Name: high fat diet feeding and high throughput triacylglyceride assay in drosophila melanogaster
Uploaded: 2017-09-13T12:30:00.000+00:00
Duration: 8 min 28 s
Description: Watch this Scientific Journal Video about High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster at JoVE.com

我们想感谢埃丽卡-泰勒编辑这份手稿。这项工作的经费来自国家卫生研究院 (P01 HL098053、P01 AG033561 和 R01 HL054732) 的赠款、导流洞、博士后研究补编 (R01 HL085481) 和研究金 (AAUW) S.B.D., 以及美国心脏协会向 S.B.D. 提供的赠款。和 R.T.B。

1. 风喂养协议 <img alt="Table 1" src="/files/ftp_upload/56029/56029table1.jpg"/> 表1。飞行食品食谱. 
这张桌子总结了不同的配料用来准备我们的控制食品。一旦制成, 10 毫升的食物被倒入瓶, 冷却和储存在4和 #176; C 用于长期存储. 
<ol> <li> 风准备 <ol> <li> 以制作1公斤高脂肪食品, 称700克预制正常飞行食品 (见 表 1 ) 和300克椰子油, 使用分析天平放入两个单独的容器中. </li>
		<li> 在微波炉中单独加热每个容器, 直到内容完全融化。将椰子油倒入苍蝇食品容器中。用搅拌器搅拌, 直到油和苍蝇的食物混合好. 
		注意: 没有大块的食物或油应该是可见的。当苍蝇的食物在微波炉里融化时, 每隔1分钟停止, 混合食物, 防止沸腾. </li>
		<li> 重新称量高脂肪食物。如果总重量小于1公斤 (700 克食物 + 300 克椰子油), 加水 (大约10毫升), 以补偿在加热过程中的蒸发, 并带回重量1公斤. </li>
		<li> 每瓶倒入约10毫升混合高脂肪食物 (约25% 瓶体积)。其次, 用棉布覆盖, 防止苍蝇和 #39; 污染。在室温下冷却1小时, 然后将小瓶转移到4和 #176; C 存储4周. 
		注意: 正常食物 (NF) 被用作控制食品。也倒入10毫升 (约25% 瓶体积) 的 NF 每瓶。对于风食品, 用椰子油取代30% 的 NF 食品。对于控制和实验条件, 苍蝇得到相同数量的 NF 和风 (每瓶10毫升的食物). </li>
	</ol> </li> </ol> <img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56029/56029fig1.jpg"/> 图 1 。风在 果蝇 中喂养. 
计划显示不同的哺养的步为苍蝇在控制食物 (正常饮食没有加法椰子油-NF) 或风 (与椰子油的加法)。整个过程需要10天后, 最初收集成人苍蝇。<a href="//ecsource.jove.com/files/ftp_upload/56029/56029fig1large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a> 
<ol start="2"> <li> 风喂养的 D. 腹中的风 注意: 该协议包括与任何飞线兼容的控制食物和喂养程序。控制和实验苍蝇放在含有相同数量食物的小瓶中 (NF 或风)。
	<ol> <li> 使用 CO 2 来麻醉苍蝇. 
		注: 对于新手, 请阅读参考 21 , 以了解有关引发和处理 四腹果蝇 的概述。
		<li> 收集0-5 天的老苍蝇从小瓶和转移 (通过翻转) 的苍蝇到新的小瓶含有 NF (以前温暖室温)。让苍蝇的年龄在21和 #176 增加5天; C. </li>
		<li> 在5天结束时, 从4和 #176 中取出含有风和 NF 的小瓶; C. 
		注意: 由于低温会使活苍蝇压力大, 在使用前, 让小瓶在室温下坐30分钟预热. </li>
		<li> 使用湿巾, 清洁和清除从瓶子两侧多余的液体. </li>
		<li> 插入一小块滤纸, 大约1厘米 x 3 厘米, 放入高脂肪的食物中吸收任何多余的液体. 
		注意: 这一步是防止苍蝇粘风食物的关键. </li>
		<li> 下一步, 将苍蝇转到高脂肪的食物上 (通过翻转)。把小瓶水平放置在他们的一侧, (不站立) 在21-22 和 #176; C 5 天. 
		注意: 避免高温, 因为这可以部分融化高脂肪的食物, 导致苍蝇坚持食物和死亡. </li>
		<li> 5 天后, 转 (通过翻转) 从 NF 和风的苍蝇到新的小瓶没有食物, 让他们的内脏空30分钟。接下来, 开始称量苍蝇. 
		注意: 在这个风喂养协议的关键步骤之一是准备一个混合风, 没有任何大块的苍蝇食物或浓缩油。均匀混合和冷却风到45-30 和 #176; c 是可取的, 在倒入小瓶和储存的食物在4和 #176; c. 控制和实验果蝇的适当年龄匹配是重要的和控制的营养和环境条件前风喂养。在饲养阶段, 控制和实验苍蝇必须给予相同数量的 NF 和风。切勿从过度拥挤的小瓶或瓶子中收集苍蝇 (以避免代的影响)。在 NF 和风喂养过程中, 每次每瓶最多放25只苍蝇, 以避免饮食限制或其他拥挤效果。风由30% 椰子油组成, 因此温度在22和 #176 以上; C 可能部分熔化风, 导致苍蝇黏附在油腻的食物和模子。在把苍蝇放在风上之前, 清洗小瓶以除去残留的油, 并插入滤纸以吸收食物中多余的液体。在风的苍蝇孵化期间, 小瓶被放置在他们的两侧, 为苍蝇提供一个无脂肪的水平表面. </li>
	</ol> </li> <li> 对每个基因型和性别进行衡量的苍蝇 <ol> <li>, 使用敏感比例记录 pre-labeled、空1.7 毫升管的重量. </li>
		<li> 使用苍蝇麻醉或 CO 2 , 麻醉和收集36苍蝇相同的基因型, 性别, 和喂养状态与一称量1.7 毫升管的刷子. </li>
