我们感谢阿尔弗雷德医学研究和教育局动物服务小组 (AMREP) 的工作人员对这项研究中使用的小鼠的援助和照顾, 并支持维多利亚国家的运营基础设施支持计划。政府。

所描述的所有实验都得到了阿尔弗雷德医学研究教育局动物伦理委员会 (AMREP) 的批准, 并根据澳大利亚国家卫生和医学研究理事会 (澳洲) 提供了人道护理, 以指导动物实验。动物被管理他们的规定的饮食和水ad 随意和安置在温度控制的环境 (~ 21-22 °c) 以 12 h 光和 12 h 黑暗的周期。七周大的雄性小鼠 (在 C57Bl/6J 背景下) 喂食正常的饮食 (能量含量 14.3 MJ/千克, 包括76% 焦从碳水化合物, 5% 脂肪, 19% 蛋白; 见材料表) 或高脂肪喂养组, 高脂肪饮食 (风) (能量含量19兆焦耳/千克, 包括36% 焦从碳水化合物, 43% 脂肪, 21% 蛋白, 专业饲料) 3 周。体重和身体成分测量使用 EchoMRI 机每周, 而代谢监测分析发生在一个蛤后3周的饮食。
1. 人体成分分析仪程序
注意:为了优化功能, 本协议中使用的 4-1 的 EchoMRI 应包含在空气温度稳定且不波动的房间内。理想情况下, 应该不断地监测这一点。如果可能的话, 机器的移动和电源的中断也应该避免。如果电源已中断并且系统必须重新启动, 则允许至少 2-3 小时的计算机预热, 然后再使用它。在开始之前, 请确保您佩戴的是正确的个人防护设备。
<ol><li>在扫描小鼠之前, 在人体成分分析仪机上进行系统测试。这包括使用校准标准 (称为菜籽油系统测试样本 (成本)) 来测试仪器的精度, 并确保其准确性没有漂移。<ol>
<li>打开系统软件, 然后单击 "系统测试工具栏" 按钮或同时按 "Alt + Y"。</li>
		<li>在计算机执行系统测试之前, 请等待提醒, 以验证是否已将正确的成本 (在本例中为鼠标特定成本) 放在系统的龙门内 (图 1)。一旦证实这确实是事实, 接受进行测试, 这将需要几分钟完成。</li>
	</ol>
</li>
	<li>系统测试通过后, 继续进行扫描。<ol>
<li>如果系统测试失败, 请重复系统测试。</li>
		<li>如果机器继续超出范围 (表明发生了偏差), 则可能需要校准以纠正这种情况。按照购买时提供的用户手册中的提示或说明完成此操作。如果问题仍然存在, 请检查手动3或向制造商的支持团队报告问题, 并寻求进一步的指导。</li>
	</ol>
</li>
	<li>把老鼠放在一个小动物标本夹 (长筒) 里, 把它们放在机器里。要做到这一点, 把持有人水平, 拿起鼠标, 并插入到气缸头的开头。慢慢地, 小心地把持有者的垂直位置, 使鼠标在气缸底部, 并准备分析。</li>
	<li>一旦在持有者内, 插入分隔符以限制鼠标在测量期间的移动。在某些情况下, 有非常活跃的老鼠, 可能需要用指尖握住分隔符。 注意: 在最初分析前, 让老鼠熟悉标本持有者的位置, 以减轻压力。使用一个红色的动物标本持有者也可以减少潜在的压力反应, 因为老鼠觉得他们在黑暗中。</li>
	<li>在软件中, 选择一个文件夹 (文件夹工具栏) 以保存数据并创建文件名。</li>
	<li>如有必要, 通过增加扫描的初始累积数, 减少脂肪和精益测量中的随机噪声量。一旦软件启动, 主堆积就被设定为一般日常使用的推荐默认值;除非有特定的原因更改这些参数, 否则默认设置将给用户提供必要的精度级别。</li>
	<li>如果没有兴趣获得免费水和总水的数据, 通过选择选项卡说不, 关闭水阶段。这样做将大大缩短扫描时间, 提高吞吐量。</li>
