高脂膳食喂养和高通量三测定在果蝇中的含量
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Talboys Ball dropper/bead Dispenser | Talboys | #: 930150 | |
| Talboys High Throughput Homogenizer | Talboys | #: 930145 | |
| Grinding Balls, Stainless Steel | OPS Diagnostics, LLC | # GBSS 156-5000-01 | 5000 balls |
| Masterblock 96 Well deep Microplates | Greiner Bio-One | # T-3058-1 | case of 80 plates |
| Greiner 96 well microplate flat bottom | Sigma Aldrich | # M4436 | 40 plates |
| Greiner CapMat for sealing multiwell plates | Sigma Aldrich | # C3606 | 50 sealing plates |
| Reagent Reservoirs | Thomas Scientific | # 1192T71 | 12/PK |
| Thermo Scientific Finnpipette 4661040 | Thermo Scientific | # 4661040 | 1-10 ul multipipette |
| Thermo Scientific Finnpipette 4661070 | Thermo Scientific | # 4661070 | 30-300ul multipipette |
| Thermo Scientific Finnpipette 4661020 | Thermo Scientific | #4661020 | 10-100ul multipipette |
| Multichannel tips | Denville Scientific Inc | # P3131-S | for 10 uL pipette |
| Multichannel tips | Denville Scientific Inc | # P3133-S | for 200 uL pipette |
| Multichannel tips | Denville Scientific Inc | #P1125 | for 100 uL pipette |
| Forceps | Roboz Surgical | # 5 Dumonts | Super fine forceps |
| Mettler Toledo Excellence XS Analytical Balance Mfr# XS64 | Cole-Parmer scientific experts | # EW-11333-00 | |
| Metler Toledo Excellence XS Toploading Balance | Cole-Parmer scientific experts | # EW-11333-49 | |
| 96-Well microplate Centrifuge | Hettich Zentrifugen | # Rotina 420R | |
| Microplate Reader | Molecular devices | # SpectraMax 190 | |
| Lab-Line Bench Top Orbit Environ Shaker Incubator | Biostad | # 3527 | |
| Infinity Triglycerides reagent | Thermo Scientific | # TR22421 | |
| Triglyceride Standard | Stanbio | #2103 – 030 | |
| Quick Start Bradford Protein Assay | Bio-RAD | # 500-0205 | 1x dye Reagent |
| Coconut oil | Nutiva | # 692752200014 | 15 0z jar |
| PBS 10X | Thermo Scientific | # AM9625 | 500 ml |
| Triton X-100 | Sigma Aldrich | # 9002-93-1 | |
| Gas-permeable Foil | Macherey-Nagel | # 740675 | 50 pieces |
| filter Paper | VWR | # 28317-241 | Pack of 100 |
| Drosophila vials | Genesee Scientific | Cat #: 32-116SB | |
| Quick Start Bovine Serum Albumin Standard | Bio-Rad | # 5000206 | |
| FlyNap Anesthetic | Carolina | # 173025 | 100 mL |
| Kimwipes Low-Lint | Uline | # S-8115 | 1-Ply, 4.4 x 8.4" |
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