		<li> 用同样的敏感秤称量装有苍蝇的管子. </li>
		<li> 确定此公式后的平均飞行重量: 
		<img alt="Equation 1" src="/files/ftp_upload/56029/56029eq1.jpg"/> 注意: 此协议中的苍蝇数量是 36. </li>
		<li> 闪光冷冻1.7 毫升管包含苍蝇通过暴跌到液氮2分钟, 收集并把管成箱。接下来, 将这些箱子转到-80 和 #176; C (优选温度) 用于存储, 直到与标签检测一起使用. 
		注意: 液氮处于极低的温度。请使用适当的个人防护用具. </li>
	</ol> </li> </ol> 2。标记化验 <ol> <li> 准备标记标准浓度 <ol> <li> 准备500毫升 PBT (PBS 1X, 0.05% 海卫). </li>
		<li> 使用0.5 毫升管和标签解决方案 ( 材料表 ), 准备100和 #181; 空白的 L (仅 PBT) 和100和 #181; 六标准标签浓度 (2 和 #181; g/和 #181; 1 和 #181; g/和 #181; 0.5 和 #181; g/和 #181; 0.25 和 #181; g/和 #181;0.125、#181; #181;0.0625、#181、g、#181、L) 与 PBT. </li>
		<li> 将管子放在冰上, 然后继续研磨苍蝇. </li>
	</ol> </li> <li> 研磨苍蝇 <ol> <li> 从-80 和 #176 中取出冻结的苍蝇; C (步骤 1.3)。把装有苍蝇的管子在冰上传输. </li>
		<li> 首先, 将 2 mm 金属研磨球放入96井球分配器中, 使用小画笔除去多余的球. </li>
		<li> 将96井研磨板倒置放在球分配器的顶部, 然后翻转将金属球直接转移到研磨板中。每个井应该有一个球. </li>
		<li> 使用多通道吸管, 将600和 #181 的 PBT 放入96井研磨板的每排. </li>
		<li> 用镊子, 每井加3只苍蝇。说明96井磨板每排/井中蝇类的基因型、性别和食物状况. </li>
		<li> 将瓶盖紧放在96井研磨板上。胶带可以放在顶部, 以确保没有泄漏. </li>
		<li> 将96井研磨板正确放置在研磨机中。将机器设置为最高速度, 并按下 #34; 运行和 #34; 研磨苍蝇3分钟. </li>
		<li> 3 分钟后, 停止研磨并进行板离心: centrifuge 为15分钟, 在 4000 x g, 4 和 #176; C。离心后, 小心地管理研磨板, 避免将上清与颗粒混合. </li>
	</ol> </li> <li> 示例加载和标记内容确定 <ol> 采取新的96井分光光度计板。使用多通道吸管, 负载200和 #181; L 的标签试剂到每行的板块. </li>
		<li> 加载20和 #181; 我的 PBT 进入第一排的第一井, 创建一个空白。移上下混合。避免形成气泡. </li>
		<li> 加载20和 #181; 每个标准稀释 (从步骤 2.1.2) 到第一排的剩余井中, 并由移上下混合。避免形成气泡. </li>
		<li> 使用多通道吸管, 将20和 #181 的每行的上清液从研磨板的每一排移到相应的96分光光度计板上。吸管向上和向下混合好。避免形成气泡. </li>
		<li> 下一步, 将透气箔放在96井板的顶部. </li>
		<li> 在37和 #176 上孵育盘; C 为 10 min. 
		注: 如果在井中有气泡, 离心板为2分钟, 在 2000 x g, 以消除所有气泡。气泡的存在会干扰吸光度的读数. </li>
		<li> 使用兼容的微阅读器读取 550 nm 板的每个井的吸光度。其次, 跟踪标准曲线, 确定未知样品的浓度 [和 #181; g/和 #181; L]. </li>
		<li> 若要确定每平均飞行重量的标签内容量, 请使用以下公式: 
		<img alt="Equation 2" src="/files/ftp_upload/56029/56029eq2.jpg"/> 注意: 在本议定书中, 3 只苍蝇在600和 #181 中被剁碎; PBT 的 L; 因此, 平均每只苍蝇的体积是200和 #181; l. </li>
	</ol> </li> <li> 布拉德福德化验和含蛋白质含量的标签内容的规范化 <ol> <li> 准备七100和 #181; 牛血清白蛋白 (BSA) 标准稀释 (75、#181; 100 和 #181; g/毫升; 150 和 #181; g/毫升; 200 和 #181; g/毫升; 250#181;500、#181;1000, #181, g/毫升) 与 PBT. </li>
		<li> 采取新的96井板和负载200和 #181; L 的1X 布拉德福德试剂到每行的板块. </li>
		<li> 在第一行的第一个井中, 添加10和 #181; L PBT 创建一个空白。移上下混合。避免形成气泡. </li>
		<li> 添加10和 #181; 每个标准稀释到第一排余下的井中, 并由移上下混合。避免形成气泡. </li>
		<li> 使用多通道吸管, 将10和 #181; 从每行的磨板上的飞清升到相应的96井板的排。吸管向上和向下混合好。避免形成气泡. </li>
		<li> 将透气箔置于96井板的顶部. </li>
		<li> 室温下孵育板5分钟。如果在井里有气泡, 离心板为2分钟在 2000 x g 去除他们. 
		注: 气泡的存在会干扰吸光度的读数. </li>
		<li> 使用分光光度计阅读 595 nm 的空白、标准和未知样品的吸光度。使用标准曲线确定每一行中样本的浓度 [和 #181; g/和 #181; L]. </li>
		<li> 使用以下公式规范化标记内容与蛋白质水平: 
		<img alt="Equation 3" src="/files/ftp_upload/56029/56029eq3.jpg"/> </li> </ol> </li> </ol>

