Name: measurement of basal and forskolin-stimulated lipolysis in inguinal adipose fat pads
Uploaded: 2017-07-21T12:30:00.000+00:00
Duration: 7 min 59 s
Description: Watch this Scientific Journal Video about Measurement of Basal and Forskolin-stimulated Lipolysis in Inguinal Adipose Fat Pads at JoVE.com

这项工作得到AHA Award 15BGIA23250030，NIH国家综合医学科学研究所的支持，获得了第8P20 GM103432-12的奖学金和怀俄明州大学授予BT援助。

所有协议均遵循怀俄明大学的动物保健指南。 动物饲养注意:根据机构动物护理和使用委员会（IACUC）批准的方案，在研究动物设施中培育成年雄性野生型小鼠（C57BL / 6）（12至24周）。 <ol><li>从第6周开始，将小鼠分为四组，分为笼子，随机将其分配到非传染性疾病或高血压（±0.01％CAP）的喂养组，直到第38周岁。 注:CAP是在脂肪组织11，17表达TRPV1通道蛋白的激动剂。将CAP与HFD混合在罩内的搅拌机中，并将混合的混合物转移到含有1分2分的小盘中。将托盘放在-20°C的冷冻箱内。 24小时后从冷冻箱中取出含有HFD + CAP饮食的托盘，并储存于-20℃的容器中，＃176; C冷冻直到使用。 </li><li>在气候控制环境（22.8±2.0°C，45 - 50％湿度）的家庭小鼠，12/12光/黑暗​​循环， 随意获取指定的饮食和水。 </li><li>在38周结束时，剖腹腹脂肪组织并用于脂肪分解实验（2-7节）。 </li></ol> 2.准备小鼠进行实验<ol><li>通过注射氯胺酮和赛拉嗪混合物（分别为10mg / kg和80mg / kg体重）麻醉小鼠。注入0.01mL混合物/ 10g体重的小鼠。 </li><li>通过坚固的脚趾捏捏确认深度麻醉。如果有踏板反射，至少30秒后再次测试鼠标。 </li><li>使用兽医眼膏防止麻醉期间眼睛的干燥。在任何程序中不要让小鼠无人值守。 </li><li>通过注射高剂量的氯胺酮和赛拉嗪混合物注射（分别为10mg / kg和80mg / kg体重）来安乐死小鼠mixture（0.01ml / 10g体重），然后颈椎脱位。 注意:这种安乐死方法由怀俄明州大学IACUC批准。 </li></ol> 3.分离腹股沟脂肪脂肪垫<ol><li>放置左侧的鼠标（供给NCD或HFD（±CAP）32周）进行手术。 注意:通过将鼠标放在左侧，左前肢和左后肢将在解剖平台上，右前肢和右后肢将远离平台。 </li><li>用2英寸2纱布垫消毒皮肤表面，浸泡在约2.5 mL的70％乙醇中。使用手术刀穿过皮肤进行2 - 3 mm的横切，露出下面的脂肪层。 </li><li>通过皮肤使用一个1厘米切割（通过鼠标的大小），使用刚好在肋骨笼下方的手术刀穿过背部表面连接两个侧切口。 </li><li>通过小心地拖动皮肤皮肤剥去皮肤使用无菌镊子，通过不切割脂肪垫使皮下垫完好无损。这是躺在皮肤下的脂肪垫。 </li><li>使用一把剪刀从底层的肌肉和筋膜小心地解剖脂肪垫。拉下脂肪垫，因为它从下面的肌肉切开。 注意:脂肪垫的重量和尺寸取决于小鼠的类型（NCD或HFD（±CAP）-fed）。 </li><li>使用镊子将其转移到含有磷酸盐缓冲盐水（PBS）的培养皿中，直到脂肪分解实验，室温（〜15分钟）。 </li><li>如步骤3.2-3.6所述，从鼠标右侧分离脂肪垫。 </li></ol> 4.基底甘油从腹股脂肪垫释放<ol><li>使用锋利的剪刀将约20毫克脂肪组织切成5至8片。 </li><li> 在200μL孵育培养基（含有2％脂肪酸的Dulbecco&#39;s Modified Eagel&#39;s Medium（DMEM））中孵育切割的腹股脂肪垫游离牛血清白蛋白（BSA）），37℃，5％CO 2和95％湿度60分钟。 <ol><li>收集培养基并在-80℃冷冻进行脂解分析。 注意:切片用于脂质提取后蛋白质浓度的测定。 </li><li>在镊子的帮助下将切片的脂肪块转移到1mL提取溶液（氯仿:甲醇（2:1，v / v）和1％冰乙酸中）中，并在37℃下在100℃下剧烈摇动下孵育60分钟转。丢弃脂肪提取液。 注意:此步骤将从切片中提取脂肪。丢弃脂肪提取液，因为它会干扰蛋白质的测定。 </li><li>使用镊子将组织（从步骤4.2.2）转移到含有500μL裂解溶液（含有0.1％十二烷基硫酸钠的SDS的0.3N NaOH）的无菌微量离心管中，并在55℃下剧烈摇动孵育过夜（12小时）以100rpm。 </li></ol></li><li>确定组织使用二辛可宁酸（BCA）试剂和BSA作为标准18（步骤4.2.3）的蛋白质浓度。 </li><li> 在冰上解冻冷冻培养基（步骤4.2.1），并使用游离甘油试剂测定培养基的甘油含量，如方案6和7 6所述 。 <ol><li>从使用甘油标准物19绘制的标准曲线中推断出样品中甘油的浓度，并表示脂肪分解为每毫升20毫克每毫克蛋白质的纳摩尔甘油。 </li></ol></li></ol> FSK刺激的脂肪分解<ol><li> 将含有2％无脂肪酸BSA，10μMFSK和5μMTriacsin C的200μLDMEM中的约20mg从NCD或HFD（±CAP）野生型小鼠获得的腹股脂肪垫（5至8个切片）预孵育37℃，5％CO 2和95％湿度60分钟。 </str翁&gt; <ol><li>使用镊子将组织块转移到相同的培养基中，并在37℃，5％CO 2和95％湿度下孵育另外60分钟。收集孵育培养基并储存在-80℃直到脂解分析。 注意:脂肪分解的刺激导致脂肪酸和甘油在15分钟内快速释放，其在第一小时内是线性的，之后是高原。由于刺激脂肪分解速率在FSK刺激的第一和第二小时之间较高且稳定，因此该方案在第二次孵育期后按照前述1测量了刺激状态下的脂肪分解。所以，实验步骤（5.1和5.1.1）都是必要的。 </li></ol></li><li>从腹股沟脂肪垫中提取脂肪，将镊子的切片（从NCD或HFD（±CAP）的小鼠获得）从步骤5.1转移到1mL提取溶液（氯仿:甲醇（2 :1。v /v）和1％冰醋酸），并在37℃下以100rpm剧烈摇动孵育60分钟。丢弃提取的脂肪。 </li><li>使用镊子将组织（来自步骤5.2）转移到含有500μL裂解溶液（含有0.1％SDS的0.3N NaOH）的无菌微量离心管中，并在55℃下以100rpm剧烈摇动孵育过夜（12小时）。 </li><li>分别如步骤4.3和4.4所述确定组织的蛋白质浓度和甘油含量。 </li></ol> 6.制备游离甘油试剂<ol><li>将琥珀色试剂19在琥珀色玻璃小瓶中的40mL去离子水中重新配置，在瓶上放置一个塞子，通过倒置10次混合。不要摇晃混合。 </li><li>通过用铝箔完全覆盖小瓶，将小瓶在4°C内保存在防光的冰箱内。 </li><li>继续制备甘油标准品（第7节）。 </LI&gt; </ol>甘油标准品的制备和甘油含量的测定<ol><li>通过用65μL去离子水稀释35μL的供应原料，制成1mM的储备液。 注意:制造商提供的甘油标准品是2.8 mM甘油。 </li><li>采取五个1厘米路径长度一次性甲基丙烯酸比色皿。使用标记为0，1.25，2.5，5和10 nmol的标记标记比色皿。 </li><li>将0,2.25,2.5,5和10μL的1mM甘油标准品加入到各自标记的比色皿中。用去离子水将每个比色杯的体积补充至10μL。 </li><li>通过将10μL样品加入到新鲜的500μL预标记离心管中的90μL去离子水中，对所有样品进行1至10次稀释（来自步骤4.4或5.1.1）。将10μL稀释的样品加入标有相应样品标识号的比色皿中。 </li><li>打开UV-VIS分光光度计并设置波形长度至540 nm。 </li><li>将游离甘油试剂保持在室温，将小瓶（步骤6.2）保持在室温15分钟。 </li><li>设置一系列标记的比色皿为空白"0"nmol标准（步骤7.3），标准（步骤7.3）和样品（步骤7.4）。 </li><li>将各10μL标准品和样品加入各自标记的比色杯中。使用"0"nmol标准为空白。 </li><li>在每个比色皿中加入0.8 mL游离甘油试剂，使用1 mL移液管含有甘油标准液。 </li><li>用1.5cm方形塑料石蜡膜覆盖比色皿，将反应杯内容物3次混合，室温放置10分钟。 </li><li>将比色杯置于UV-VIS分光光度计中，记录吸光度（波长540 nm）。 </li><li>使用X轴上的标准浓度（nmol）和Y轴上记录的吸光度绘制标准曲线。 </li><li>计算gl的浓度使用在步骤7.12中绘制的标准曲线进行外推，样品中的yerol（nmol）。 </li><li>将稀释因子10（参见步骤7.4）和20（总体积）的样品浓度（nmol /比色杯）乘以</li><li>将每个样品中甘油的浓度（步骤7.13）除以通过BCA方法计算的蛋白质的mg。 注意:由于腹股沟脂肪垫样品孵育60分钟，表示结果为甘油/ mg蛋白/ h的nmol。 </li></ol>

