Name: assessing whole-body lipid-handling capacity in mice
Uploaded: 2020-11-24T14:00:00.000+00:00
Duration: 7 min 57 s
Description: Watch this Scientific Journal Video about Assessing Whole-Body Lipid-Handling Capacity in Mice at JoVE.com

这项工作得到国家卫生研究院（NIH）的支持，授予R00-DK114498，美国农业部（美国农业部）向Y.Z提供3092-51000-062。

根据贝勒医学院（BCM）机构动物护理和使用委员会批准的动物护理和实验协议，动物被安置在标准化条件下。动物被喂食标准或特殊的饮食，水广告脂质，并保持12小时的日夜循环。
1. 测量禁食血清脂
<ol><li>下午5点以后将老鼠转移到一个新的笼子里，并快速免费取水，过夜（实验前大约16个小时的禁食）。通宵禁食确保完全排空小鼠的胃肠道。 注意:老鼠在禁食时吃粪便，所以食物提取不能确保它们被充分禁食。</li>
	<li>第二天早上，抓住老鼠的尾巴，把它放在水面上。轻轻地拉回鼠标尾部，将鼠标放在抑制器中。将鼻子约束重新训练鼠标的运动，同时仍然允许鼠标呼吸。拧紧鼻子约束的旋钮。监控胸部运动，确保动物正常呼吸，并尽量减少鼠标在抑制器中停留的时间。</li>
	<li>在自由移动的鼠标的尾部静脉中做一个表面切口（尼克），从切口中抽出25μL的血液，制成玻璃毛细血管（填充大约1/3的毛细血管），而不抑制鼠标。迅速将血液吹入微中心管中。</li>
	<li>使用型粉停止出血，在笼子里补充饲料，并确保小鼠没有出现压力的迹象。</li>
	<li>完成所有小鼠的血液抽取。</li>
	<li>让血液凝固，使其在室温下不受干扰1小时。在冷藏台式微中心中，在 4 °C 下以 2，000 x g 旋转凝固的血液样本 10 分钟，并转移超高血清（血清）进行分析。 注:血清可在 +20 °C 下存储数周，直到分析。对于长期存储，将血清保持在 +70°C。</li>
	<li>使用制造商提供的协议分析每个脂质代谢物。</li>
</ol>2. 口服脂内耐受性测试
<ol><li>下午5点以后，称量小鼠，计算第二天交给它们的脂内体积。然后，将老鼠转移到一个新的笼子里，并禁食它们过夜（16小时）。</li>
	<li>第二天早上，将老鼠的尾巴放在一个笼子里，以帮助识别它们在随后的出血步骤中。</li>
	<li>在尾部静脉上划一个刻痕，从切口中抽出 15 μL 的血液到玻璃毛细血管中（填充大约 1/5 的毛细血管），并快速将血液吹入 T = 0 血清的微中心管中。 注意:除非小鼠出现过度出血，否则无需在检测过程中止血。</li>
	<li>Gavage 小鼠使用 18G 盖瓦奇针的 20% 内脂，每克体重为 15 μl，使用禁食前体重。将每只鼠标堆叠 1 分钟。 注:称量动物，确定适当的咽针大小。一般来说，18号的咽针适合25克>小鼠，20-22号的咽针更适合较小的动物。擦伤老鼠，抓住肩膀上的皮肤，把动物的头抱到位。将咽针放入口腔，然后沿着上味轻轻推进，直到食道到达。管子应该通过没有阻力。一旦达到适当的深度，内脂可以慢慢管理。在插入针头时，在管理脂内后，轻轻取出相同角度的针头。将动物送回笼子，并监测有呼吸困难或其他不适的迹象。 注:缺乏口腔咽部或尾部出血技术经验的研究人员可以将每只小鼠堆叠2分钟甚至更长时间。</li>
