这项研究得到了维也纳市市长的医疗科学基金和Österreichische 德国 Laboratoriumsmedizin 和 Klinische 瓦的支持。

这里描述的所有方法都已得到维也纳医科大学动物护理和使用委员会的批准, 并根据欧洲实验动物科学协会联合会 (FELASA) 进行。请注意, 本议定书所述的所有程序都应在机构和政府批准之后以及在技术上精通的工作人员执行。
1. 风喂养小鼠
注意: 在12小时的光/暗循环中保持所有 C57BL/6J 小鼠, 可以免费获得食物和水。
<ol><li>在6周的年龄, 将老鼠放在风 (40-60% 脂肪卡路里) 上8-12 周以诱发肥胖, 同时喂养精益对照组低脂饮食 (LFD) (10% 脂肪卡路里)。</li>
	<li>每周确定小鼠的体重。重量曲线应显示在两组相似的模式, 在风-美联储组更高的斜率。</li>
</ol>2. OGTT
注: 如果在 OGTT 每15分钟内选择血液取样时间点, 则应同时执行最多15只小鼠的实验, 以便每只老鼠至少有1分钟的处理时间。
<ol><li>OGTT 前一天的准备工作<ol>
<li>将小鼠转移到一个有新鲜床上用品的笼子里, 在测试前一夜将它们快速送到 (14 小时), 同时确保小鼠能够获得饮用水 (例如, 在第二天早上下午6:00 将食物移除, 在上午8:00 开始时间)。 注意: 禁食小鼠过夜是标准的方法, 但较短的快速 (5-6 小时) 是更具生理作用的小鼠 (参见讨论了解详细信息)。</li>
	</ol></li>
	<li>实验当天的准备工作 (但在实验之前)<ol>
<li>准备10毫升的20% 葡萄糖溶液 (溶解 d-(+)-葡萄糖在蒸馏水)。 注: 所有用于动物的试剂必须是药理级和无菌的。</li>
		<li>为等离子收集准备一个96井板, 通过为每个取样时间点和每只老鼠填充一井, 与5µL NaEDTA (0.5 M EDTA, pH 8.0 在0.9% 氯化钠, 存贮在 RT)。在实验中, 把这个盘子储存在冰上。 注意: 有关详细的核对清单, 请参见补充图 1 。</li>
	</ol></li>
	<li>测量所有小鼠的体重并用永久性标记标记它们的尾巴, 以使小鼠易于区分 (例如, 鼠标 1 = 1 破折号, 鼠标 2 = 2 破折号,等)。</li>
	<li>葡萄糖测量和血液取样 (图 2)<ol>
<li>小心地用锋利的剪刀 (图 2中的 "变体 A") 切断 1-2 mm 的尾尖。在服用新的血液样本进行血糖测定之前, 一定要先清除掉第一滴血液以避免溶血或组织液的污染。画一个小的血液样本 (〜3µL) 的测量基础的血糖水平 (= 时间点 0) 与血糖。 注意: 检查并调整血糖上的测试条的电荷数。 注: 作为一种替代采血方法, 尼克在图 2中用锋利的手术刀刀片 ("变体 B") 的鼠标侧尾静脉。侧尾静脉通常是访问大约1/3 沿尾巴长度从尾巴尖端, 向基地的尾巴, 为多个样本。推荐使用局部麻醉膏。在动物被送回笼子之前, 至少三十年代在软组织上应用手指按压来阻止血液流动。</li>
		<li>收集血液样本 (大约30µL) 使用新鲜毛细管 (保持毛细血管管水平)。用吸管把吸管顶端放在毛细管末端的顶部, 并小心地将收集到的血液放入96孔板的井中, 同时避免气泡, 从而清空毛细管管。对所有小鼠重复这个过程-一次一个。 注: 作为血液收集的替代品通过毛细管, 使用的吸管调整到正确的体积 (如, 30 µL) 收集血液, 或收集一滴血从尾部石蜡膜, 并吸管到 EDTA 溶液。严格避免石油果冻与血液或血糖试纸的接触, 因为它可能影响随后的葡萄糖和胰岛素测量。 注意: OGTT 对老鼠很有压力: 瘦老鼠在一夜之间会失去大约15% 的体重。此外, 不同时间点的血液取样会导致大量失血。为了便于采血, 可以用凡士林小心地按摩老鼠尾。 注: 机构指南可限制在规定时间内采集的血液的允许数量。取样量和 timepoints 应调整到不超过允许的最大值。应使用小鼠的体重来计算所允许的总血液提取量。</li>
	</ol></li>
	<li>根据体重计算葡萄糖溶液的体积 (1 克葡萄糖/千克体重; 这可以增加到3克/千克), 由口服胃对每只老鼠进行管理。例如, 一个体重为30克的老鼠需要150µL 20% 葡萄糖溶液来管理30毫克葡萄糖。 注: 以葡萄糖为基础的剂量对鼠标的重量是标准程序。如果身体成份数据是可利用的, 葡萄糖的剂量为 OGTT 应该根据稀薄的身体质量计算 (参见讨论为细节)。</li>
	<li>葡萄糖管理<ol>
<li>准备一切在整个实验期间 (定时器, 实验记录表, 葡萄糖监测和试纸, 毛细血管, 注射器, 葡萄糖溶液, 96-井板, 手术刀, 计算器, 平衡, 永久性标记, 工作台纸,吸管与小费, 和手套)。</li>
		<li>对于葡萄糖的应用, 通过牢牢抓住它的颈背来抑制老鼠。对颈部周围的皮肤施加足够的硬度, 防止老鼠扭出束缚, 并适当地把头向后倾斜。还要确保鼠标能正常呼吸。 注: 一旦葡萄糖管理开始, 良好的时间管理是非常重要的。</li>
		<li>小心地管理葡萄糖溶液 (基于步骤 2.5) 直接进入胃使用喂养针。小心地将喂食针通过口朝食道。让老鼠吞下针头: 针头完全下沉到小鼠的下食管/胃里。然后注入葡萄糖溶液 (图 3a)。<ol>
<li>如果遇到任何阻力, 或如果动物立即挣扎, 撤回针, 并重新定位它。在第一次胃后立即启动计时器, 并在1分钟间隔内将葡萄糖管理给所有其他小鼠。 注意: 将一滴葡萄糖溶液直接从喂食针放到老鼠嘴里可能会有帮助, 这会刺激舔舐和吞咽, 从而方便插入针头。插入喂食针时不要施加压力, 因为这可能会严重伤害动物。</li>
		</ol>
</li>
	</ol></li>
	<li>15分钟后, 测量血糖水平与血糖, 并另外采取血液样本 (〜30µL) (详见详细说明在步骤 2.4) 的每一个鼠标在相同的顺序, 因为他们被注射。 注意: 时间管理是非常重要的;尽可能使用与胃相同的时间间隔。让小鼠尽可能自由地移动, 并在整个过程中限制最小化以减少压力, 这可能会改变结果。用一只手的牛奶尾巴, 并收集与其他血液。</li>
	<li>根据预期结果重复步骤 2.7 (例如, 在30、45、60、90、120、150和葡萄糖管理后180分钟)。如果选定的时间点超过120分钟, 确保小鼠能够获得饮用水。确保老鼠总是能接触到饮用水。当实验完成后, 将小鼠返回到装有食物和水的笼子里。 注意: OGTT 对老鼠来说很累人。因此, 在进行下一次代谢测试之前至少要等待1周, 例如 ITT。</li>