	<li>通过选择 "开始扫描" 或按键盘上的 F5 启动扫描。输入有关该动物的所有相关数据 (例如,动物 ID、体质量、等) 并按 "确定" 或 F5 开始扫描, 这将需要大约1分钟。</li>
	<li>获得数据后, 从机器上取出含有鼠标的动物支架, 将动物放回笼子里。一旦所有动物都经过扫描, 导出数据进行进一步分析和归类。</li>
	<li>使用前后, 根据制造商的指示彻底清洁动物的持有者。由于这些持有人是由丙烯酸塑料建造, 应避免异丙醇和乙醇, 因为它们可能导致开裂的持有人和/或迅速恶化的持有人, 从而增加了破碎的可能性。相反, 要么使用暖洗碗水溶液, 或者, 如果需要进一步消毒剂, 使用 F10 (1:125 稀释) 或其他消毒剂或清洁喷雾剂 (见材料表), 然后擦掉。</li>
</ol>2. 代谢动物监测系统程序
注: 系统需要2小时的预热和稳定。如果机器已关闭, 必须打开它, 使氧化锆电池加热到725摄氏度。此外, 我们通常在进入动物监测系统前一天在人体成分分析仪中放置小鼠, 以避免任何束缚压力的问题。
<ol><li>确保连接到动物监控系统的计算机开启并打开控制程序。从 "工具" 菜单中选择 "Oxymax 实用程序" 选项以启动泵。</li>
	<li>用适当的水填满水瓶, 权衡并检查小鼠的健康状况, 组织食物。如果测量系统中的食物摄入量, 考虑对食物进行粉粉。通过压抑弹簧加载的平台, 把食物塞进料斗, 填满食物料斗。确保食品料斗和水瓶完全充分, 以确保有足够的食物和水, 以持续分配的实验时间。</li>
	<li>检查 drierite/干燥剂的状态;如果使用颜色指示器, 它应该是蓝色的, 因此干燥, 但如果它是粉红色/紫色, 它有明显的吸湿性, 应更换或顶部。</li>
	<li>检查氨陷阱和苏打石灰的条件, 并在需要时更换。如果氨陷阱一次连接两个, 当第二个陷阱显示颜色变化的迹象, 替换第一个。CO2偏移量的增加也意味着需要更换苏打石灰。 注: 干燥剂可以在烤箱中烘干并重新使用, 但是我们遵循系统制造商的建议, 每次都要用新鲜的。</li>
	<li>组装房间。要做到这一点, 把食物料斗放在天平上, 然后把房间放在顶部, 然后用穿孔的平台将它插入房间的地板上。小心地将鼠标放在腔内, 并将系统的盖子与前面和后面的夹子固定在一起, 然后在定位水瓶和紧固之前安全地安装。作为预防措施, 重新检查所有室盖、鼠标和水 (图 2A-d)。 注意: 根据被检查的老鼠的大小, 可能有必要调整食物料斗上方的空间高度, 这样老鼠就可以接触到食物, 但没有足够的空间让它们直接睡在喂食器的顶端。</li>
	<li>由于建议在每次试验前校准气体传感器, 校准系统。<ol>
<li>使用已知成分的气体 (0.5% CO2, 20.5% O2, 平衡氮气)。通过调节器和软管将校准气罐连接到系统。打开并确保油箱输出压力读数为 5-10 psi。 注: 一些系统将有第二罐, 软管和调节器使用纯氮气作为 "抵消" 气体。我们操作的系统使用苏打石灰生成 CO2自由空气。</li>
		<li>按照步骤校准 O2和 CO2传感器。从 "工具" 菜单中选择 "校准", 并依次校准 O2和 CO2。在校准前确保 1) 样品和参考流量为 0.400LPM, 2) 氧化锆 O2传感器温度为725°c (@ 1 °c), 3) 样品和参考干燥器和空气泵, 和 4) 校准气体附加和打开。</li>
		<li>如果需要, 在校准 o2传感器时, 稍微调整氧化锆氧传感器前面的偏移控制, 以实现 O2比值值 1.0000 (@ 0.0002)。这是为了确保它在可接受的范围内 (在计算机屏幕上的软件显示中以绿色字体突出显示)。</li>