	<li>在 T = 1、2、3、4、5 和 6 小时抽血:通过尾部出血每只小鼠抽取 15 μL 的血液（1/5 毛细管），并迅速将血液吹入微中心管中。</li>
	<li>在室温下以 2，000 x g 旋转血液样本，在微中心中旋转 10 分钟。将超高纳特（包括浮动脂肪层）转移到 PCR 管中存储。超高纳特可以在 +20 °C 下存储数周，直到分析。 注:超高等离子体应该是等离子体。如果某些样品在离心时已经凝固，则不会影响甘油三酯测量。</li>
	<li>最后一次采血后，停止使用型粉出血，在笼子里补充饲料，并确保小鼠没有表现出极度紧张的迹象。</li>
	<li>装载 2 μL 甘油三酯标准，并将超分子收集到 96 井板中。</li>
	<li>加入 200 μL 甘油三酯试剂，让板在 37 °C 下孵育 5 分钟，用于颜色开发。</li>
	<li>测量实验室板读卡器中 500 nm 的吸血量，参考波长为 660 nm，并计算样本的浓度。</li>
</ol>3. β3 肾上腺受体激动器 CL 316，243 刺激利波利西斯检测
<ol><li>准备 CL 316，243 作为无菌盐水中 5 毫克/mL （50 倍） 的库存溶液，并储存在 ±20°C，直到使用。</li>
	<li>在早晨，称量小鼠，计算实验所需的稀释CL 316，243溶液量。小鼠将得到10μL每克体重稀释CL 316，243，为1毫克/千克体重的最后剂量。</li>
	<li>将老鼠转移到一个新的笼子里，免费取水，并禁食4小时。</li>
	<li>使用盐水从 50 倍库存中制作足够的 1x CL 316，243 溶液。1x CL 316，243 溶液的最终浓度为 0.1 毫克/mL。使用盐水进行对照组。</li>
	<li>标记存放在同一笼子里的老鼠的尾巴，以便在出血步骤中轻松识别。</li>
	<li>在尾部静脉中划出一个刻痕，从切口中抽取 15 μL 的血液到玻璃毛细血管中（填充大约 1/5 的毛细血管），并快速将血液吹入微中心管中，用于 T = 0 样本。 注意:除非小鼠出现过度出血，否则无需在检测过程中止血。</li>
	<li>以 10 μL/g 体重的体量，在腹地注射稀释的 CL 316，243 溶液（或如果包含在实验中）。将每只鼠标堆叠 1 分钟。每60分钟的实验最多使用5只小鼠，或为双人小组使用10只小鼠。</li>
	<li>在 T = 5、15、30、60 分钟抽血:通过尾部出血每只小鼠抽取 15 微升血液（1/5 毛细血管）。</li>
	<li>最后一次采血后，停止使用型粉出血，在笼子里补充饲料，并确保小鼠没有表现出极度紧张的迹象。</li>
	<li>在冷藏微中心中，在 4 °C 下以 2，000 x g 旋转血液样本 5 分钟。将超纳特转移到 PCR 管进行存储。超高纳特可以在 +20°C 下存储数周，直到分析。</li>
	<li>负载 1 μL 的 2 倍串序稀释甘油标准 （0.156， 0.312， 0.625， 1.25 和 2.5 毫克/毫升三聚氨酯当量浓度）， 并收集超分子到 96 井板.加入100微升免费甘油试剂，让板在37°C孵育5分钟，使颜色形成。</li>
	<li>使用实验室板读卡器测量 540 nm 的吸血量，并计算样品的浓度。</li>
</ol>