	<li>实验后, 离心机血液样本在 2500 x g, 30 分钟, 4 ° c。将上清液 (等离子) 转移到板的空井, 并将其贮存在-20 ° c 直到分析。<ol>
<li>如果存在, 则记录样本的溶血 (见3节)。</li>
	</ol></li>
	<li>使用商业上可用的 ELISA 试剂盒确定血浆胰岛素水平 (请参见材料表), 按照制造商的说明进行操作。 注: 根据禁食状态以及对被调查小鼠的新陈代谢情况, 在这种化验过程中可能出现困难: 隔夜空腹胰岛素水平 (时间点 0) 非常低, 因此接近检测限度。为了避免这个问题, 建议的血浆体积的数量加倍, 相应地将 ELISA 法的结果减半。另一方面, 如果小鼠在 OGTT 时达到胰岛素峰值, 特别是在风喂养的小鼠, 胰岛素水平可能超过检测限度: 稀释样本 (例如, 10 倍与 0.9% NaCl) 和重复 ELISA 检测。血浆样品中的溶血可能导致胰岛素的降解, 导致读数值的减少。降解取决于样品中的时间、温度和血红蛋白浓度。始终保持溶血样品冷或冰, 以减少胰岛素的退化。</li>
</ol>3. ITT
注意: 在执行 ITT 时, 同样的预防措施也必须适用于 OGTT (处理老鼠、血液、血糖和凡士林等)。例如, 所有的注射应在15分钟内进行1分钟的间隔, 如果15只老鼠进行并行测试。对于 ITT, 随后收集的血液样本毛细管管是可选的。
<ol><li>实验前的准备<ol>
<li>在注射胰岛素前至少2小时的快速小鼠, 同时确保小鼠能够饮用饮用水 (例如, 在上午8:00 时取出食物, 在2-5 小时后测试小鼠)。</li>
		<li>稀释胰岛素 1:1, 000 在0.9% 氯化钠 (股票: 100 u/毫升胰岛素; 工作浓度 0.1 u/毫升), 并准备20% 葡萄糖 (d-(+) 葡萄糖溶解在蒸馏水中), 以管理, 如果老鼠变得低血糖。 注意: ITT 通常是在短时间内进行, 以避免低血糖, 否则可能发生在夜间禁食动物。所有用于动物的试剂都必须是药理级和无菌的。</li>
	</ol>
</li>
	<li>测量小鼠的体重, 标记尾巴, 用锋利的剪刀剪尾尖, 并测量2.4 步中 OGTT 的基本血糖水平。</li>
	<li>胰岛素注射液<ol>
<li>注射胰岛素腹腔 (0.75 U 胰岛素/千克体重, 预先计算), 用颈背法抑制小鼠。</li>
		<li>使用新鲜的, 无菌27或30针为每种动物, 以避免不适和任何注射现场感染的风险。 注意: 皮肤的消毒可以延长胰岛素的持续时间, 因此会对动物造成额外的干扰。因此, 不建议这样做。</li>
		<li>将小鼠头部向下倾斜, 露出动物腹侧。将无菌针放在动物腹部右下象限30°角的斜面上 (图 3b)。在第一个鼠标插入后立即启动计时器。 注: 低剂量 ITTs (0.1 U/千克) 可进行特别评估肝胰岛素敏感性。至于 OGTT, 根据体重计算注射量是标准的程序, 而根据瘦体质量的剂量是首选, 如果身体组成的数据是可用的。</li>
	</ol>
</li>
	<li>测量所选时间点的血糖水平 (例如, 在15、30、45、60和90分钟之后)。 注: 由于胰岛素在小鼠13中有短时间的10分钟, 胰岛素治疗后的晚期差异 (如如, 2 小时后) 可能不反映胰岛素作用的直接作用。管理20% 葡萄糖溶液, 以防老鼠变成低血糖 (血糖水平低于35毫克/dL), 并有死亡的危险。</li>
	<li>在最后的时间点后, 将小鼠放回他们准备好大量食物和水的笼子里。</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+approaches+for+assessing+insulin+sensitivity+and+resistance+in+vivo:+advantages,+limitations,+and+appropriate+usage.">Current approaches for assessing insulin sensitivity and resistance in vivo: advantages, limitations, and appropriate usage.</a> Am J Physiol Endocrinol Metab. 294 (1), E15-E26 (2008).</li><li>McGuinness, O. P., Ayala, J. E., Laughlin, M. R., Wasserman, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+experiment+in+centralized+mouse+phenotyping:+the+Vanderbilt+experience+and+recommendations+for+evaluating+glucose+homeostasis+in+the+mouse.">NIH experiment in centralized mouse phenotyping: the Vanderbilt experience and recommendations for evaluating glucose homeostasis in the mouse.</a> Am J Physiol Endocrinol Metab. 297 (4), E849-E855 (2009).</li><li>Pacini, G., Omar, B., Ahren, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+and+models+for+metabolic+assessment+in+mice.">Methods and models for metabolic assessment in mice.</a> J Diabetes Res. 2013, 986906 (2013).</li><li>Polonsky, K. S., Rubenstein, A. 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随着糖尿病和相关疾病在世界人口中的高患病率, 对疾病的分子机制、预防和治疗的研究有很强的要求19。该协议描述了建立风小鼠的成熟方法, 一种用于代谢研究的健壮动物模型, 以及 OGTT 和 ITT 的传导, 它们是评估全身代谢改变的有力工具。如胰岛素抵抗。本文所提出的方法可能有助于研究疑似基因、环境因素以及药理、膳食、物理或基因治疗对全身葡萄糖代谢的作用<sup class="x...