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作者没有什么可透露的。

肥胖诱导小鼠可能需要几个月19,20。在苍蝇中, 这种风喂养协议允许在几天或更短的时间内诱导过量的脂肪堆积, 导致脂肪堆积仅在18小时后增加 (参见图 2)。风哺养以被描述的协议增加葡萄糖内容12并且减少钢脂肪酶和 PGC-1 表达24。这与成人果蝇的禁食形成对比, 导致脂肪和葡萄糖含量的快速下降25、26和增加的钢表达式24。同时, 提升钢或PGC-1级别可防止风引起的肥胖14、24。虽然30% 风已经使用在这个协议, 喂养苍蝇与 3%, 7%, 或15% 脂肪饮食诱发肥胖的剂量依赖性的时尚12, 也增加了风喂养的持续时间 (图 3)。我们还发现, 饲喂单一脂肪酸的主要成分的椰子油,即, 14% 月桂酸或5% 肉豆蔻酸, 导致显著升高脂肪堆积的12。
加速代谢反应与这些苍蝇在一个风方案是相关的减少寿命27在哺乳动物的观察,28, 但和 #62; 80% 的苍蝇生存在过去20天风27。由保守的细胞和分子过程控制脂质和葡萄糖代谢的快速诱导脂肪积累有利于许多肥胖相关的研究, 如糖尿病或毒性心肌病10, 12,13,14,15,16。在代谢失衡和相关的心脏功能障碍的结果中, 关键的发现可能会使人类进行比较性的转化研究。
风喂养协议与其他果蝇物种和与黑腹果蝇共享类似饮食的昆虫模型兼容。这种风喂养协议可以适当地适应生物如C. 线虫或其他昆虫需要不同的 ' 正常 ' 食物媒介。30% 风不适合用于需要高温的实验;然而, 减少椰子油或单一脂肪酸的浓度可能是足够的替代品12。
这个标签协议是基于 PBS, 一个无毒的缓冲, 可以方便地处理和实验没有油烟罩, 与以前使用的有机溶剂 (甲醇/氯仿或乙醚) 的脂提取和量化的15,29,30. 此缓冲区的另一个优点是它可以根据相同的初始匀浆对蛋白质含量进行规范化, 使小组织样品中的脂肪测定容易实现, 并为研究器官提供新的机会相声在个体代谢稳态。同样, 在研磨后获得的同样的最初的飞行匀浆, 可以用来进行平行的葡萄糖和糖原检测, 允许研究相同生物样品中的脂质和葡萄糖代谢的变化。此标记协议比以前的协议15、2930更省力或更耗时, 且吞吐量相当高, 使用96井格式, 可以快速测试接近100示例一小时内该协议也可用于量化脂肪含量的其他饮食条件下, 包括高糖饮食13,31, 饮食限制和饥饿, 以及老化研究, 以了解年龄相关的变化脂肪代谢。
肥胖、代谢综合征和相关病症在历史上是空前的高, 对人类健康造成灾难性的后果1,2。联合风协议和高通量标签分析提供了一个独特的平台, 使用模型生物体, 如果蝇, 以迅速诱导肥胖和屏幕的遗传因素, 或天然和合成化合物, 以更好地理解新陈代谢失调最终, 这可能会极大地促进科学发现的发展, 开发新的治疗或治疗代谢性疾病。