		<li>成功 O2和 CO2传感器校准后, 关闭校准气瓶并断开软管与调节器的连接。校准后, O2用于参考空气 (大气) 应改为 20.92 (00.02)。如果校准超出公差, 请重复, 并参阅制造商的故障排除指南。如果失败, 请与制造商联系以获取进一步的说明。</li>
	</ol></li>
	<li>进行实验设置。从实验菜单中选择 "实验文件打开"。选择适当的模板 (例如,鼠标)。在实验菜单的 "设置" 下, 定义应记录的实验参数 (例如,鼠标 ID、权重、组、等) 取消选择不使用的任何分庭, 并选择要保存实验的位置。</li>
	<li>通过在实验菜单中选择 "运行", 确保测量食物摄取量和开始捕获数据时 tared。数据的捕获时间长短取决于表型、动物隔离的机构指南和系统使用情况。 注: 在我们的手, 实验是例行运行48小时, 与前24小时作为驯化的新环境和第二个24小时用于数据分析。数据收集期是根据调查员希望保持老鼠单独居住和依赖动物伦理认可的时间而定的。或者, 如果有规定, 在进入系统和连接之前, 老鼠可能被适应在分庭。在使用12室系统时, 每室每隔13分钟测量一次。</li>
	<li>定期检查和监测在老鼠在系统中获得的结果, 以确保动物福利和收集适当的数据。任何问题都可以在现阶段予以确认并予以纠正。每天早晚在系统中检查每个鼠标。</li>
	<li>检查数据文件页顶部的 "新陈代谢" 选项卡, 以查看每只鼠标在耗氧量、汇率和能源消耗方面实时收集的数据。同时, 分束和食物消耗数据可以分别位于活动和喂食标签中。检查 "O2 " 是否在读取大约 20.90-20.94, "CO2在" 大约 0.040-0.050, 汇率在0.7 和1之间, 并且流量在 0.5-0.6 升/分钟之间是恒定的。</li>
	<li>定期检查老鼠是否能获得食物和水, 而且它们都在消耗它们。确保他们没有显示任何遇险迹象, 如挖在穿孔地板。此外, 还要监视显示的结果。</li>
	<li>在分配的实验时间完成后, 从实验菜单中选择 "停止" 并导出结果 (如 csv 文件、文件 > 导出 > 生成主题 csv) 进行分析。</li>
	<li>检查老鼠的健康状况, 权衡它们, 然后返回他们的家庭笼子。<ol>
<li>老鼠在分离后可能会互相敌视, 所以当他们再次被安置在一起时, 就进行监控。</li>
		<li>拆卸笼子, 从料斗中取出多余的食物, 然后从笼子里排出任何粪便、尿液和食物。将瓶子和小排量浸入稀释的 T-bac 溶液中, 浸泡并清洁稀释漂白剂溶液中的其它成分。用清水冲洗, 然后离开空气干燥。</li>
	</ol></li>
	<li>用软件计算新陈代谢参数。该软件利用许多等式提供最终数据输出4。 用于计算耗氧量和二氧化碳产量: 耗氧量: VO2 (山泥倾泻)= vio2i v o o2o;二氧化碳生产: VCO2 (山泥倾泻)= Vo2o-Vico2i 位置: Vi = 输入通风率 (山泥倾泻), Vo = 输出通风速率 (山泥倾泻), o2i = o2集中在输入, o2o = o2浓度在输出, co2i = co2在输入时集中, co2o = CO2在输出中的浓度。 用于计算汇率:汇率 = VCO2 /VO2。请注意, 蛋白质氧化没有被测量, 因此没有调整的汇率。 能源支出计算: 能源支出: CV = 3.815 + 1.232 * 汇率 热量 (大卡/小时)) = CV * VO2.其中: CV 是热值 (热量与耗氧量的关系)。这是源自 "营养科学的元素", 称为 Lusk 表, 由格雷厄姆 Lusk。</li>
</ol>