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A link between regulation of the thermogenic and lipid oxidation pathways in the brown fat cell.</a> J Biol Chem. 276 (2), 1486-1493 (2001).</li><li>Leung, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capsaicin+as+an+anti-obesity+drug.">Capsaicin as an anti-obesity drug.</a> Prog Drug Res. 68, 171-179 (2014).</li><li>Hursel, R., Westerterp-Plantenga, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermogenic+ingredients+and+body+weight+regulation.">Thermogenic ingredients and body weight regulation.</a> Int J Obes (Lond). 34 (4), 659-669 (2010).</li><li>Kang, J. H., 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+capsaicin+reduces+obesity-induced+insulin+resistance+and+hepatic+steatosis+in+obese+mice+fed+a+high-fat+diet.">Dietary capsaicin reduces obesity-induced insulin resistance and hepatic steatosis in obese mice fed a high-fat diet.</a> Obesity (Silver Spring). 18 (4), 780-787 (2010).</li><li>Winkler, B., Steele, R., Altszuler, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+glycerol+uptake+to+plasma+glycerol+concentration+in+the+normal+dog.">Relationship of glycerol uptake to plasma glycerol concentration in the normal dog.</a> Am J Physiol. 216 (1), 191-196 (1969).</li><li>Dugan, C. E., Cawthorn, W. P., MacDougald, O. A., Kennedy, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+microfluidic+enzyme+assays+for+simultaneous+detection+of+lipolysis+products+from+adipocytes.">Multiplexed microfluidic enzyme assays for simultaneous detection of lipolysis products from adipocytes.</a> Anal Bioanal Chem. 406 (20), 4851-4859 (2014).</li></ol>