在这个时代, 肥胖及其相关的经济负担是一个世界性的问题1。每三美国人中就有两个超重或肥胖与相关的心脏疾病, 在成年人口的主要死因2。需要采用新的有效方法来充分研究代谢综合征的遗传和分子成分, 并利用模型生物体进行调控。因此, 我们选择果蝇果蝇模型, 因为它与哺乳动物共享最基本的生物过程, 包括老鼠和人类3,4,5,6。在进化过程中,果蝇的基因组是高度保守的, 但总体上要小得多, 基因重复性和代谢复杂度更低, 这使得它非常适合理解许多人类疾病所牵连的基本机制4,7,8. 另外, 由脂肪组织、肠道和胰腺所进行的特征过程在苍蝇中代表, 并在葡萄糖和脂代谢中调解调节功能, 例如, 与人相似9, 10,11。此外, 在人类控制肥胖, 胰岛素抵抗和糖尿病的基本分子通路在果蝇12,13,14中的功能保存,15,16. 象更高的有机体,果蝇有跳动的心脏在发展期间由相似的过程形成对那哺乳动物心脏3,17。因此, 开发一个可靠的风喂养协议和高通量标签化验, 适应有效的筛选目的使用的遗传工具盒的果蝇, 提供了一个重要的手段来研究和理解的基本遗传基础潜在的复杂代谢疾病。
风食品本身是由标准的实验室飞行食品补充椰子油, 其中主要是组成的饱和脂肪酸已知与代谢综合征18。当在哺乳动物模型中诱发肥胖, 例如啮齿目动物, 可能需要月19,20, 我们的优化风喂养协议在果蝇有效和性增加个体脂肪含量的问题天12,14。该协议, 与高通量标记化验, 允许有效的大规模筛选的影响, 遗传因素, 环境影响和药物候选者发现新的调节脂肪代谢。因此, 这些协议可能与理解和/或对抗肥胖和肥胖相关的人类病症有关。
该喂养方案具有通用性, 可用于研究单饱和脂肪酸和不饱和脂肪酸的代谢和功能作用。使用此高通量标签检测不仅限于黑腹果蝇, 而且可以适应各种具有角质层或硬细胞外基质的小模型生物体 (如如、其他果蝇种、线虫和其他新兴的无脊椎动物模型有机体) 在不同的环境、遗传或生理条件下测量脂肪含量, 在发育的任何阶段, 成年或代谢疾病的阶段。标签法是基于比色测量的一系列酶反应, 将标签降解成游离脂肪酸, 甘油, 甘油 3-磷酸盐和最后的 H2O2 , 反应与 4-基安 (4--和 35-dichloro-2-hydroxybenzene 磺酸 (35 地区) 生产一种用 96-井分光光度计测量的红色产品。