<ol>
	<li>Dixon, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+chylomicron+uptake+into+lacteals.">Mechanisms of chylomicron uptake into lacteals.</a> Annals of the New York Academy of Sciences. 1207, 52-57 (2010).</li><li>Nuno, J., de Oya, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoprotein+lipase:+review.">Lipoprotein lipase: review.</a> Revista Cl&#237;nica Espa&#241;ola. 170 (3-4), 83-87 (1983).</li><li>Williams, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+processes+that+handle+--+and+mishandle+--+dietary+lipids.">Molecular processes that handle -- and mishandle -- dietary lipids.</a> Journal of Clinical Investigation. 118 (10), 3247-3259 (2008).</li><li>Burla, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MS-based+lipidomics+of+human+blood+plasma:+a+community-initiated+position+paper+to+develop+accepted+guidelines.">MS-based lipidomics of human blood plasma: a community-initiated position paper to develop accepted guidelines.</a> Journal of Lipid Research. 59 (10), 2001-2017 (2018).</li><li>Umpleby, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormone+measurement+guidelines:+Tracing+lipid+metabolism:+the+value+of+stable+isotopes.">Hormone measurement guidelines: Tracing lipid metabolism: the value of stable isotopes.</a> Journal of Endocrinology. 226 (3), 1-10 (2015).</li><li>Mitchell, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+paired+sibling+analysis+of+the+beta-3+adrenergic+receptor+and+obesity+in+Mexican+Americans.">A paired sibling analysis of the beta-3 adrenergic receptor and obesity in Mexican Americans.</a> Journal of Clinical Investigation. 101 (3), 584-587 (1998).</li><li>Mahoney, L. B., Denny, C. A., Seyfried, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caloric+restriction+in+C57BL/6J+mice+mimics+therapeutic+fasting+in+humans.">Caloric restriction in C57BL/6J mice mimics therapeutic fasting in humans.</a> Lipids in Health and Disease. 5, 13 (2006).</li><li>Hayek, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+fat+increases+high+density+lipoprotein+(HDL)+levels+both+by+increasing+the+transport+rates+and+decreasing+the+fractional+catabolic+rates+of+HDL+cholesterol+ester+and+apolipoprotein+(Apo)+A-I.+Presentation+of+a+new+animal+model+and+mechanistic+studies+in+human+Apo+A-I+transgenic+and+control+mice.">Dietary fat increases high density lipoprotein (HDL) levels both by increasing the transport rates and decreasing the fractional catabolic rates of HDL cholesterol ester and apolipoprotein (Apo) A-I. Presentation of a new animal model and mechanistic studies in human Apo A-I transgenic and control mice.</a> Journal of Clinical Investigation. 91 (4), 1665-1671 (1993).</li><li>Hogarth, C. A., Roy, A., Ebert, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+evidence+for+the+absence+of+a+functional+cholesteryl+ester+transfer+protein+gene+in+mice+and+rats.">Genomic evidence for the absence of a functional cholesteryl ester transfer protein gene in mice and rats.</a> Comparative Biochemistry and Physiology - Part B: Biochemistry &amp; Molecular Biology. 135 (2), 219-229 (2003).</li><li>Tall, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+cholesterol+ester+transfer+protein+and+relationship+to+coronary+artery+disease+risk.">Functions of cholesterol ester transfer protein and relationship to coronary artery disease risk.</a> Journal of Clinical Lipidology. 4 (5), 389-393 (2010).</li><li>Singh, A. K., Singh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triglyceride+and+cardiovascular+risk:+A+critical+appraisal.">Triglyceride and cardiovascular risk: A critical appraisal.</a> Indian Journal of Endocrinology and Metabolism. 20 (4), 418-428 (2016).</li><li>Miller, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triglycerides+and+cardiovascular+disease:+a+scientific+statement+from+the+American+Heart+Association.">Triglycerides and cardiovascular disease: a scientific statement from the American Heart Association.</a> Circulation. 123 (20), 2292-2333 (2011).</li><li>Dron, J. S., Hegele, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+Hypertriglyceridemia.">Genetics of Hypertriglyceridemia.</a> Frontiers in Endocrinology (Lausanne). 11, 455 (2020).</li><li>Dole, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+relation+between+non-esterified+fatty+acids+in+plasma+and+the+metabolism+of+glucose.">A relation between non-esterified fatty acids in plasma and the metabolism of glucose.</a> Journal of Clinical Investigation. 35 (2), 150-154 (1956).</li><li>Bartelt, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+adipose+tissue+activity+controls+triglyceride+clearance.">Brown adipose tissue activity controls triglyceride clearance.</a> Nature Medicine. 17 (2), 200-205 (2011).</li><li>de Souza, C. J., Burkey, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta+3-adrenoceptor+agonists+as+anti-diabetic+and+anti-obesity+drugs+in+humans.">Beta 3-adrenoceptor agonists as anti-diabetic and anti-obesity drugs in humans.</a> Current Pharmaceutical Design. 7 (14), 1433-1449 (2001).</li><li>Braun, K., Oeckl, J., Westermeier, J., Li, Y., Klingenspor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-adrenergic+control+of+lipolysis+and+thermogenesis+in+adipose+tissues.">Non-adrenergic control of lipolysis and thermogenesis in adipose tissues.</a> Journal of Experimental Biology. 221, (2018).</li></ol>