在发达世界, 由于缺乏体力活动和加工食品的过量消费, 肥胖和糖尿病已经达到了流行病的程度, 这是由于迅速的城市化、工业化和全球化所造成的影响。虽然研究胰岛素抵抗和它的疾病, 如高脂血症和动脉粥样硬化, 已经在过去的几十年中获得突出, 复杂的生物机制, 调节代谢在健康和疾病仍然不完全了解, 仍然迫切需要新的治疗方法来预防和治疗这些疾病1。
胰岛素, 它的 counter-regulatory 激素胰高血糖素作为主要调节细胞能量供应和营养素平衡, 从而也保持适当的系统性血糖浓度2。葡萄糖本身是胰腺β细胞分泌胰岛素的主要刺激之一, 而其他营养素、体液因子以及神经输入则进一步改变了这种反应。胰岛素通过促进过剩的血糖扩散到肌肉和脂肪细胞, 并进一步激活糖酵解以及蛋白质或脂肪酸的合成, 从而触发了联邦政府的合成代谢过程。此外, 胰岛素抑制糖, 抑制肝脏葡萄糖的输出。慢性过量能量消耗和 meta-inflammation 导致高和周围胰岛素抵抗由于胰岛素受体表达的下调以及下游信号通路的改变, 从而导致受损对胰岛素介导的葡萄糖处理的敏感性以及对肝脏葡萄糖分泌不足的抑制作用3,4,5,6。
广泛的动物模型与遗传, 营养, 或实验性诱导疾病已被证明是优秀的工具, 研究胰岛素抵抗的分子机制和各种形式的糖尿病以及其伴随的疾病7.一个典型的例子是广泛使用, 并建立了良好的风诱导小鼠模型, 这是特点是快速体重增加, 由于饮食摄入与降低代谢效率相结合, 导致胰岛素抵抗8,9. 在动物模型和人类中, 空腹血糖和胰岛素水平的升高, 以及对葡萄糖管理的耐受性受损, 常被用于胰岛素抵抗和其他葡萄糖系统改变的指标代谢.因此, 监测血糖和胰岛素水平的基础状态或刺激后, 容易接近读数。
本议定书概述了风喂养小鼠的产生, 以及两种常用的方法, 口服葡萄糖耐受试验 (OGTT) 和胰岛素抵抗试验 (ITT), 这是有用的表征代谢表型和调查葡萄糖代谢的改变。我们详细地描述了 OGTT, 它评估了口服葡萄糖负荷和胰岛素分泌的处理时间。此外, 我们还提供了关于如何通过监测血糖浓度来对胰岛素的反应来检查全身胰岛素的行为的指导。本文中描述的协议已被广泛采用, 并已在多个研究中使用10、11、12。除了轻微的修改, 这可能有助于增加成功, 我们提供了实验设计和数据分析的指导方针, 以及有用的提示, 以避免潜在的陷阱。本文所描述的协议可以是非常强大的工具, 以研究遗传, 药理, 饮食, 和其他环境因素对全身葡萄糖代谢的影响, 并对其相关的疾病, 如胰岛素抵抗。除了刺激葡萄糖或胰岛素, 其他各种化合物可用于刺激, 根据个人研究的目的。虽然在这份手稿的范围之外, 许多其他下游应用可以在抽取的血样上进行, 例如对血糖和胰岛素以外的血液值进行分析 (例如、血脂和脂蛋白图谱) 以及详细代谢标志物的分析 (例如, 通过定量实时聚合酶链式反应 (PCR), 免疫印迹分析, 酶联免疫吸附试验 (ELISA))。进一步的流式细胞仪和荧光活化细胞分类 (组) 可用于研究不同单细胞种群的影响, 同时也可用于不分析。
总的来说, 我们提供了一个简单的协议来生成一个风诱导的小鼠模型, 同时进一步描述了研究全身代谢变化的两个强有力的方法, OGTT 和 ITT, 这可以成为有用的工具, 研究疾病的发病机制和开发新的治疗方法, 特别是在代谢相关疾病领域, 如胰岛素抵抗 & 糖尿病。

