in obese mice
Uploaded: 2021-11-11T13:00:00
Description: Watch this Scientific Journal Video about Determining Basal Energy Expenditure and the Capacity of Thermogenic Adipocytes to Expend Energy in Obese Mice at JoVE.com

ML is funded by the Department of Medicine at UCLA, pilot grants from P30 DK 41301 (UCLA:DDRC NIH) and P30 DK063491 (UCSD-UCLA DERC).

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 &#176;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)
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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...

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 organism1. The Standard condition was classically defined as heat production occurring in an awake but resting adult, not ingesting or digesting food, at thermoneutrality and without any stress1. The Basal Metabolic Rate (BMR) or basal energy expenditure in mice is referred to as the SMR but in individuals ingesting and digesting food under mild thermal stress (ambient temperatures 21-22 &#176;C)1. The challenges and difficulties of directly measuring heat production made indirect calorimetry, namely calculating heat production from oxygen consumption measurements, to become the most popular approach to determine the BMR. Calculating the BMR from oxygen consumption is possible because the oxidation of nutrients by mitochondria to synthesize ATP is responsible for 72% of the total oxygen consumed in an organism, with 8% of total oxygen consumption also occurring in mitochondria but without generating ATP (uncoupled respiration)1. The majority of the remaining 20% of oxygen consumed can be attributed to nutrient oxidation in other subcellular locations (peroxisomal fatty acid oxidation), anabolic processes, and reactive oxygen species formation1. Thus, in 1907, Lusk established an equation, based on empirical measurements, widely used to transform oxygen consumption and CO2 production into energy dissipation as heat. In humans, the brain accounts for ~25% of the BMR, the musculoskeletal system for ~18.4%, the liver for ~20 %, the heart for ~10%, and the adipose tissue for ~3-7%2. In mice, the tissue contribution to BMR is slightly different, with the brain representing ~6.5%, the skeletal muscle ~13%, the liver ~52%, the heart ~3.7%, and adipose tissue ~5%3.
Remarkably, the biochemical reactions defining the BMR are not fixed and change in response to different needs, such as external work (physical activity), development (tissue growth), internal stresses (counteracting infections, injuries, tissue turnover), and changes in ambient temperature (cold defense)1. Some organisms actively recruit processes to generate heat in cold exposure, implying that heat produced by metabolism is not just an accidental byproduct. Instead, evolution selected regulatory mechanisms that could specifically upregulate heat production by changing the rate of metabolic reactions1. Thus, these same oxygen consumption measurements can be used to determine the capacity of an organism to generate heat in response to cold.
Two major processes contribute to heat generation upon cold exposure. The first one is shivering, which generates heat by increasing mitochondrial oxidative phosphorylation and glycolysis in muscle to cover the physical work done by involuntary muscle contraction. Therefore, cold exposure will increase oxygen consumption in muscles1. The second is Non-Shivering Thermogenesis, which occurs through an increase in oxygen consumption in brown and beige adipocytes (BA). Dissipation of energy into heat in BA is mediated by the mitochondrial uncoupling protein 1 (UCP1), which allows proton re-entry into the mitochondrial matrix, decreasing the mitochondrial proton gradient. The dissipation of the mitochondrial proton gradient by UCP1 increases heat production by the elevation in electron transfer and oxygen consumption and the energy released by proton dissipation per se without generating ATP (uncoupled). Moreover, thermogenic BA can recruit additional mechanisms that elevate oxygen consumption without causing a large dissipation in the proton gradient, by activating futile oxidative ATP synthesis and consumption cycles. The metabolic cages described here, namely the CLAMS-Oxymax system from Columbus Instruments, offer the possibility to measure energy expenditure at different ambient temperatures. However, to determine BA thermogenic capacity using whole-body oxygen consumption measurements, one needs to: (1) eliminate the contribution of shivering, and other non-BA metabolic processes to energy expenditure, and (2) specifically activate BA thermogenic activity in vivo. Thus, a second protocol describes how to selectively activate BA in vivo using pharmacology in anesthetized mice at thermoneutrality (30 &#176;C), with anesthesia and thermoneutrality limiting other non-BA thermogenic processes (i.e., physical activity). The pharmacological strategy to activate BA is treating mice with the &#946;3-adrenergic receptor agonist CL-316,246. The reason is that cold exposure promotes a sympathetic response releasing norepinephrine to activate &#946;-adrenergic receptors in BA, which activates UCP1 and fat oxidation. Furthermore, &#946;3-adrenergic receptor expression is highly enriched in adipose tissue in mice.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CLAMS-Oxymax System</td>
			<td>Columbus Instruments</td>
			<td>CLAMS-center feeder-ENC</td>
			<td>Including enviromental enclosure and Zirconia oxygen sensor</td>
		</tr>
		<tr>
			<td>Desktop PC with Oxymax Software</td>
			<td>HP/Columbus</td>
			<td>N/A</td>
			<td>PC needed to be purchased separately</td>
		</tr>
		<tr>
			<td>Drierite jug (Calcium Sulfate with Cobalt Chloride Indicator)</td>
			<td>Fisher Scientific</td>
			<td>23-116681</td>
			<td>Needed to dry the gas entering the oxygen sensor, humidity can damage the sensor</td>
		</tr>
		<tr>
			<td>NMR for body composition</td>
			<td>Echo-MRI</td>
			<td>Echo-MRI 100</td>
			<td>Measure lean and fat mass in alive mice. It is necessary for ANCOVA analyses.</td>
		</tr>
		<tr>
			<td>CL-316-243</td>
			<td>Sigma</td>
			<td>C5976</td>
			<td>Injected to the mice subcutaneously to activate thermogenesis</td>
		</tr>
		<tr>
			<td>High fat diet</td>
			<td>Research Diets</td>
			<td>D12266B</td>
			<td>Provided to the mice prior and during measurements</td>
		</tr>
		<tr>
			<td>Pentobarbital/Nembutal</td>
			<td>Pharmacy at DLAM</td>
			<td>N/A</td>
			<td>Anesthesia for the mice</td>
		</tr>
		<tr>
			<td>Primary standard grade gas (tank and regulator)</td>
			<td>Praxair</td>
			<td>NI CD5000O6P-K/PRS 2012-2331-590</td>
			<td>20.50% Oxygen, 0.50% CO2 balanced with nitrogen used for calibration</td>
		</tr>
	</tbody>
</table>