<table><tbody><tr><td>Talboys Ball dropper/bead Dispenser</td><td>Talboys</td><td>#: 930150</td><td></td></tr><tr><td>Talboys High Throughput Homogenizer</td><td>Talboys</td><td>#: 930145</td><td></td></tr><tr><td>Grinding Balls, Stainless Steel&amp;nbsp;</td><td>OPS Diagnostics, LLC</td><td># GBSS 156-5000-01</td><td>5000 balls</td></tr><tr><td>Masterblock 96 Well deep Microplates</td><td>Greiner Bio-One</td><td># T-3058-1</td><td>case of 80 plates</td></tr><tr><td>Greiner&amp;nbsp; 96 well microplate flat bottom</td><td>Sigma Aldrich</td><td># M4436</td><td>40 plates</td></tr><tr><td>Greiner CapMat for sealing multiwell plates</td><td>Sigma Aldrich</td><td># C3606</td><td>50 sealing plates</td></tr><tr><td>Reagent Reservoirs&amp;nbsp;</td><td>Thomas Scientific</td><td># 1192T71</td><td>12/PK</td></tr><tr><td>Thermo Scientific Finnpipette 4661040</td><td>Thermo Scientific</td><td># 4661040</td><td>1-10 ul multipipette</td></tr><tr><td>Thermo Scientific Finnpipette 4661070</td><td>Thermo Scientific</td><td># 4661070</td><td>30-300ul multipipette</td></tr><tr><td>Thermo Scientific Finnpipette 4661020</td><td>Thermo Scientific</td><td>#4661020</td><td>10-100ul multipipette</td></tr><tr><td>Multichannel tips</td><td>Denville Scientific Inc</td><td># P3131-S</td><td>for 10 uL pipette</td></tr><tr><td>Multichannel tips</td><td>Denville Scientific Inc</td><td># P3133-S</td><td>for 200 uL pipette</td></tr><tr><td>Multichannel tips</td><td>Denville Scientific Inc</td><td>#P1125</td><td>for 100 uL pipette</td></tr><tr><td>Forceps&amp;nbsp;</td><td>Roboz Surgical</td><td># 5 Dumonts</td><td>Super fine forceps</td></tr><tr><td>Mettler Toledo Excellence XS Analytical Balance Mfr# XS64</td><td>Cole-Parmer scientific experts</td><td># EW-11333-00</td><td></td></tr><tr><td>Metler Toledo Excellence XS Toploading Balance</td><td>Cole-Parmer scientific experts</td><td># EW-11333-49</td><td></td></tr><tr><td>96-Well microplate Centrifuge</td><td>Hettich Zentrifugen</td><td># Rotina 420R</td><td></td></tr><tr><td>Microplate Reader</td><td>Molecular devices</td><td># SpectraMax 190</td><td></td></tr><tr><td>Lab-Line Bench Top Orbit Environ Shaker Incubator</td><td>Biostad</td><td># 3527</td><td></td></tr><tr><td>Infinity Triglycerides reagent</td><td>Thermo Scientific</td><td># TR22421</td><td></td></tr><tr><td>Triglyceride Standard</td><td>Stanbio</td><td>#2103 - 030</td><td></td></tr><tr><td>Quick Start Bradford Protein Assay</td><td>Bio-RAD</td><td># 500-0205</td><td>1x dye Reagent</td></tr><tr><td>Coconut oil</td><td>Nutiva</td><td># 692752200014</td><td>15 0z jar</td></tr><tr><td>PBS 10X</td><td>Thermo Scientific</td><td># AM9625</td><td>500 ml</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td># 9002-93-1</td><td></td></tr><tr><td>Gas-permeable Foil</td><td>Macherey-Nagel</td><td># 740675</td><td>50 pieces</td></tr><tr><td>filter Paper</td><td>VWR</td><td># 28317-241</td><td>Pack of 100</td></tr><tr><td>Drosophila vials</td><td>Genesee Scientific</td><td>Cat #: 32-116SB</td><td></td></tr><tr><td>Quick Start Bovine Serum Albumin Standard</td><td>Bio-Rad</td><td># 5000206</td><td></td></tr><tr><td>FlyNap Anesthetic</td><td>Carolina</td><td># 173025</td><td>100 mL</td></tr><tr><td>Kimwipes Low-Lint</td><td>Uline</td><td># S-8115</td><td>1-Ply, 4.4 x 8.4&quot;</td></tr></tbody></table>

high fat diet feeding and high throughput triacylglyceride assay in drosophila melanogaster