<table><tbody><tr><td>Mouse strain: C57BL/6J</td><td>The Jackson Laboratory</td><td>664</td><td>LFD/HFD</td></tr><tr><td>Accu Chek Performa - Glucometer</td><td>Roche</td><td>6870228</td><td>OGTT/ITT</td></tr><tr><td>Accu Chek Performa - Strips</td><td>Roche</td><td>6454038</td><td>OGTT/ITT</td></tr><tr><td>D-(+)-Glucose solution</td><td>Sigma-Aldrich</td><td>G8769</td><td>OGTT</td></tr><tr><td>Actrapid - Insulin</td><td>Novo Nordisk</td><td>417642</td><td>ITT</td></tr><tr><td>Reusable Feeding Needles</td><td>Fine Science Tools</td><td>#18061-22</td><td>OGTT; 22 gauge (-24 gauge for young mice)</td></tr><tr><td>Omnifix-Fine dosing syringes</td><td>Braun</td><td>9161406V</td><td>OGTT/ITT</td></tr><tr><td>Sterican Insulin needle (30G x 1/3&quot;; &amp;oslash; 0.30 x 13 mm)</td><td>Braun</td><td>304000</td><td>ITT; lean mice</td></tr><tr><td>Sterican (G 27 x 3/4&quot;; &amp;oslash; 0.40 x 20 mm)&amp;nbsp;&amp;nbsp;</td><td>Braun</td><td>4657705</td><td>ITT; mice on HFD</td></tr><tr><td>96 Well PCR Plates, non-skirted, flexible</td><td>Braintree Scientific, Inc.</td><td>SP0016</td><td>OGTT</td></tr><tr><td>Ultrasensitive Mouse Insulin ELISA kit</td><td>Crystam Chem</td><td>90080</td><td>OGTT</td></tr><tr><td>Rodent Diet with 60% kcal% fat</td><td>Research Diets Inc</td><td>D12492</td><td>mice on HFD</td></tr><tr><td>Rodent Diet with 10% kcal% fat.</td><td>Research Diets Inc</td><td>D12450B</td><td>mice on LFD</td></tr><tr><td>BRAND micro haematocrit capillary</td><td>Sigma-Aldrich</td><td>BR749321</td><td>OGTT/ITT</td></tr><tr><td>Vaseline - creme</td><td>Riviera</td><td>P1768677</td><td>OGTT/ITT</td></tr></tbody></table>

