搜索

Measurement of Resting Energy Metabolism in Mice Using Oxymax Open Circuit Indirect Calorimeter
采用Oxymax 开放环路间接能量测定仪测量小鼠中的静息能量新陈代谢   

评审
匿名评审
引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Nature Medicine
Aug 2014

Abstract

Indirect calorimeter is a powerful tool to monitor resting energy metabolism through the measurement of oxygen (O2) consumption and carbon dioxide (CO2) production. From the measurement of VO2 and VCO2, the respiratory exchange ratio (RER) can be calculated to assess energy fuel utilization and energy expenditure (Evan et al., 2012). Previously, indirect calorimeter has been widely used in metabolic disease research in mice to reveal the potential roles of specific genes or treatments in regulating energy metabolism (for example: Bi et al., 2014; Feng et al., 2014). Here, we described a protocol to evaluate the resting energy metabolism of C57BL/6 mice during dark and light cycles using the Oxymax Open Circuit indirect calorimeter.

Keywords: Brown adipose tissue (棕色脂肪组织), Skeletal muscle (骨骼肌), Oxygen consumption (耗氧), Insulin resistance (胰岛素抵抗), Diabetes (糖尿病)

Materials and Reagents

  1. Adult mice (C57BL/6 male mice at 3-month old were used for data acquisition in this protocol, but male or female mice of other genetic backgrounds or strains, at different ages can be used)
  2. Food (normal chow diet or high fat diet) and water (ad lib)
  3. Compressed gas mixture with the components of 4,929 PPM CO2, 20.47% O2 and Balance N2

Equipment

  1. Oxymax Open Circuit Indirect Calorimeter (Columbus Instruments, model: Open Circuit Indirect Calorimeter) (Figure 1)


    Figure 1. Open circuit indirect calorimeter components

  2. Computer with software provided by the manufacture (Columbus Instruments, model: Oxymax v4.91 )

Procedure

  1. Turn on the Oxymax instrument and computer, allowing the system to warm up for 2 h;
  2. Start the Oxymax v4.91 program;
  3. Perform CO2 calibration;
    1. Select calibration in the program, turn on the gas tank and set it to 5-10 psi;
    2. Press the CO2 button in “calibration” window (Figure 2a). Gain should be close to 1 after calibration (Figure 2b);
  4. Perform O2 calibration;
    1. Press the O2 button in “calibration” window (Figure 2a);
    2.  Desired O2 level is listed in the first prompt window (Figure 2d). Use the fine and coarse knobs (Figure 2c) to adjust the O2 level to desired level;
    3. Turn off the gas tank after finishing the O2 calibration.


      Figure 2. CO2 and O2 calibration

  5. Setup a new experiment, choose the chambers to be used, input the Identification number and weight of mice to be measured (Figure 3), only one mouse is allowed per chamber;


    Figure 3. Input of mouse information

  6. Setup the measurement schedule including number of Intervals, time of cage settle, cage measure, reference settle, reference measure, and reference method (Figure 4).


    Figure 4. Setup schedule for measurement. “0” for intervals means the experiment will run indefinitely until stopped by the user. “8” for reference methods means the experiment will measure the reference air after every 8 subjects.

  7. Place the mice in the corresponding calorimetric chambers, standard cages are used (Figure 5);


    Figure 5. Test chamber for mice (standard cage)

  8. Fill the cage with sufficient food and water for a period of 24 h. Ensure that food and water are available ad libitum (Figure 5);
  9. Check if there is any leakage in the system with software as follows:
    Open Tools → Select Sample Pump from Oxymax utility  →  Ensure Test in Valve open and N2 and Ref Air/Cal Valve closed → Turn Sample Pump ON → Select the chamber to be tested in Expansion Interface Disconnect drier input tubing from tested chamber → put the finger over it, If there is no system leaks, the ball on the front of the system sample pump will drop to 0; If not, check all air fittings to assure an air-tight connection and test it again (Figure 6).


    Figure 6. Gas leakage check

  10. Run the experiment for 24 h and export the data in Excel file format, which include the data listed as below:
    1. O2 consumption = (VO2 input) – (VO2 output), ml/kg/h;
    2. CO2 production = (VCO2 output) – (VCO2) input, ml/kg/h;
    3. Respiratory exchange ratio (RER) = VCO2/VO2 Ratio;
    4. Energy Expenditure (Heat production) = calorific value (Cv ) x VO2 =(3.815 + 1.232 x RER) x VO2, cal/h;
  11. Close the experiment and return the mice to their home cages;
  12. Turn off the system and clean the calorimetry with water and appropriate disinfectant.