作者没有什么可以披露的。

TGL的击穿过程为甘油和脂肪酸被by ATGL 9 basal脂解期间催化和由蛋白质的阵列编排包括腺苷酸环化酶/ PKA-依赖性途径的激脂解21，22，23在活化。脂解作用增强增加脂肪酸的血浆水平为运输和能源使用24。脂肪酸被线粒体吸收为乙酰CoA，用于产生能量。 脂肪分解的程度和程度可以通过脂肪组织的甘油释放来有效测量（ 表1和图1 ）。 TGL的水解产生脂肪酸和甘油。以前的研究表明，膳食补充的CAP显着增强了基础以及FSK刺激的甘油释放E从腹股沟和棕色脂肪垫11,17。增加脂解作用的脂肪酸产量增加可作为产生热和产热的饲料，因为CAP也显着增加了腹股沟脂肪垫中线粒体UCP-1的表达。一致的这一概念，先前的研究表明，联合呼吁增强过氧化物酶体增殖激活受体α（PPARα）的腹股沟脂肪垫的表达，其在线粒体脂肪酸氧化和中UCP-1 25，26的转录上调了关键作用， 27 。因此，计算机辅助编组设备也在HFD喂养的小鼠28,29增加的代谢活性，产热和能量消耗。此外，HFD喂养抑制了刺激的脂肪分解，而CAP增加了基础和FSK刺激的PKA依赖性脂肪分解以及增强的胰岛素敏感性30 。因此， 如图2中的模型所描述的那样，解释说CAP可以增加基础和刺激的脂肪分解而不引起胰岛素抵抗，因为多余的脂肪被燃烧以通过线粒体UCP-1上调产生热量。 本文介绍了测量白色脂肪组织中甘油释放的方法。虽然有几种方法，使用甘油的稳定同位素是可用的，它们需要或者高效液相色谱法或气相色谱法/质谱法用于测量的15，16。此外，甘油放射性示踪剂测量与非特异性31相关 。替代方法包括确定涉及脂解的脂肪酶和调节蛋白的mRNA和蛋白质水平。 d也可使用比色法确定循环中长链脂肪酸的浓度。这里描述的方法在分析脂肪组织中的甘油释放方面非常有效，因为可以使用能够进行荧光和吸光度测量的平板读数器进行。此外，本文中描述的简化的甘油释放分析方法提供了改进的稳定性和精度32 。 应该采用标准操作程序，因为这种方法包括外科技术来分离脂肪垫。该协议建议使用无脂肪的BSA。这可能导致气泡形成，这将干扰吸光度读数。因此，处理样品时应注意防止气泡。除非在议定书中建议采取这样的步骤，否则应注意不要在BSA处理过程中摇动或反转样品。此外，腹股沟脂肪的脂肪提取步骤至关重要，因为脂肪的存在会干扰机体h腹股沟脂肪组织蛋白测定。另外，在制备甘油标准品时，重要的是通过倒置混合内容物，并且不摇动小瓶。 这里描述的方法可用于成功分离腹股沟脂肪垫用于多种测定，包括脂肪分解测量。为腹股沟脂肪垫描述的脂肪分解方案可以容易地扩展到其他组织中的脂肪分解测量。该方法针对各种脂肪垫以及前脂肪细胞进行了优化。 总之，本文介绍了测量从NCD或HFD（±CAP）的小鼠分离的腹股沟脂肪脂肪垫中的基础和FSK刺激的甘油释放的方法。所提供的数据表明，CAP的TRPV1激活增加了基础和FSK刺激的脂肪分解，并防止脂肪在脂肪组织中的积累。这些数据表明TRPV1蛋白在调节中的关键作用n在白色脂肪组织中的脂肪分解。