study of in vivo glucose metabolism in high-fat diet-fed mice using oral glucose tolerance test (ogtt) and insulin tolerance test (itt)

肥胖是2型糖尿病发病的最重要的单一危险因素, 这种疾病的特点是抗胰岛素刺激的葡萄糖摄取和全身糖代谢的严重失代偿。尽管在了解葡萄糖代谢方面取得了相当大的进展, 但它在健康和疾病方面的调控的分子机制仍然 under-investigated, 而新的预防和治疗糖尿病的方法是迫切需要的。饮食衍生葡萄糖刺激胰腺分泌胰岛素, 这是主要调节器的细胞合成过程中的美联储状态, 从而平衡血糖水平保持系统的能量状态。慢性过度触发 meta-inflammation, 导致周围胰岛素受体相关信号的改变, 从而降低对胰岛素介导的葡萄糖处理的敏感性。这些事件最终导致空腹血糖和胰岛素水平升高以及葡萄糖耐受减少, 这反过来又成为胰岛素抵抗的重要指标。在这里, 我们提出了一个协议的产生和代谢特征的高脂饮食 (风) 喂养小鼠作为一个常用的模型, 饮食诱导胰岛素抵抗。我们详细说明了口服葡萄糖耐受试验 (OGTT), 它监测的周围处理的口服葡萄糖负荷和胰岛素分泌随着时间的推移。此外, 我们提出了一个协议的胰岛素耐受性测试 (ITT), 以监测全身胰岛素行为。这些方法和它们的下游应用代表了强有力的工具来表征小鼠的一般代谢表型, 以及专门评估葡萄糖代谢的变化。它们可能在胰岛素抵抗、糖尿病和肥胖的广泛研究领域中特别有用, 以便更好地了解发病机制, 并测试治疗干预的效果。