Representative data


Figure 7. O2 consumption, CO2 production, RER and Energy Expenditure of 3-month old C57BL6 mice during light and dark cycle.
The daily rhythms of metabolic parameters were recorded under a 12 h-light (open bar) and 12 h-dark cycle (black bar) (Left). Data were presented as Means ± SE (n = 5) during light and dark cycle (right). *, p < 0.05; **, p < 0.01 analyzed by the Student’s t-test (comparison of mean values between the light and dark cycles).

Notes

  1. Begin data collection of mice after 1-day of acclimation in the metabolic chambers;
  2. Install the Oxymax system under a constant environmental temperature (22 °C) and 12 h light (6 am-6 pm), 12 h dark cycle (6 pm-6 am);
  3. VO2 and VCO2 were increased by approximately 15% and 28% in dark cycle, respectively;
  4. RER was increased from 0.90 (light cycle) to 0.99 (dark cycle), suggesting a shift in macronutrient source from a mix of fat + carbohydrates to predominant carbohydrates in the dark cycle (the VCO2/VO2 ratio of fatty acid oxidation is 0.7 and carbohydrates oxidation is 1.0);
  5. Energy expenditure was 20% greater in dark than light cycle, with the most active phase of mice being between 7 pm-12 pm;
  6. Indirect calorimetry is a versatile system to investigate alternations of metabolic rate under different conditions. For example:
    1. To compare metabolic homeostasis and energy expenditure in wild type and mutant mice fed with normal chow diet or high-fat-diet;
    2. To investigate changes in metabolic rate with aging, a potential indicator of improved health status.
    3. Oxymax open circuit indirect calorimeter can also be incorporated with other chamber systems, such as activity, body mass, feeding, drinking, food access control, running wheel, urine collection, sleep detection, body core temperature and heart rate to fulfill different experimental designs.

References

  1. Bi, P., Shan, T., Liu, W., Yue, F., Yang, X., Liang, X. R., Wang, J., Li, J., Carlesso, N., Liu, X. and Kuang, S. (2014). Inhibition of Notch signaling promotes browning of white adipose tissue and ameliorates obesity. Nat Med 20(8): 911-918.
  2. Even, P. C. and Nadkarni, N. A. (2012). Indirect calorimetry in laboratory mice and rats: principles, practical considerations, interpretation and perspectives. Am J Physiol Regul Integr Comp Physiol 303(5): R459-476.
  3. Feng, B., Jiao, P., Helou, Y., Li, Y., He, Q., Walters, M. S., Salomon, A. and Xu, H. (2014). Mitogen-activated protein kinase phosphatase 3 (MKP-3)-deficient mice are resistant to diet-induced obesity. Diabetes 63(9): 2924-2934.

简介

间接热量计是通过测量氧(O 2)消耗和二氧化碳(CO 2)生产来监测静息能量代谢的有效工具。 根据VO 2和VCO 2的测量,可以计算呼吸交换比(RER)以评估能量燃料利用和能量消耗(Evan et al。 ,2012)。 以前,间接热量计已经广泛用于小鼠的代谢疾病研究中,以揭示特定基因或治疗在调节能量代谢中的潜在作用(例如:Bi等人,2014; et al。,2014)。 在这里,我们描述了一个协议,以评估C57BL/6小鼠的休息能量代谢在黑暗和光周期使用Oxymax开路间接量热仪。

关键字:棕色脂肪组织, 骨骼肌, 耗氧, 胰岛素抵抗, 糖尿病

材料和试剂

  1. 成年小鼠(3月龄的C57BL/6雄性小鼠用于该方案中的数据采集,但是可以使用其他遗传背景的雄性或雌性小鼠或不同年龄的雄性小鼠)
  2. 食物(正常饮食或高脂肪饮食)和水(自由饮食)
  3. 具有4,929PPM CO 2,20.47%O 2和余量N 2的组分的压缩气体混合物

设备

  1. Oxymax开路间接热量计(哥伦布仪器,型号:开路间接热量计)(图1)


    图1.开路间接量热器组件

  2. 具有由制造商提供的软件的计算机(Columbus Instruments,型号:Oxymax v4.91)

程序

  1. 打开Oxymax仪器和计算机,让系统预热2小时;
  2. 启动Oxymax v4.91程序;
  3. 执行CO 2 校准;
    1. 在程序中选择校准,打开气罐并将其设置为5-10 psi;
    2. 在"校准"窗口中按CO 2 按钮(图2a)。校准后增益应接近1(图2b);
  4. 执行O 2 校准;
    1. 在"校准"窗口(图2a)中按O 2 按钮;
    2.  所需的O 2 级别列在第一个提示窗口中(图2d)。使用 细调和粗调旋钮(图2c)以将O 2级调节到期望的 级别;
    3. 在完成O <2>校准后关闭气罐。