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作者没有什么可透露的。

关键步骤
本文所述的协议提供了一个例子, 其中的方法, 以测量身体成分和各种代谢参数的小鼠使用身体成分分析仪和代谢动物监测系统。对于这两种技术, 至关重要的是要确保机器的工作是最佳的, 而且要做到这一点, 研究人员必须对人体成分分析仪进行系统测试, 并校准到已知的代谢气体成分。动物监测系统使用前的设备。这将确保结果更加一致, 并有机会发现?...

新陈代谢支撑着正常细胞、器官和全身生理学的许多方面。因此, 在各种病理的设置中, 新陈代谢的改变可能直接导致潜在的状况, 或者可能会受到负面影响, 作为病理的副作用。传统上, 代谢研究和能量平衡研究集中在肥胖和相关的疾病领域, 如胰岛素抵抗、糖尿病前、葡萄糖不耐受、心血管疾病和糖尿病。鉴于世界范围内这种情况不断升级, 以及这些条件所造成的个人、社会和经济代价, 这项研究是有必要的。因此, 制定预防战略和针对肥胖的新疗法是世界各地研究实验室的一个持续目标, 这些研究主要依赖于临床前老鼠模型。
虽然称重小鼠对体重的增加或损失提供了可靠的评估, 但它并没有提供构成全身成分 (脂肪质量、精益质量、游离水以及毛皮和爪子等其他成分) 的不同成分的分解。在研究完成后, 脂肪垫的重量在老鼠死后提供了一个准确的测量不同的脂肪库, 但只能提供一个单一的时间点的数据。因此, 经常需要招收多个群体, 以调查随着时间的推移, 肥胖的发展, 大大增加动物数量, 时间和成本。使用双能量 X 射线骨密度测定 (DEXA) 提供了一种方法来评估身体脂肪和瘦组织的内容, 并允许研究员获得数据的纵向方式。然而, 这一过程要求老鼠麻醉1, 反复的麻醉可能会影响脂肪组织的积聚或影响新陈代谢调节的其他方面。EchoMRI 利用核磁共振 relaxometry 测量脂肪和精益质量、游离水和总含水量。这是可以实现的, 因为不同的组织成分之间的对比, 在时间, 振幅和空间分布的产生的无线电频率允许划定和定量的每一种组织类型的差异。这种技术是有利的, 因为它是非侵入性的, 快速的, 简单的, 不需要麻醉或辐射, 而且, 重要的是, 已积极验证抗化学分析2。
肥胖的一个关键考虑和相关的研究是能量平衡等式。而脂肪堆积比纯粹的能量更复杂 (食物摄入) 与能量 (能量消耗) 相比, 它们是能够测量的重要因素。每日能量开支是一共四个不同的组分: (1) 基本的能量开支 (休息的新陈代谢率);(2) 由于食品消耗的热效应, 能源支出;(3) 体温所需的能量;(4) 体力活动所消耗的能量。当能源消耗产生热量时, 用一种动物 (称为直接热量计) 来测量热量, 可以用来评估能源支出。或者, 对 o2和 co2的受启发和过期浓度的测量, 允许确定全体 O2消耗量和 co2生产, 可作为间接测量的一种方法 (间接热计量) 加热生产, 从而计算能源消耗。食物摄入量的增加或能源支出的减少将使老鼠容易发胖, 观察这些参数的变化可以为特定的肥胖模型提供可能的行动机制的有用信息。一个相关的代谢参数的兴趣是呼吸交换比 (汇率), 一个指标的基质/燃料 (即,碳水化合物或脂肪) 的比例正在进行新陈代谢和被用来产生能量。因此, 测量食物摄入量 (能量消耗) 与体力活动水平相结合, O2消费、汇率和能源支出可以提供对机体代谢剖面的广泛理解。收集这些数据的一种方法是使用一个综合的实验动物监测系统 (蛤), 它是基于间接量热法测量能量消耗, 并具有确定物理活动水平的额外能力 (光束断裂) 和食物摄取通过秤纳入测量室。
在本议定书中, 我们提供了一个直接的描述, 使用人体成分分析仪来评估小鼠体内成分和代谢动物监测系统, 以衡量新陈代谢的各个方面。将讨论这些技术的考虑和限制, 以及建议的分析、解释和数据表示方法。