作者没有什么可透露的。

这三个分析描述了实验室中强有力的功能，并考虑了几个关键问题。夜间禁食是确定禁食血清脂质水平和口腔脂内耐受性测试所必需的。对于口腔脂内耐受性测试，在室温下旋转血液以尽量减少脂肪层的形成至关重要，尤其是在 1 小时和 2 小时的时间点:重要的是不要丢弃这个脂肪层，如果它形成。确保将超高肽与脂质层一起转移，并轻轻地将它们混合在一起，以进行甘油三酯测定。
<p class="jov...

碳水化合物和脂质是能量代谢的两大基质。异常脂质代谢导致许多人类疾病，包括II型糖尿病、心血管疾病、脂肪肝疾病和癌症。膳食脂质，主要是甘油三酯，通过肠道吸收到淋巴系统，并进入心脏1附近的血小龙静脉循环。脂质由血液中的脂蛋白颗粒携带，脂肪酸脂肪通过脂蛋白脂酶在肌肉和脂肪组织2等外周器官的作用而释放。剩余的富含胆固醇的残留颗粒由肝脏3清除。老鼠在实验室中被广泛用作研究脂质代谢的研究模型。拥有全面的遗传工具集和相对较短的繁殖周期，它们是研究脂质如何被吸收、合成和代谢的有力模型。
由于脂质代谢的复杂性，复杂的脂质学研究或同位素示踪研究通常用于量化脂质物种或脂质相关代谢通量和命运的集合4，5。这给没有专门设备或专业知识的研究人员带来了巨大的挑战。在本文中，我们提出了三个测试，在使用具有技术挑战性的技术之前，这些测试可以作为初始测试。它们是小鼠的非终端程序，因此非常有助于识别脂质处理能力的潜在差异和缩小受影响的过程。
首先，测量禁食血清脂质分子可以帮助确定小鼠的整体脂质特征。老鼠应该禁食，因为许多脂质物种在饭后会上升，而且增加的程度受到饮食成分的强烈影响。许多脂质分子，包括总胆固醇、甘油三酯和非酯化脂肪酸（NEFA），都可以使用商业套件和可读取吸血的板读器进行测量。
其次，口服脂质耐受性测试将脂质处理能力评估为吸收和代谢的净效应。口服的脂内导致循环甘油三酯水平激增（1至2小时），之后血清甘油三酯水平恢复到基底水平（4至6小时）。此检测提供了有关鼠标处理外源性脂质的程度的信息。心脏、肝脏和棕色脂肪组织是甘油三酯的活跃消费者，而白色脂肪组织将其储存为能量储备。这些功能的变化将导致测试结果的差异。
最后，促进脂溶解以调动储存的脂质被认为是减肥的可能策略。脂肪组织中的β3-肾神经受体信号通路在脂肪细胞脂解中起着重要作用，人类遗传学已经识别出与肥胖相关的3-肾上腺受体中功能丧失的多态性Trp64Arg。CL 316，243，一种特异且有效的β-肾上腺素受体激动剂，刺激脂肪组织脂解和甘油释放。评估鼠标对CL 316，243的反应可以提供关于化合物的开发、改进和理解的宝贵信息。
总的来说，这些测试可以用作小鼠脂质代谢状态变化的初始屏幕。它们是为仪器和试剂的可访问性而选择的。通过这些检测结果，研究人员可以形成动物代谢适应的整体图景，并决定更复杂和更有针对性的方法。