    图2. CO 2 和O 2 校准

    5.设置一个新实验,选择要使用的腔室,输入要测量的小鼠的标识号和重量(图3),每个腔室只允许一只小鼠;


    图3.输入鼠标信息

    6.设置测量时间表,包括间隔数,笼式沉降时间,笼式测量,参考沉降,参考测量和参考方法(图4)。


    图4.测量的安排计划"0"表示实验将无限期运行,直到用户停止。参考方法的"8"意味着实验将在每8个受试者之后测量参考空气。

    7.将小鼠放入相应的量热室中,使用标准笼(图5);


    图5.小鼠测试室(标准笼)

    8.用足够的食物和水给笼子充满24小时。确保食物和水随时可用(图5);
    9.使用软件检查系统是否有任何泄漏,如下所示:
    打开工具→从 Oxymax实用程序 选择 → 确保阀门打开和 N2 和参考空气/校准阀门关闭→打开样品泵在测试室中断开干燥器输入管道→将手指放在其上,如果没有系统泄漏,则系统样品泵前部的球将下降到0;如果没有,检查所有空气配件,以确保气密连接并再次测试(图6)。


    图6.气体泄漏检查

    10.运行实验24小时,并导出Excel文件格式的数据,其中包括如下所列的数据:
    1. ml/kg/h;
      输入(VO 2输出),ml/kg/h;
    2. (VCO 2输入) - (VCO 2)输入,ml/kg/h;
    3. 呼吸交换比(RER)= VCO 2 /VO <2>比率;
    4. 能量消耗(热产生)=热值(Cv)×VO 2 =(3.815 + 1.232×RER)×VO 2,cal/h;
    11.关闭实验并将小鼠送回他们的笼子;
    12.关闭系统,用水和适当的消毒剂清洁量热计

代表数据


图7.在光照和黑暗循环期间3个月大的C57BL6小鼠的O 2消耗,CO 2生产,RER和能量消耗。每日节律在12小时光照(开放条)和12小时 - 黑暗循环(黑色条)(左)下记录代谢参数。在光照和黑暗循环期间(右),数据表示为平均值±SE(n/n = 5)。 *, p 0.05; **, p 通过Student's t 测试(亮和暗循环之间的平均值的比较)分析。

笔记

  1. 开始在代谢室中适应1天后收集小鼠的数据;
  2. 在恒定的环境温度(22℃)和12小时光(6 am-6 pm),12小时黑暗周期(6 pm-6 am)下安装Oxymax系统;
  3. VO 2和VCO 2在暗循环中分别增加了约15%和28%;
  4. RER从0.90(光周期)增加到0.99(暗周期),表明在黑暗周期中,大量营养素源从脂肪+碳水化合物的混合物转移到主要碳水化合物(VCO 2 脂肪酸氧化的比率为0.7,碳水化合物氧化为1.0);
  5. 能量消耗在黑暗中比光周期高20%,小鼠的最活跃期在下午7点至12点之间;
  6. 间接热量学是一种通用的系统,用于研究不同条件下代谢率的变化。例如:
    1. 比较野生型的代谢动态平衡和能量消耗 和喂以正常饮食或高脂肪饮食的突变小鼠;
    2. 调查老年代谢率的变化,这是改善健康状况的潜在指标
    3. 也可以并入Oxymax开路间接热量计 与其他室系统,如活动,体重,喂食, 饮用,食物存取控制,跑轮,尿液收集,睡眠 检测,身体核心温度和心率完成不同 实验设计。

参考文献

  1. Bi,P.,Shan,T.,Liu,W.,Yue,F.,Yang,X.,Liang,XR,Wang,J.,Li,J.,Carlesso,N.,Liu, ,S.(2014)。 抑制Notch信号可促进白色脂肪组织褐变,并改善肥胖。 Nat Med 20(8):911-918
  2. 甚至,P.C。和Nadkarni,N.A。(2012年)。 实验室小鼠和老鼠的间接热量测定:原则,实践考虑,解释和观点。 Physics Regul Integr Comp Physiol 303(5):R459-476。
  3. Feng,B.,Jiao,P.,Helou,Y.,Li,Y.,He,Q.,Walters,M.S.,Salomon,A.and Xu,H。 丝裂原活化蛋白激酶磷酸酶3(MKP-3)缺陷型小鼠对饮食诱导的肥胖。 糖尿病 63(9):2924-2934
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Nie, Y., Gavin, T. P. and Kuang, S. (2015). Measurement of Resting Energy Metabolism in Mice Using Oxymax Open Circuit Indirect Calorimeter. Bio-protocol 5(18): e1602. DOI: 10.21769/BioProtoc.1602.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要谷歌账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。