<table><tbody><tr><td>4 in 1 system</td><td>EchoMRI</td><td>4 in 1 system</td><td>Whole body composition analyser</td></tr><tr><td>Canola oil test sample (COSTS)</td><td>EchoMRI</td><td>Mouse-specific (contact company for cat number)</td><td></td></tr><tr><td>Animal specimen holder&amp;nbsp;</td><td>EchoMRI</td><td>103-E56100R</td><td></td></tr><tr><td>Delimiter&amp;nbsp;</td><td>EchoMRI</td><td>600-E56100D</td><td></td></tr><tr><td>12 chamber system</td><td>Columbus Instruments</td><td>Custom built</td><td>Metabolic Caging System; includes control program</td></tr><tr><td>Drierite</td><td>Fisher Scientific</td><td>238988</td><td>CLAMS consumable</td></tr><tr><td>Calibration gas tank</td><td>Air Liquide</td><td>Mixed to order</td><td>Gas calibration (0.5% CO&lt;sub&gt;2&lt;/sub&gt;, 20.5% O&lt;sub&gt;2&lt;/sub&gt;, balance nitrogen).&amp;nbsp;</td></tr><tr><td>Normal chow diet</td><td>Specialty Feeds</td><td>Irradiated mouse and rat diet</td><td></td></tr><tr><td>High fat diet</td><td>Specialty Feeds</td><td>SF04-001</td><td></td></tr><tr><td>Balance</td><td>Mettler Toledo</td><td>PL202-S</td><td>Balance for weighing mice</td></tr><tr><td>TexQ Disinfectant spray</td><td>TexWipe</td><td></td><td></td></tr><tr><td>Hydrogen Peroxide cleaning solution</td><td>TexWipe</td><td>TX684</td><td></td></tr></tbody></table>

body composition and metabolic caging analysis in high fat fed mice

身体成分的改变 (脂肪或精益质量), 代谢参数, 如全身耗氧量, 能量消耗, 和基质的利用, 以及食物摄取和体力活动等行为可以提供重要的信息, 关于疾病的潜在机制。考虑到身体成分和新陈代谢对肥胖的发展及其后继后遗症的重要性, 必须在临床前期研究中对这些参数进行准确的测量。在过去几十年中, 技术的进步使人们有可能以无侵入性和纵向的方式在啮齿动物模型中得出这些措施。因此, 这些代谢措施在评估基因操作 (例如挖空或转基因小鼠、病毒性击倒或基因过度表达)、实验药物/复合筛查和膳食的反应时证明是有用的,行为或身体活动干预。在这里, 我们描述了用于测量身体成分和代谢参数使用动物监测系统在喂食和高脂肪饲料喂养小鼠的协议。

在图 3中看到的结果显示, 在高脂肪喂养时, 身体成分参数的典型变化是通过 EchoMRI 测量的。在基线中, 任何测量参数 (图 3A-f) 都没有差别。然而, 在仅仅1周的高脂肪喂养后, 风组的体重、脂肪质量和脂肪质量百分比显著增加 (图 3A、B、D)。在3周的饮食干预中, 这两组人?...

Watch this Scientific Journal Video about Body Composition and Metabolic Caging Analysis in High Fat Fed Mice at JoVE.com

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

高脂喂养小鼠体内成分及代谢锁定分析

本协议描述了使用人体成分分析仪和代谢动物监测系统来表征小鼠体内成分和代谢参数。以高脂喂养诱发肥胖模型为例, 为这些技术的应用提供借鉴。

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

body-composition-and-metabolic-caging-analysis-in-high-fat-fed-mice

Research

JoVE Journal

Medicine

15.3K 次观看.  Baker Heart and Diabetes Institute. 本协议描述了使用人体成分分析仪和代谢动物监测系统来表征小鼠体内成分和代谢参数。以高脂喂养诱发肥胖模型为例, 为这些技术的应用提供借鉴。

视频: Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

A Protocol for Housing Mice in an Enriched Environment

Environmental enrichment (EE) is a housing environment for mice that boosts mental and physical health compared to standard laboratory housing. Our recent studies demonstrate that environmental enrichment decreases adiposity, increases energy expenditure, resists diet induced obesity, and causes cancer remission and inhibition in mice. EE typically consists of larger living space, a variety of &#8216;toys&#8217; to interact with, running wheels, and can include a number of other novel environmental changes. All of this fosters a more complex social engagement, cognitive and physical stimulations. Importantly, the toy location and type of toy is changed regularly, which encourages the mice to adapt to a frequently changing and novel environment. Many variables can be manipulated in EE to promote health effects in mice. Thus these approaches are difficult to control and must be properly managed to successfully replicate the associated phenotypes. Therefore, the goal of this video is to demonstrate how EE is properly set up and maintained to assure a complex, challenging, and controlled environment so that other researchers can easily reproduce the protective effects of EE against obesity and cancer.