图 1说明了在饮食上对小鼠进行代谢分的示意图时间表。在大约6周的年龄, 老鼠应该放在一个风, 而 LFD 组可以作为对照组。重要的是, 体重应该每周确定, 以观察是否有预期的体重增加。任何形式的压力 (例如、噪音或攻击性的男性行为) 都会影响体重增加, 应立即消除。每组小鼠的饮食实验应包括至少10小鼠, 因为这些饮食-实验是耗时的, 和异?...

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利用口服葡萄糖耐量试验OGTT和胰岛素耐量试验ITT研究高脂饮食喂养小鼠的体内葡萄糖代谢- 博士论文医学|视频

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

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)

本文描述了高脂喂养小鼠的生成和代谢特性, 作为饮食诱导的胰岛素抵抗和肥胖的模型。它还有详细的协议来执行口服葡萄糖耐受试验和胰岛素耐受试验, 监测葡萄糖代谢的全身变化在体内。

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

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67.9K 次观看.  Medical University of Vienna. 本文描述了高脂喂养小鼠的生成和代谢特性, 作为饮食诱导的胰岛素抵抗和肥胖的模型。它还有详细的协议来执行口服葡萄糖耐受试验和胰岛素耐受试验, 监测葡萄糖代谢的全身变化在体内。

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

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 ...

科研

医学

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic factors affecting glucose uptake. The measurement of intracellular fluorescence is a widely used method to test the uptake of fluorescently-labeled glucose (FD-glucose) in cells in vitro, or for imaging glucose-consuming tissues in vivo. This assay assesses glucose uptake at a chosen time point. The intracellular analysis assumes that the metabolism of FD-glucose is slower than that of endogenous glucose, which participates in catabolic and anabolic reactions and signaling. However, dynamic glucose metabolism also alters uptake mechanisms, which would require kinetic measurements of glucose uptake in response to different factors. This article describes a method for measuring extracellular FD-glucose depletion and validates its correlation with intracellular FD-glucose uptake in cells and tissues ex vivo. Extracellular glucose depletion may be potentially applicable for high-throughput kinetic and dose-dependent studies, as well as identifying compounds with glycemic activity and their tissue-specific effects.

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

Extracellular depletion of fluorescently labeled glucose correlates with glucose uptake and could be used for high-throughput screening of glucose uptake in excised organs and cell cultures.

细胞外葡萄糖消耗作为细胞和组织离体葡萄糖摄取的间接测量

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic ...

生物化学

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

腹腔葡萄糖耐受试验、肺功能测定和肺固定研究肥胖和代谢障碍对肺部结局的影响

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

免疫与感染

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 ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

持续药物输注系统对小鼠脑内下丘脑胰岛素信号的研究

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

高脂喂养小鼠体内成分及代谢锁定分析

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

Acclimation Prior to an Intraperitoneal Insulin Tolerance Test to Mitigate Stress-Induced Hyperglycemia in Conscious Mice

The insulin tolerance test is commonly used in metabolic studies to assess whole body insulin sensitivity in rodents. It is a relatively simple test that involves measurement of blood glucose levels over time following a single intraperitoneal injection of insulin. Given that it is performed in the conscious state and blood is often collected via a tail snip, it has the potential to elicit a stress response from animals due to anxiety associated with handling and blood collection. As such, a stress-induced rise in blood glucose can occur, making it difficult to detect and interpret the primary endpoint measure, namely an insulin-mediated reduction in blood glucose. This has been seen in many mouse strains, and is quite common in diabetic db/db mice, where glucose levels can increase, rather than decrease, after insulin administration. Here, we describe a method of acclimating mice to handling, injections and blood sampling prior to performing the insulin tolerance test. We find that this lowers stress-induced hyperglycemia and results in data that more accurately reflects whole body insulin sensitivity.