脂肪组织为所需的产热3,4脂肪2和脂肪酸氧化存储能量。通过饮食摄取的脂肪酸与脱蛋白一起包装到乳糜微粒中，并通过血液循环递送到身体的不同组织。虽然大多数细胞在体内储存能量的储备，脂肪组织储存多余的能量为脂肪5,6。脂肪组织中的脂肪分解是由复杂过程调节的，脂肪分解的分子细节仍然模糊7 。 脂肪分解是通过脂肪甘油三酯脂肪酶（ATGL） 8将存储在脂肪组织中的甘油三酯（TGL）水解以产生甘油和脂肪酸（FA）的过程。基础和刺激脂肪分解的改变是肥胖的特征。 basal脂肪分解由ATGL活化9调节，其将TGL转化为二酰基甘油（DAG），随后水解成单酰甘油（MAG）。 通过腺苷酸环化酶激活激素敏感性脂肪酶（HSL）激活环磷酸腺苷（cAMP）依赖性蛋白激酶A（PKA）刺激并引起脂肪分解。因此，脂肪分解，基础和刺激的测量对于分析涉及该过程的蛋白质的活性是重要的。另外，解开脂肪分解的分子调控可能有利于开发针对肥胖的新型治疗策略10 。因为刺激脂肪分解和脂肪酸氧化的分子是降低储存在贮库中的脂肪的潜在候选者，所以重要的是采用鲁棒的重现性测定法。 以前发表的数据表明，CAP在白色脂肪组织中表达的TRPV1蛋白的激活增强了基础和FSK（腺苷酸环化酶激活剂） - 腹股沟脂肪垫中的刺激性脂肪分解11 。以前的研究还表明，通过CAP长期激活TRPV1的激活PKA 12。因为PKA的活化刺激脂解13，14，测量从NCD或HFD（±CAP）32周供给各自的饮食后喂食的小鼠分离的腹股沟脂肪垫基础和PKA依赖性刺激脂解作用将验证TRPV1的作用激活脂解。 本文介绍了确定基础和刺激的脂肪分解的有效方法。虽然这采用glycerol和繁琐的高效液相色谱法或气相色谱法/质谱法测量15，16是可用的放射性同位素等方法，这种方法提供了一种更直接的，简单且成本有效的确定脂肪组织脂肪分解的技术。

<table><tbody><tr><td>Capsaicin</td><td>Sigma, USA</td><td>M2028</td><td>TRPV1 agonist</td></tr><tr><td>Forskolin</td><td>Sigma, USA</td><td>F6886</td><td>Adenylyl cyclase activator</td></tr><tr><td>DMEM</td><td>GE healthcare and life sciences, UT, USA</td><td>SH30081.01</td><td></td></tr><tr><td>High fat diet</td><td>Research diets, New Brunswick, USA</td><td>D12492</td><td>Abbreviated as HFD</td></tr><tr><td>Tris</td><td>Amresco, USA</td><td>O497</td><td></td></tr><tr><td>Sodium chloride</td><td>Thermofisher Scientific</td><td>BP358-212</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma, USA</td><td>D6750</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma, USA</td><td>D9163</td><td></td></tr><tr><td>Sodium orthovanadate</td><td>Sigma, USA</td><td>S6508</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma, USA</td><td>P8340</td><td></td></tr><tr><td>Free Glycerol reagent</td><td>Sigma USA</td><td>F6428</td><td></td></tr><tr><td>DMSO</td><td>Sigma, USA</td><td>D8779</td><td></td></tr><tr><td>Triacsin C</td><td>Sigma, USA</td><td>T4540</td><td>Acyl CoA transferase inhibitor</td></tr><tr><td>Bovine serum albumin</td><td>Sigma, USA</td><td>A7030</td><td></td></tr><tr><td>Chloroform</td><td>Sigma Aldrich</td><td>31998-8</td><td></td></tr><tr><td>Methanol</td><td>Thermofisher Scientific, USA</td><td>A412-1</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Amresco, USA</td><td>O583</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma, USA</td><td>L3371</td><td></td></tr><tr><td>Bicinchoninic acid reagent</td><td>Sigma, USA</td><td>BCA1-1KT</td><td></td></tr><tr><td>UV-VIS Spectrophotometer</td><td>Pharmacia Biotech, NJ, USA</td><td>Ultrospec 2000</td><td></td></tr><tr><td>Normal chow diet</td><td>Labdiet.com</td><td>500I</td><td>abreviated as NCD</td></tr><tr><td>C57BL/6 mice</td><td>Jackson Laboratory, CT, USA</td><td>Stock number000664</td><td>wild type mice</td></tr><tr><td>Parafilm</td><td>Heathrow Scientific, USA</td><td>HS 234526A</td><td></td></tr><tr><td>Glycerol standard</td><td>Sigma, USA</td><td>G7793</td><td></td></tr></tbody></table>

measurement of basal and forskolin-stimulated lipolysis in inguinal adipose fat pads