Environmental enrichment for mice requires a complex and challenging setup, as well as comprehensive husbandry and handling techniques to assure robust metabolic and anti-cancer effects in the mice. This protocol provides detailed procedures to reproduce the above mentioned effects in mice.

在丰富环境的协议房屋小鼠

Environmental enrichment (EE) is a housing environment for mice that boosts mental and physical health compared to standard laboratory housing. Our recent ...

科研

行为学

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

在自觉的，奔放的小鼠正常血糖高胰岛素 - 夹钳

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

医学

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

利用X射线计算机断层扫描在小鼠肥胖模型的FAT卷的分割和测量

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

优化分析<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;脂肪肝

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to insulin-stimulated glucose uptake and a gross decompensation of systemic glucose metabolism. Despite considerable progress in the understanding of glucose metabolism, the molecular mechanisms of its regulation in health and disease remain under-investigated, while novel approaches to prevent and treat diabetes are urgently needed. Diet derived glucose stimulates the pancreatic secretion of insulin, which serves as the principal regulator of cellular anabolic processes during the fed-state and thus balances blood glucose levels to maintain systemic energy status. Chronic overfeeding triggers meta-inflammation, which leads to alterations in peripheral insulin receptor-associated signaling and thus reduces the sensitivity to insulin-mediated glucose disposal. These events ultimately result in elevated fasting glucose and insulin levels as well as a reduction in glucose tolerance, which in turn serve as important indicators of insulin resistance. Here, we present a protocol for the generation and metabolic characterization of high-fat diet (HFD)-fed mice as a frequently used model of diet-induced insulin resistance. We illustrate in detail the oral glucose tolerance test (OGTT), which monitors the peripheral disposal of an orally administered glucose load and insulin secretion over time. Additionally, we present a protocol for the insulin tolerance test (ITT) to monitor whole-body insulin action. Together, these methods and their downstream applications represent powerful tools to characterize the general metabolic phenotype of mice as well as to specifically assess alterations in glucose metabolism. They may be especially useful in the broad research field of insulin resistance, diabetes and obesity to provide a better understanding of pathogenesis as well as to test the effects of therapeutic interventions.

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

The current article describes the generation and metabolic characterization of high-fat diet-fed mice as a model of diet-induced insulin resistance and obesity. It further features detailed protocols to perform the oral glucose tolerance test and the insulin tolerance test, monitoring whole-body alterations of glucose metabolism in vivo.

口服葡萄糖耐受试验 (OGTT) 和胰岛素耐受试验 (ITT) 对高脂饮食喂养小鼠体内葡萄糖代谢的影响研究 (英文)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

生化和高通量的微观评估的脂肪含量<em&gt;秀丽隐杆线虫</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

生物学

Palatable Western-style Cafeteria Diet as a Reliable Method for Modeling Diet-induced Obesity in Rodents

Obesity is rapidly increasing in incidence in developed and developing countries and is known to induce or exacerbate many diseases.&#160;The health burden of obesity and its comorbid conditions highlight the need for better understanding of its pathogenesis, yet ethical constraints limit studies in humans. To this end externally valid models of obesity in laboratory animals are essential for the understanding of being overweight and obesity.&#160;While many species have been used to model the range of changes that accompany obesity in humans, rodents are most commonly used. Our laboratory has developed a western-style cafeteria diet that consistently leads to considerable weight gain and markers of metabolic disease in rodents. The diet exposes rodents to a variety of highly palatable foods to induce hyperphagia, modeling the modern western food environment. This diet rapidly induces weight gain and body fat accumulation in rats allowing for the study of effects of overeating and obesity. While the cafeteria diet may not provide the same control over macronutrient and micronutrient profile as purified high-fat or high-fat, high-sugar diets, the cafeteria diet typically induces a more severe metabolic phenotype than that observed with purified diets and is more in line with metabolic disturbances observed in the overweight and obese human population.