<meta charset="utf8"></head><body>腹膜内胰岛素耐量试验前的驯化以减轻清醒小鼠应激诱导的高血糖

Stress-induced elevations in glucose levels can confound interpretation of data derived from a conscious intraperitoneal insulin tolerance test in mice. In this article, we describe a method to acclimate the mice to handling, injections and blood sampling prior to performing the insulin tolerance test in order to limit stress-induced hyperglycemia.

腹膜内胰岛素耐量试验前的驯化以减轻清醒小鼠应激诱导的高血糖

The insulin tolerance test is commonly used in metabolic studies to assess whole body insulin sensitivity in rodents. It is a relatively simple test that ...

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 ...

生物学

小鼠高血糖和低血糖夹的手术安装

Hyperglycemic Clamp and Hypoglycemic Clamp in Conscious Mice

Diabetes mellitus (DM) is caused by insufficient insulin release from the pancreatic &#946;-cells (Type1 DM) and insulin sensitivity in muscles, liver, and adipose tissues (Type2 DM). Insulin injection treats DM patients but leads to hypoglycemia as a side effect. Cortisol and catecholamines are released to activate glucose production from the liver to recover hypoglycemia, called counter-regulatory responses (CRR). In DM research using rodent models, glucose tolerance tests and 2-deoxy-glucose injection are used to measure insulin release and CRR, respectively. However, blood glucose concentrations change persistently during experiments, causing difficulties in assessing net insulin release and CRR. This article describes a method in which blood glucose is kept at 250 mg/dL or 50 mg/dL in conscious mice to compare the release of insulin and CRR hormones, respectively.
Polyethylene tubing is implanted in the mice's carotid artery and jugular vein, and the mice are allowed to recover from the surgery. The jugular vein tubing is connected to a Hamilton syringe with a syringe pump to enable insulin or glucose infusion at a constant and variable rate. The carotid artery tubing is for blood collection. For the hyperglycemic clamp, 30% glucose is infused into the vein, and blood glucose levels are measured from the arterial blood every 5 min or 10 min. The infusion rate of 30% glucose is increased until the blood glucose level becomes 250 mg/dL. Blood is collected to measure insulin concentrations. For hypoglycemic clamp, 10 mU/kg/min insulin is infused together with 30% glucose, whose infusion rate is variable to maintain 50 mg/dL of blood glucose level. Blood is collected to measure counter-regulatory hormones when both glucose infusion and blood glucose reach a steady state. Both hyperglycemic and hypoglycemic clamps have the same surgical procedure and experimental setups. Thus, this method is useful for researchers of systemic glucose metabolism.

作者聚焦:使用经济高效的高血糖和低血糖钳夹技术研究小鼠大脑中的血糖稳态

A hyperglycemic clamp is used for measuring insulin release with a maintained higher blood glucose concentration. A hypoglycemic clamp is for measuring glucose production induced by counter-regulatory responses. Both methods use the same surgical procedure. Here, we present a clamp technique to assess systemic glucose metabolism.

清醒小鼠的高血糖钳夹和低血糖钳夹

Diabetes mellitus (DM) is caused by insufficient insulin release from the pancreatic &#946;-cells (Type1 DM) and insulin sensitivity in muscles, liver, ...

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)

摘要

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在自觉的，奔放的小鼠正常血糖高胰岛素 - 夹钳

细胞外葡萄糖消耗作为细胞和组织离体葡萄糖摄取的间接测量

腹腔葡萄糖耐受试验、肺功能测定和肺固定研究肥胖和代谢障碍对肺部结局的影响

优化分析

持续药物输注系统对小鼠脑内下丘脑胰岛素信号的研究

高脂喂养小鼠体内成分及代谢锁定分析

腹膜内胰岛素耐量试验前的驯化以减轻清醒小鼠应激诱导的高血糖

腹膜内胰岛素耐量试验前的驯化以减轻清醒小鼠应激诱导的高血糖

小鼠间间间禁食的代谢效应评估 2:1 间歇禁食

清醒小鼠的高血糖钳夹和低血糖钳夹