心脏病是全世界人类死亡的头号原因。许多研究表明, 肥胖与人类心脏功能失调之间有密切的联系, 但是需要更多的工具和研究来更好地阐明所涉及的机制。一个多世纪以来, 基因高度驯服的果蝇模型在发现重要基因和分子通路方面起到了很大的作用, 证明它们在物种中是高度保守的。许多生物过程和疾病机制在功能上被保存在飞行中, 如发育 (如, 身体计划, 心脏), 癌症和神经退行性疾病。最近, 关于肥胖和继发性疾病的研究, 如模型生物体中的心脏病, 在确定参与人体代谢综合征的关键调控因子方面发挥了极为关键的作用。
在这里, 我们建议使用这种模式的有机体作为一个有效的工具, 以诱导肥胖,即, 过度脂肪积累, 并制定一个有效的协议, 以监测脂肪含量的形式, 标记积累。除了高度保守的, 但不太复杂的基因组, 苍蝇也有短寿命的快速试验, 结合 cost-effectiveness。本文提供了一个详细的协议为高脂肪饮食 (风) 喂养在果蝇诱导肥胖和高通量三 (标签) 测定的相关增加脂肪含量, 目的是高度重现性和有效的 large-scale 基因或化学筛选。这些协议提供了新的机会来有效地调查肥胖所涉及的监管机制, 并为药物发现研究提供一个标准化的平台, 用于快速测试候选药物对发展或预防肥胖、糖尿病及相关代谢疾病。

在四腹果蝇中, 与其他物种的情况一样, 雄性和雌性之间存在性异形,22。众所周知, 雌性比男性更大, 腹部中的脂肪比22多。为了测试我们的协议的有效性, 我们进行了标记分析, 以确定标准实验室野生 (w1118) 中的男女之间的标签内容差异。数据显示, 女性的全身脂肪比男性的要多 (图 2A, B)。数据还表明, 该化验是稳定的, 没有变化的标签量化随着时间的推移 (最多50分钟后孵化), 并没有变化取决于生物样本的大小 (3 或5苍蝇)。
风消耗已被证明是导致肥胖的人和鼠标19,20,23。为了测试我们的喂养协议的有效性, 我们用我们的风和控制喂食雌性苍蝇进行了标记化验。我们发现, 风在果蝇中的消耗会导致随时间逐渐累积的脂肪含量增加 (图 3A-c)。另一个重要的发现是, 在只有18小时的风喂养后 (图 3C), 我们能够在这些苍蝇中诱发脂肪含量的显著增加。这些结果表明, 这种遗传模型系统是一个理想的工具, 加速研究寻找新的调节风诱导肥胖。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56029/56029fig2.jpg"> 
图 2.标记化验在男性和女性苍蝇. 2周的老苍蝇的正常饮食收集, 按性别分组 (男性和女性), 权衡和地面 (3 或5苍蝇每井) 标签分析。按照本文所述的步骤进行标记分析。每个样品的吸光度在 550 nm 在不同的时点 (0 分钟, 5 分钟, 20 min 和 50 min) 被读取, 以确定随着时间的推移标记量化的最终波动, 不同种群大小 (3 和5苍蝇) 的脂肪含量变化 ( w1118苍蝇, 和男性和女性的标签内容的差异。结果表明, 雌性的脂肪堆积量大于其雄性 (A-b), 标记测量在反应孵化37° c (ab) 后不波动50分钟。此外, 使用3或5只苍蝇 (A-B) 的标记检测之间的平均标记级别保持不变。数据显示为平均± SEM. 统计学: 无显著差异。<a href="//ecsource.jove.com/files/ftp_upload/56029/56029fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56029/56029fig3.jpg"> 
图 3.风对脂肪堆积的影响. 
a-b:在正常或风上2周的老雌蝇收集5天, 并进行标记化验以确定脂肪含量。标记内容是以权重 (A) 或蛋白质级别 (B) 进行规范化的。数据表明, 风消费导致增加脂肪含量的两种方法的正常化。C:在正常/控制食品 (NF) 和18小时、1天或2天的风上, 对2周的老雌蝇进行标记含量检测。研究结果显示, 从18小时到2天的接触风, 标记水平有了显著的提高。数据显示为平均± SEM. 统计: 学生 t 测试。* p & #60; 0.05、** 和 #60; 0.01、*** 和 #60; 0.001。<a href="//ecsource.jove.com/files/ftp_upload/56029/56029fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster at JoVE.com

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

高脂膳食喂养和高通量三测定在果蝇中的含量

这是一个高脂肪饮食喂养协议, 以诱发肥胖在果蝇, 一个模型, 了解的基本分子机制牵连脂。它还提供了一种高通量三测定方法, 用于测量在各种饮食、环境、遗传或生理条件下的果蝇中的脂肪积累和潜在的其他 (昆虫) 模型。

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

high-fat-diet-feeding-high-throughput-triacylglyceride-assay

Nanyang Technological University

Research

JoVE Journal

Genetics

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

11.2K 次观看.  Sanford Burnham Prebys Medical Discovery Institute. 这是一个高脂肪饮食喂养协议, 以诱发肥胖在果蝇, 一个模型, 了解的基本分子机制牵连脂。它还提供了一种高通量三测定方法, 用于测量在各种饮食、环境、遗传或生理条件下的果蝇中的脂肪积累和潜在的其他 (昆虫) 模型。