<table><tbody><tr><td>20% Intralipid</td><td>Sigma Aldrich</td><td>I141</td><td></td></tr><tr><td>BD Slip Tip Sterile Syringes 1ml</td><td>Shaotong</td><td>B07F1KRMYN</td><td></td></tr><tr><td>CL 316,243 Hydrate</td><td>Sigma-Aldrich</td><td>C5976</td><td></td></tr><tr><td>Curved Feeding Needles (18 Gauge)</td><td>Kent Scientific</td><td>FNC-18-2-2</td><td></td></tr><tr><td>Free Glycerol Reagent</td><td>Sigma Aldrich</td><td>F6428</td><td></td></tr><tr><td>Glycerol Standard Solution</td><td>Sigma</td><td>G7793</td><td></td></tr><tr><td>HR SERIES NEFA-HR(2)COLOR REAGENT A</td><td>Fujifilm Wako Diagnostics</td><td>999-34691</td><td></td></tr><tr><td>HR SERIES NEFA-HR(2)COLOR REAGENT B</td><td>Fujifilm Wako Diagnostics</td><td>991-34891</td><td></td></tr><tr><td>HR SERIES NEFA-HR(2)SOLVENT A</td><td>Fujifilm Wako Diagnostics</td><td>995-34791</td><td></td></tr><tr><td>HR SERIES NEFA-HR(2)SOLVENT B</td><td>Fujifilm Wako Diagnostics</td><td>993-35191</td><td></td></tr><tr><td>Matrix Plus Chemistry Reference Kit</td><td>Verichem</td><td>9500</td><td></td></tr><tr><td>Micro Centrifuge Tubes</td><td>Fisher Scientific</td><td>14-222-168</td><td></td></tr><tr><td>Microhematrocrit Capillary Tube, Not Heparanized</td><td>Fisher Scientific</td><td>22-362-574</td><td></td></tr><tr><td>NEFA STANDARD SOLUTION</td><td>Fujifilm Wako Diagnostics</td><td>276-76491</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Boston Bioproducts</td><td>BM-220</td><td></td></tr><tr><td>Thermo Scientific Triglycerides Reagent</td><td>Fisher Scientific</td><td>TR22421</td><td></td></tr><tr><td>Total Cholesterol Reagents</td><td>Thermo Scientifi</td><td>TR13421</td><td></td></tr></tbody></table>

assessing whole-body lipid-handling capacity in mice

评估脂质代谢是评估代谢功能的基石，它被认为是活体代谢研究的关键。脂质是许多不同的分子的一类，其合成和代谢涉及许多通路。需要一个起点来评估营养和肥胖研究的脂质血脂。本文描述了三种简单易行的方法，这些方法几乎不需要专业知识或实践来掌握，并且大多数实验室都可以将其改编为筛查小鼠的脂质代谢异常。这些方法是（1）测量几个禁食血清脂质分子使用商业套件（2）通过口服脂质耐受性测试，以评估饮食脂质处理能力，（3）评估对药物化合物CL 316，243的反应，在小鼠。这些方法将共同提供小鼠脂质处理能力的高水平概述。

我们用三段摘录显示，每次检测都提供了有关小鼠脂质代谢的宝贵信息。对于C57BL/6J雄性小鼠来说，从8周大开始接受8周高脂肪饮食（HFD）喂养的挑战，总胆固醇水平显著升高，而血清甘油三酯和NEFA则没有（表1），这表明血液中的甘油三酯和NEFA不是主要受膳食脂肪挑战的调节。在第二组小鼠中，C57BL/6J和C57BL6/N子系统从8周大开始喂养HFD8周。他们的血清甘油三酯水平在口腔脂内挑战后?...

