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.