视频: High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and lowered activity levels. These multifactorial diseases arise from a combination of genetic, environmental, and behavioral factors. One such complex disease is Metabolic Syndrome (MetS), which is a cluster of metabolic disorders, including hypertension, hyperglycemia, and abdominal obesity. Exercise and dietary intervention are the primary treatments recommended by doctors to mitigate obesity and its subsequent metabolic diseases. Exercise intervention, in particular aerobic interval training, stimulates favorable changes in the common risk factors for Type 2 Diabetes Mellitus (T2DM), Cardiovascular Disease (CVD), and other conditions. With the influx of evidence describing the therapeutic effect exercise has on metabolic health, establishing a system that models exercise in a controlled setting provides a valuable tool for assessing the effects of exercise in an experimental context. Drosophila melanogaster is a great tool for investigating the physiological and molecular changes that result from exercise intervention. The flies have short lifespans and similar mechanisms of metabolizing nutrients when compared to humans. To induce exercise in Drosophila, we developed a machine called the TreadWheel, which utilizes the fly&#39;s innate, negative geotaxis tendency to gently induce climbing. This enables researchers to perform experiments on large cohorts of genetically diverse flies to better understand the genotype-by-environment interactions underlying the effects of exercise on metabolic health.

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The TreadWheel uses rotational motion to gently induce exercise in adult Drosophila melanogaster by exploiting flies&#39; innate, negative geotaxis. It allows for analysis of the interactions between exercise and factors, such as genotype, sex, and diet, and their effect on physiological and molecular assays to assess metabolic health.

TreadWheel:果蝇轻度诱导运动的间歇训练协议

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and ...

科研

遗传学

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, and issues commands to start or stop feeding. Deciphering the processes underlying the decision-making of timely and moderate feeding carries major implications in our understanding of physiological and psychological disorders related to feeding control. Simple, quantitative, and robust methods are required to measure the food ingestion of animals after experimental manipulation, such as forcibly increasing the activities of certain target neurons. Here, we introduced dye-labeling-based feeding assays to facilitate the neurogenetic study of feeding control in adult fruit flies. We review available feeding assays, and then describe our methods step-by-step from setup to analysis, which combine thermogenetic and optogenetic manipulation of neurons controlling feeding motivation with dye-labeled food intake assay. We also discuss the advantages and limitations of our methods, compared with other feeding assays, to help readers choose an appropriate assay.

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Quantitative food-intake assays with dyed food provide a robust and high-throughput means to evaluate feeding motivation. Combining the food consumption assay with thermogenetic and optogenetic screens is a powerful approach to investigate the neural circuits underlying appetite in adult Drosophila melanogaster.

在果蝇中结合定量食物摄入量测定和强迫激活神经元研究食欲

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, ...

行为学

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental factors, and the toxicological and pharmacological effects of numerous substances. Few methods currently implemented are amenable to high throughput measurement. The Microplate Feeder Assay (MFA) was developed for quantifying the consumption of liquid food for individual flies using absorbance. In this assay, flies consume liquid food medium from select wells of a 1536-well microplate. By incorporating a dilute tracer dye into the liquid food medium and loading a known volume into each well, absorbance measurements of the well acquired before and after consumption reflect the resulting change in volume (i.e., volume consumed). To enable high throughput analysis with this method, a 3D-printed coupler was designed that allows flies to be sorted individually into 96-well microplates. This device precisely orients 96- and 1536-well microplates to give each fly access to up to 4 wells for consumption, thus enabling food preference quantification in addition to regular consumption. Furthermore, the device has barrier strips that toggle between open and closed positions to allow for controlled containment and release of a column of samples at a time. This method enables high throughput measurements of consumption of aqueous solutions by many flies simultaneously. It also has the potential to be adapted to other insects and to screen consumption of nutrients, toxins, or pharmaceuticals.

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

The microplate feeder assay offers an economical, high throughput method for quantifying liquid food consumption in Drosophila. A 3D-printed device connects a 96-well microplate in which flies are housed to a 1536-well microplate from which flies consume a feeding solution with a tracer dye. The solution volume decline is measured spectrophotometrically.