脂解是脂肪组织中作为甘油三酯存储的脂质水解成甘油和脂肪酸的过程。本文介绍了从正常食物饮食（NCD），高脂肪饮食（HFD）或含有0.01的高脂肪饮食（HFD）的野生型小鼠分离的腹股沟脂肪垫中测量基础和毛喉素（FSK）刺激的脂肪分解的方法百分比的辣椒素（CAP;瞬时受体潜在的香草素亚科1（TRPV1）激动剂）32周。本文所述的用于进行离体脂解的方法采用Schweiger 等人 1我们提供了一个详细的方案，通过紫外 - 可见（UV / VIS）分光光度法测量甘油水平。这里描述的方法可用于成功分离腹股沟脂肪垫用于脂肪分解测量以获得一致的结果。腹股沟脂肪垫描述的方案可以容易地扩展到其他组织中的脂肪分解。

为了评估CAP对基础和受刺激的脂肪分解的影响，本研究测量了从NCD或HFD（±CAP）野生型小鼠分离的腹股沟脂肪脂肪垫中的脂肪分解。腹股沟脂肪垫的基础和FSK刺激的脂肪分解的代表性结果在表I中给出。在Triacsin C存在下，基础和FSK刺激的甘油释放，其抑制酰基辅酶A合成酶并阻止TGL的再生。 如图1所示，HFD抑制了FSK刺激的脂肪分解，CAP增加了基础和FSK刺激的脂肪分解。使用Triacsin C抑制TGL从甘油和脂肪酸11的再生。所有数据表示为均值±SEM使用单因素方差分析进行组间比较，并使用Student t检验进行事后分析。样品量已设定<html>以确定一组中结果变量的平均值是否与另一组中的平均值显着不同。 p值&lt;0.05被认为具有统计学意义。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55625/55625fig1.jpg" /> 图1:CAP增加基底和FSK刺激的甘油释放在腹股脂肪垫。条形图表示从NCD或HFD（±CAP）野生型小鼠分离的腹股沟脂肪垫中的基础和FSK（10μM）刺激的甘油释放（nmol / mg蛋白/ h）的平均值±SEM。 **表示n = 6时p值&lt;0.05的统计学显着性。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55625/55625fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 5 / 55625fig2.jpg"/&gt; 图2:甘油三酯水解（TGL）导致游离脂肪酸（FFA）和甘油的产生。 FFA经历线粒体β-氧化。这与解偶联蛋白1（UCP-1）的上调一起刺激产热。甘油释放通过文中描述的方案测量。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55625/55625fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 脂解 （nmoles甘油/ mg蛋白/ h） </td><td> NCD （n = 6） </td><td> HFD （n = 6） </td><td> HFD + CAP （n = 6） </td></tr><tr><td>基础</td><td> 16.22±1.28 </td><td> 17.16±1.48 </td><td> 52.07±2.66 </td></tr><tr><td> FSK刺激 （用Triacsin C） </td><td> 54.88±3.27 </td><td> 41.23±4.43 </td><td> 72.04±4.16 </td></tr></tbody></table> 表1:CAP对基底和FSK刺激的脂肪分解在腹股脂肪垫中的作用。在从NCD，HFD或HFD + CAP喂养的野生型小鼠获得的腹股沟脂肪脂肪组织中测量的平均基础和FSK刺激的甘油释放±SEM。

Watch this Scientific Journal Video about Measurement of Basal and Forskolin-stimulated Lipolysis in Inguinal Adipose Fat Pads at JoVE.com

Measurement of Basal and Forskolin-stimulated Lipolysis in Inguinal Adipose Fat Pads

该方案描述了从正常食物饮食（NCD）或高脂肪饮食（HFD）±辣椒素喂养的野生型小鼠获得的腹股沟脂肪垫中测定基础和毛喉素刺激的脂肪分解的方法。作为脂肪分解的指标，从腹股沟脂肪脂肪垫测量甘油释放。