determining basal energy expenditure and the capacity of thermogenic adipocytes to expend energy in obese mice

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.

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...

Watch this Scientific Journal Video about Determining Basal Energy Expenditure and the Capacity of Thermogenic Adipocytes to Expend Energy in Obese Mice at JoVE.com

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

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

Energy expenditure measurements are necessary to understand how changes in metabolism can lead to obesity. Basal energy expenditure can be determined in ...

This protocol allows a researcher to determine whether there are differences in the energetic balance of mice, which can help identifying the physiological processes responsible for a change in body weight. It can also be used to determine the capacity of brown fat to expend energy. This technique quantifies the oxygen consumed and the CO2 produced by an individual mouse in a controlled environment.It allows users to calculate the energy expenditure in this individual mouse and determine whether different treatments or genetic manipulations can change energy expenditure. To begin, open the software controlling the enclosure and the airflow and let the software test the computer's communication with the equipment. Once communication is established, click file, then open experiment configuration and select the experiment configuration pre-designed by the vendor or set up from a previous assay.Next, click on experiment, then click on properties to open the experiment properties window. In the properties window, set up the parameters of the environmental enclosure including the ambient temperature and 12-hour light cycles. Then click on experiment, followed by setup to open the experiment setup window where the parameters of each metabolic cage are defined.Next, add a pre-weighed amount of food to the feeders in the metabolic cages housing eight-week-old female mice. Also, place the water bottles and check that the bottles are sealed correctly and do not leak. After 24 hours, weigh the food remaining in the cages and weigh the mice.Two to three days after the mice have been housed in the system, start indirect calorimetry and activity measurements. First, calibrate the CLAMS systems oxygen and carbon dioxide zirconia-based detector using a calibration gas of known composition. After turning on and ensuring that the tank output pressure is between 5 to 10 pounds per square inch, open the calibration utility software for calibrating and testing the gas sensors.Click on experiment, then calibrate and press start. Wait for the sensors to be tested and for the software to ask the user to turn the knobs of the gas sensor until the value of oxygen identity is one. Click next when the step is complete.After measuring the body weight and composition of the mice, start the oxygen, carbon dioxide, and activity measurements by clicking on experiment and run. After a minimum of 48 hours, stop the experiment by clicking experiment, then stop. To export the data, click on file, then export and export all subjects as a CSV file.During the dark phase, mice have higher oxygen consumption and carbon dioxide production, and thus have higher energy expenditure. Mice on a regular diet and a fed state with food ingestion occurring in the dark cycle are characterized by respiratory exchange range ratio values close to one, indicating a preference to use carbohydrates. During the light cycle when mice mostly sleep and thus fast, there is a shift to fat oxidation with RER values being closer to 0.7.Accordingly, physical activity measured as XYZ laser beam break counts increases during the dark phase and decreases during the light phase. High-fat diet feeding increases body weight and fat mass without changing the lean mass. High-fat diet-fed mice ate more kilocalories per day, mainly due to higher caloric density per gram of food.However, physical activity is similar between the chow and high-fat diet-fed mice even during the dark period. Lower respiratory coefficient ratio values of high-fat diet-fed mice indicate a preference to use fat as the primary substrate for oxidation. Oxygen consumption, but not carbon dioxide production, increases in high-fat diet-fed mice, which results in a significant increase in energy expenditure per mouse.Energy expenditure is increased by a beta-3 agonist injection, mainly as a result of the adrenergic activation of thermogenic adipocytes, which raises oxygen consumption, carbon dioxide production, and thus energy expenditure itself. To measure energy expenditure with the CLAMS, the calibration must be completed and body weight should be measured to perform the ANCOVA analysis. Also, food intake must be measured in both the adaptation period and during measurements.The adaptation period is over when mice recover the initial food intake and body weight. This procedure can also be used to test the acute effects of drugs on mouse activity and behavior. We also use this system in a collaborative study that demonstrated which population of neurons controlled This technique is essential to study diseases related to weight gain and for identifying processes controlling nutrient preference as well as physical activity, thermogenesis, and energy balance.

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