This protocol describes the use of a highly palatable, western-style cafeteria diet to model overeating and obesity in rodents. Here, we provide a detailed outline of food selection, preparation and measurement, and explain methodological factors that assist in generating a robust and reproducible phenotype.

美味的西式自助餐厅饮食作为模拟啮齿动物饮食诱发肥胖症的可靠方法

Obesity is rapidly increasing in incidence in developed and developing countries and is known to induce or exacerbate many diseases.&#160;The health ...

Assessing Whole-Body Lipid-Handling Capacity in Mice

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a class of many different molecules with many pathways involved in their synthesis and metabolism. A starting point for evaluating lipid hemostasis for nutrition and obesity research is needed. This paper describes three easy and accessible methods that require little expertise or practice to master, and that can be adapted by most labs to screen for lipid-metabolism abnormalities in mice. These methods are (1) measuring several fasting serum lipid molecules using commercial kits (2) assaying for dietary lipid-handling capability through an oral intralipid tolerance test, and (3) evaluating the response to a pharmaceutical compound, CL 316,243, in mice. Together, these methods will provide a high-level overview of lipid handling capability in mice.

This paper provides three easy and accessible assays for assessing lipid metabolism in mice.

评估小鼠全身脂质处理能力

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a ...

Identification and Dissection of Diverse Mouse Adipose Depots

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, uncoupling of metabolism to generate heat, and secretion of adipokines to regulate whole-body homeostasis and immune responses. Emerging research is identifying important regional differences in the developmental, molecular, and functional profiles of adipocytes located in discrete depots throughout the body. Different properties of the depots are medically relevant since metabolic diseases often demonstrate depot-specific effects. This protocol will provide investigators with a detailed anatomic atlas and dissection guide for the reproducible and accurate identification and excision of diverse mouse adipose tissues. Standardized dissection of discrete adipose depots will allow detailed comparisons of their molecular and metabolic characteristics and contributions to local and systemic pathologic states under various nutritional and environmental conditions.

Adipocytes exist in discrete depots and have diverse roles within their unique microenvironments. As regional differences in adipocyte character and function are uncovered, standardized identification and isolation of depots is crucial for advancement of the field. Herein, we present a detailed protocol for the excision of various mouse adipose depots.

各种鼠标脂肪库的识别和解剖

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, ...

Assessment of the Metabolic Effects of Isocaloric 2:1 Intermittent Fasting in Mice

Intermittent fasting (IF), a dietary intervention involving periodic energy restriction, has been considered to provide numerous benefits and counteract metabolic abnormalities. So far, different types of IF models with varying durations of fasting and feeding periods have been documented. However, interpreting the outcomes is challenging, as many of these models involve multifactorial contributions from both time- and calorie-restriction strategies. For example, the alternate day fasting model, often used as a rodent IF regimen, can result in underfeeding, suggesting that health benefits from this intervention are likely mediated via both caloric restriction and fasting-refeeding cycles. Recently, it has been successfully demonstrated that 2:1 IF, comprising 1 day of fasting followed by 2 days of feeding, can provide protection against diet-induced obesity and metabolic improvements without a reduction in overall caloric intake. Presented here is a protocol of this isocaloric 2:1 IF intervention in mice. Also described is a pair-feeding (PF) protocol required to examine a mouse model with altered eating behaviors, such as hyperphagia. Using the 2:1 IF regimen, it is demonstrated that isocaloric IF leads to reduced body weight gain, improved glucose homeostasis, and elevated energy expenditure. Thus, this regimen may be useful to investigate the health impacts of IF on various disease conditions.

The current article describes a detailed protocol for isocaloric 2:1 intermittent fasting to protect and treat against obesity and impaired glucose metabolism in wild-type and ob/ob mice.

小鼠间间间禁食的代谢效应评估 2:1 间歇禁食

Intermittent fasting (IF), a dietary intervention involving periodic energy restriction, has been considered to provide numerous benefits and counteract ...

高脂喂养小鼠体内成分及代谢锁定分析

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