测量基底和福斯柯林刺激的脂肪分解在腹股沟脂肪脂肪垫

Lipolysis is a process by which the lipid stored as triglycerides in adipose tissues are hydrolyzed into glycerol and fatty acids. This article describes ...

measurement-basal-forskolin-stimulated-lipolysis-inguinal-adipose-fat

Nanyang Technological University

Research

JoVE Journal

Biochemistry

9.6K 次观看.  University of Wyoming. 该方案描述了从正常食物饮食（NCD）或高脂肪饮食（HFD）±辣椒素喂养的野生型小鼠获得的腹股沟脂肪垫中测定基础和毛喉素刺激的脂肪分解的方法。作为脂肪分解的指标，从腹股沟脂肪脂肪垫测量甘油释放。 

视频: Measurement of Basal and Forskolin-stimulated Lipolysis in Inguinal Adipose Fat Pads

Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis

Considering that adipose tissue (AT) is an endocrine organ, it can influence whole body metabolism. Excessive energy storage leads to the dysregulation of adipocytes, which in turn induces abnormal secretion of adipokines, triggering metabolic syndromes such as obesity, dyslipidemia, hyperglycemia, hyperinsulinemia, insulin resistance and type 2 diabetes. Therefore, investigating the molecular mechanisms behind adipocyte dysregulation could help to develop novel therapeutic strategies. Our protocol describes methods for evaluating the molecular mechanism affected by hypoxic conditions of the AT, which correlates with adipocyte apoptosis in adult mice. This protocol describes how to analyze AT in vivo through gene expression profiling as well as histological analysis of adipocyte differentiation, proliferation and apoptosis during hypoxia exposure, ascertained through staining of hypoxic cells or HIF-1&#945; protein. Furthermore, in vitro analysis of adipocyte differentiation and its responses to various stimuli completes the characterization of the molecular pathways behind possible adipocyte dysfunction leading to metabolic syndromes.

Adipose tissue (AT) can influence whole body homeostasis, therefore understanding the molecular mechanisms of adipocyte differentiation and function is of importance. We provide a protocol for gaining new insights into these processes by analyzing adipocyte homeostasis, differentiation and hypoxia exposure as a model for induced adipocyte apoptosis.

成人脂肪细胞生物体的数字调控机制，通过分化与凋亡的动态平衡

Considering that adipose tissue (AT) is an endocrine organ, it can influence whole body metabolism. Excessive energy storage leads to the dysregulation of ...

科研

发育生物学

A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health. Still, there are significant limitations to available primary culture models, all of which have failed to faithfully recapitulate the adipose microenvironment or extend WAT viability beyond two weeks. The lack of a reliable primary culture model severely impedes research in WAT metabolism and drug development. To this end we have utilized NIH's standards of a microphysiologic system to develop a novel platform for WAT primary culture called 'SWAT' (sandwiched white adipose tissue). We overcome the natural buoyancy of adipocytes by sandwiching minced WAT clusters between sheets of adipose-derived stromal cells. In this construct, WAT samples are viable over eight weeks in culture. SWAT maintains the intact ECM, cell-to-cell contacts, and physical pressures of in vivo WAT conditions; additionally, SWAT maintains a robust transcriptional profile, sensitivity to exogenous chemical signaling, and whole tissue function. SWAT represents a simple, reproducible, and effective method of primary adipose culture. Potentially, it is a broadly applicable platform for research in WAT physiology, pathophysiology, metabolism, and pharmaceutical development.

White adipose tissue (WAT) has critical deficiencies in its current primary culture models, hindering pharmacological development and metabolic studies. Here, we present a protocol to produce an adipose microphysiological system by sandwiching WAT between sheets of stromal cells. This construct provides a stable and adaptable platform for primary WAT culture.

人体脂肪 Microphysiologic 平台: 夹心白色脂肪组织

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health.  Still, there are significant limitations to available primary ...