用于果蝇消费量化的高吞吐量微板馈线分析

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

高通量检测，以检查个体产蛋首<em&gt;果蝇</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

神经科学

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

一个简单的方法来衡量在改建悬赏寻求行为使用<em&gt;果蝇</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

毛细管馈线分析测量的摄食<em&gt;果蝇</em

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

果蝇幼虫和成年脑的代谢分析

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

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

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating the neuronal regulation of these behaviors is essential for the understanding of the physiological and molecular mechanisms underlying nutritional homeostasis. The use of genetically tractable animal models such as worms, flies, and fish greatly facilitates these types of studies. In the last decade, the fruit fly Drosophila melanogaster has been used as a powerful animal model by neurobiologists investigating the neuronal control of feeding and foraging behaviors. While undoubtedly valuable, most studies examine adult flies. Here, we describe a protocol that takes advantage of the simpler larval nervous system to investigate neuronal substrates controlling feeding behaviors when larvae are exposed to diets differing in their protein and carbohydrates content. Our methods are based on a quantitative colorimetric no-choice feeding assay, performed in the context of a neuronal thermogenetic-activation screen. As a read-out, the amount of food eaten by larvae over a 1 h interval was used when exposed to one of the three dye-labeled diets that differ in their protein to carbohydrates (P:C) ratios. The efficacy of this protocol is demonstrated in the context of a neurogenetic screen in larval Drosophila, by identifying candidate neuronal populations regulating the amount of food eaten in diets of different macronutrient quality. We were also able to classify and group the genotypes tested into phenotypic classes. Besides a brief review of the currently available methods in the literature, the advantages and limitations of these methods are discussed and, also, some suggestions are provided about how this protocol might be adapted to other specific experiments.

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Described here is a protocol that enables the colorimetric quantification of the amount of food eaten within a defined interval of time by Drosophila melanogaster larvae exposed to diets of different macronutrient quality. These assays are conducted in the context of a neuronal thermogenetic screen.

使用 德罗索菲拉· 拉瓦在恒温遗传神经元屏幕上摄入的宏量营养素的量化

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating ...

A Rapid Food-Preference Assay in Drosophila

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate their food environment. The fruit fly, Drosophila melanogaster, is a genetically tractable model organism that is widely used to decipher the molecular, cellular, and neural underpinnings of food preference. To analyze fly food preference, a robust feeding method is needed. Described here is a two-choice feeding assay, which is rigorous, cost-saving, and fast. The assay is Petri-dish-based and involves the addition of two different foods supplemented with blue or red dye to the two halves of the dish. Then, ~70 prestarved, 2-4-day-old flies are placed in the dish and&#160;allowed to choose between blue and red foods in the dark for about 90 min. Examination of the abdomen of each fly is followed by the calculation of the preference index. In contrast to multiwell plates, each Petri dish takes only ~20 s to fill and saves time and effort. This feeding assay can be employed to quickly determine whether flies like or dislike a particular food.

A Rapid Food-Preference Assay in Drosophila

We present a protocol for a two-choice feeding assay for flies. This feeding assay is fast and easy to run and is suitable not only for small-scale laboratory research, but also for high-throughput behavioral screens in flies.

果蝇的快速食物偏好分析

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate ...

高脂膳食喂养和高通量三测定在果蝇中的含量

摘要

探索更多视频

此视频中的章节

TreadWheel:果蝇轻度诱导运动的间歇训练协议

在果蝇中结合定量食物摄入量测定和强迫激活神经元研究食欲

用于果蝇消费量化的高吞吐量微板馈线分析

高通量检测，以检查个体产蛋首

一个简单的方法来衡量在改建悬赏寻求行为使用

毛细管馈线分析测量的摄食

果蝇幼虫和成年脑的代谢分析

生化和高通量的微观评估的脂肪含量

使用德罗索菲拉· 拉瓦在恒温遗传神经元屏幕上摄入的宏量营养素的量化

果蝇的快速食物偏好分析

高脂膳食喂养和高通量三测定在果蝇中的含量

摘要

探索更多视频

此视频中的章节

TreadWheel:果蝇轻度诱导运动的间歇训练协议

在果蝇中结合定量食物摄入量测定和强迫激活神经元研究食欲

用于果蝇消费量化的高吞吐量微板馈线分析

高通量检测，以检查个体产蛋首

一个简单的方法来衡量在改建悬赏寻求行为使用

毛细管馈线分析测量的摄食

果蝇幼虫和成年脑的代谢分析

生化和高通量的微观评估的脂肪含量

使用 德罗索菲拉· 拉瓦在恒温遗传神经元屏幕上摄入的宏量营养素的量化

果蝇的快速食物偏好分析

使用德罗索菲拉· 拉瓦在恒温遗传神经元屏幕上摄入的宏量营养素的量化