Watch this Scientific Journal Video about Assessing Whole-Body Lipid-Handling Capacity in Mice at JoVE.com

Assessing Whole-Body Lipid-Handling Capacity in Mice

本文为评估小鼠脂质代谢提供了三种简单易行的检测。

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 ...

assessing-whole-body-lipid-handling-capacity-in-mice

Research

JoVE Journal

Medicine

3.9K 次观看.  Baylor College of Medicine. 本文为评估小鼠脂质代谢提供了三种简单易行的检测。

视频: Assessing Whole-Body Lipid-Handling Capacity in Mice

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

确定小鼠肝脏中的胆汁密度

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

科研

发育生物学

Shotgun Lipidomics of Rodent Tissues

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have made lipidomics a powerful analytical tool for monitoring tissue lipidome compositions in homeostatic as well as disease states. This paper presents a step-by-step protocol for a shotgun lipid analysis method that supports the simultaneous detection and quantification of a few hundred molecular lipid species in different tissue and biofluid samples at high throughput. This method leverages automated nano-flow direct injection of a total lipid extract spiked with labeled internal standards without chromatographic separation into a high-resolution mass spectrometry instrument. Starting from sub-microgram amounts of rodent tissue, the MS analysis takes 10 min per sample and covers up to 400 lipids from 14 lipid classes in mouse lung tissue. The method presented here is well suited for studying disease mechanisms and identifying and quantifying biomarkers that indicate early toxicity or beneficial effects within rodent tissues.

Shotgun mass spectrometry-based lipidomics delivers a sensitive quantitative snapshot of a broad spectrum of lipid classes simultaneously in a single measurement from various rodent tissues.

啮齿动物组织的霰弹枪脂质组学

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have ...

生物化学

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 ...

医学

Quantification of Atherosclerosis in Mice

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic inflammatory disease. Experimental atherosclerosis studies have elucidated the role of cholesterol and inflammation in the disease process. This has led to successful clinical trials with pharmaceutical agents that reduce clinical manifestations of atherosclerosis. Careful and well-controlled experiments in mouse models of the disease could further elucidate the pathogenesis of the disease, which is not fully understood. Standardized lesion analysis is important to reduce experimental variability and increase reproducibility. Determining lesion size in aortic root, aortic arch, and brachiocephalic artery are common endpoints in experimental atherosclerosis. This protocol provides a technical description for evaluation of atherosclerosis at all these sites in a single mouse. The protocol is particularly useful when material is limited, as is frequently the case when genetically modified animals are being characterized.

Murine models of atherosclerosis are useful tools to investigate pathogenic pathways on a molecular level, but require standardized quantification of lesion development. This protocol describes an optimized method to determine lesion size in the major arterial vessels including the aortic root, aortic arch, and brachiocephalic artery.

小鼠动脉粥样硬化的定量

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic ...

免疫与感染

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 ...

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

Imaging and Analysis of Oil Red O-Stained Whole Aorta Lesions in an Aneurysm Hyperlipidemia Mouse Model

Apolipoprotein E (Apoe)- or low density lipoprotein receptor (Ldlr)-deficient hyperlipidemic mice are the two most commonly used models for atherosclerosis research. They are used to study the impact of a various genetic factors and different cell types on atherosclerotic lesion formation and as well as test the development of new therapies. Isolation, excision of the whole aorta, and quantification of Oil Red O-stained atherosclerotic lesions are basic morphometric methods used to evaluate atherosclerotic burden. The goal of this protocol is to describe an optimized, step-by-step surgical method to dissect, perfuse-fix, isolate, stain, image and analyze atherosclerotic lesions in mouse aortas with Oil Red O. Because atherosclerotic lesions can form anywhere in the entire aortic tree, this whole aorta Oil Red O staining method has the advantage of evaluating lipid-laden plaques in the entire aorta and all branches in a single mouse. In addition to Oil Red O staining, fresh isolated whole aortas can be used for variety of in vitro and in vivo experiments and cell isolations.