行为学

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

本地化识别，切除脂肪小鼠油库，并

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

医学

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

人网膜和皮下脂肪组织 CD34+CD31+ 内皮细胞的分离、扩增和脂肪诱导

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

免疫与感染

Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for the design of personalized microtissues for health screening and drug testing. Current strategies are often quite complex and fail to accurately fully depict the multicellular and functional properties of thermogenic adipose tissue. Microvascular fragments, small intact microvessels comprised of arteriole, venules, and capillaries isolated from adipose tissue, serve as a single autologous source of cells that enable vascularization and adipose tissue formation. This article describes methods for optimizing culture conditions to enable the generation of three-dimensional, vascularized, and functional thermogenic adipose tissues from microvascular fragments, including protocols for isolating microvascular fragments from adipose tissue and culture conditions. Additionally, best practices are discussed, as are techniques for characterizing the engineered tissues, and sample results from both rodent and human microvascular fragments are provided. This approach has the potential to be utilized for the understanding and development of treatments for obesity and metabolic disease.

Here, we present a detailed protocol outlining the use of microvascular fragments isolated from rodent or human fat tissue as a straightforward approach to engineer functional, vascularized beige adipose tissue.

微血管碎片血管化产热脂肪组织的三维培养

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for ...

生物工程

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 ...

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

新生小鼠肩胛间脂肪组织基质血管部分棕色脂肪细胞的分化和成像

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with ...

生物学

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

Adipocytes store energy in the form of triglycerides in lipid droplets. This energy can be mobilized via lipolysis, where the fatty acid side chains are sequentially cleaved from the glycerol backbone, resulting in the release of free fatty acids and glycerol. Due to the low expression of glycerol kinase in white adipocytes, glycerol re-uptake rates are negligible, while fatty acid re-uptake is dictated by the fatty acid binding capacity of media components such as albumin. Both glycerol and fatty acid release into media can be quantified by colorimetric assays to determine the lipolytic rate. By measuring these factors at multiple time points, one can determine the linear rate of lipolysis with high confidence. Here, we provide a detailed protocol for the measurement of lipolysis in in vitro differentiated adipocytes and ex vivo adipose tissue from mice. This protocol may also be optimized for other preadipocyte cell lines or adipose tissue from other organisms; considerations and optimization parameters are discussed. This protocol is designed to be useful in determining and comparing the rate of adipocyte lipolysis between mouse models and treatments.

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

Triglyceride lipolysis in adipocytes is an important metabolic process resulting in the liberation of free fatty acids and glycerol. Here, we provide a detailed protocol to measure basal and stimulated lipolysis in adipocytes and ex vivo adipose tissue from mice.

测量体外分化的离体小鼠脂肪组织和原代前脂肪细胞的脂肪分解率

Adipocytes store energy in the form of triglycerides in lipid droplets. This energy can be mobilized via lipolysis, where the fatty acid side chains are ...

小鼠白色脂肪组织的半自动分离

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

作者聚焦：使用组织解离剂从小鼠白色脂肪组织中半自动分离基质血管组分  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

使用组织解离器从小鼠白色脂肪组织中半自动分离基质血管部分

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

测量基底和福斯柯林刺激的脂肪分解在腹股沟脂肪脂肪垫

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此视频中的章节

成人脂肪细胞生物体的数字调控机制，通过分化与凋亡的动态平衡

人体脂肪 Microphysiologic 平台: 夹心白色脂肪组织

本地化识别，切除脂肪小鼠油库，并

人网膜和皮下脂肪组织 CD34+CD31+ 内皮细胞的分离、扩增和脂肪诱导

微血管碎片血管化产热脂肪组织的三维培养

评估小鼠全身脂质处理能力

新生小鼠肩胛间脂肪组织基质血管部分棕色脂肪细胞的分化和成像

测量体外分化的离体小鼠脂肪组织和原代前脂肪细胞的脂肪分解率

使用组织解离器从小鼠白色脂肪组织中半自动分离基质血管部分

使用组织解离器从小鼠白色脂肪组织中半自动分离基质血管部分