This protocol provides a step-by-step procedure to analyze atherosclerotic burden in mice. Investigators can use this protocol to compare the abundance, location, and size of atherosclerotic lesions in different animals.

动脉瘤高脂血症小鼠模型中油红O染色全主动脉病变的成像和分析

Apolipoprotein E (Apoe)- or low density lipoprotein receptor (Ldlr)-deficient hyperlipidemic mice are the two most commonly used models for ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.

在小鼠原代肝细胞测定脂肪酸氧化和脂肪生成的

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

研究高脂血症诱导小鼠模型中的 HFpEF 病理生理学

A Murine Model of Hyperlipidemia&#45;Induced Heart Failure with Preserved Ejection Fraction

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) driven by lipotoxicity is incompletely understood. Given the urgent need for animal models that accurately mimic cardio-metabolic HFpEF, a hyperlipidemia-induced murine model was developed by reverse engineering phenotypes seen in HFpEF patients. This model aimed to investigate HFpEF, focusing on the interplay between lipotoxicity and metabolic syndrome. Hyperlipidemia was induced in wild-type (WT) mice on a 129J strain background through bi-weekly intraperitoneal injections of poloxamer-407 (P-407), a block co-polymer that blocks lipoprotein lipase, combined with a single intravenous injection of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR). Extensive assessments were conducted between 4 and 8 weeks post-treatment, including echocardiography, blood pressure recording, whole-body plethysmography, echocardiography (ECG) telemetry, activity wheel monitoring (AWM), and biochemical and histological analyses. The LDLR/P-407 mice exhibited distinctive features at four weeks, including diastolic dysfunction, preserved ejection fraction, and increased left ventricular wall thickness. Notably, blood pressure and renal function remained within normal ranges. Additionally, ECG and AWM revealed heart blocks and reduced activity, respectively. Diastolic function deteriorated at eight weeks, accompanied by a significant decline in respiratory rates. Further investigation into the double treatment model revealed elevated fibrosis, wet/dry lung ratios, and heart weight/body weight ratios. The LDLR/P-407 mice exhibited xanthelasmas, ascites, and cardiac ischemia. Interestingly, sudden deaths occurred between 6 and 12 weeks post-treatment. The murine HFpEF model offers a valuable and promising experimental resource for elucidating the intricacies of metabolic syndrome contributing to diastolic dysfunction within the context of lipotoxicity-mediated HFpEF.

作者聚焦：探索脂毒性与 HFpEF 之间的关系 

This protocol presents a detailed approach to replicating a murine model of hyperlipidemia-induced heart failure with preserved ejection fraction (HFpEF). The design combines the administration of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR) and poloxamer-407 (P-407).

射血分数保留的高脂血症诱导心力衰竭小鼠模型

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) driven by lipotoxicity is incompletely understood. Given the urgent need for ...

评估小鼠全身脂质处理能力

摘要

探索更多视频

此视频中的章节

确定小鼠肝脏中的胆汁密度

啮齿动物组织的霰弹枪脂质组学

优化分析

小鼠动脉粥样硬化的定量

高脂喂养小鼠体内成分及代谢锁定分析

生化和高通量的微观评估的脂肪含量

动脉瘤高脂血症小鼠模型中油红O染色全主动脉病变的成像和分析

在小鼠原代肝细胞测定脂肪酸氧化和脂肪生成的

射血分数保留的高脂血症诱导心力衰竭小鼠模型

射血分数保留的高脂血症诱导心力衰竭小鼠模型