高脂饮食范式斑马鱼幼体：饲养，实时成像，和食物摄入的量化
Summary
Zebrafish are emerging as a valuable model of dietary lipid processing and metabolic disease. Described are protocols of lipid-rich larval feeds, live imaging of dietary fluorescent lipid analogs, and quantification of food intake. These techniques can be applied to a variety of screening, imaging, and hypothesis driven inquiry techniques.
Abstract
Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich meal, which consists of an emulsion of chicken egg yolk liposomes created by sonicating egg yolk in embryo media. Detailed instructions are provided to screen larvae for egg yolk consumption so that larvae that fail to feed will not confound experimental results. The chicken egg yolk liposomes can be spiked with fluorescent lipid analogs, including fatty acids and cholesterol, enabling both systemic and subcellular visualization of dietary lipid processing. Several methods are described to mount larvae that are conducive to short- and long-term live imaging with both upright and inverted objectives at high and low magnification. Additionally presented is an assay to quantify larval food intake by extracting the lipids of larvae fed fluorescent lipid analogs, spotting the lipids on a thin layer chromatography plate, and quantifying the fluorescence. Finally, critical aspects of the procedures, important controls, options for modifying the protocols to address specific experimental questions, and potential limitations are discussed. These techniques can be applied not only to focused, hypothesis driven inquiries, but also to a variety of screens and live imaging techniques to study dietary lipid metabolism and the control of food intake.
Introduction
通过该肠调节膳食脂质加工机制，肝控制复合脂质合成和脂蛋白代谢和这些器官如何与中枢神经系统中工作，以控制食物摄取是不完全理解。它是生物医学兴趣的肥胖，心血管病，糖尿病和非酒精性脂肪肝疾病的当前流行的光来阐明这个生物学。在细胞培养研究和老鼠提供的大部分我们的膳食脂肪和疾病，和斑马鱼（ 斑马鱼 ）之间的机械关系的认识正在成为一种理想的模式，以配合这项工作。
斑马鱼也有类似的胃肠（GI）的器官，脂质代谢，脂蛋白运输到高等脊椎动物1,2，发展迅速，并在遗传上容易处理。幼虫斑马鱼的光学清晰度便于在体内研究中，particulař优势胃肠系统作为其胞外环境中的学习（ 即 ，胆汁，菌群，内分泌信令）几乎是不可能的模型离体进行。根据，研究相结合的遗传易处理性和有助文章的主体住斑马鱼的成像各种饮食控制（高脂肪3,4， -胆固醇5和-carbohydrate饮食6,7），与心血管疾病8的模型，糖尿病9,10，肝脂肪变性11-13，和肥胖14-16正在兴起，以提供代谢见解的主机。
过渡幼虫斑马鱼成代谢研究的一个重要方面是在其他动物模型的斑马鱼和利用斑马鱼的独特优势新颖测定的发展开发的技术的最优化。该协议提出的技术开发和优化的喂养幼虫斑马鱼梨皮D-一顿丰富的，可视化的整个身体饲料脂肪处理亚细胞分辨率，并测量食物的摄入。鸡蛋黄被选择以构成富含脂质的膳食，它包含高含量的脂肪和胆固醇（脂质组合〜鸡蛋黄，其中约5％的胆固醇，60％是甘油三酯，和35％的58％为磷脂的）。鸡蛋黄提供了比典型的商业斑马鱼微丸食物（〜15％的脂质）和优点，即它是与特定的脂肪酸种类的已知百分比的标准化供给更多的脂肪，如斑马鱼的饮食和喂养团没有被跨越实验室17标准化。此外，在蛋黄提供荧光脂质类似物可视运输和膳食脂类18的积聚，图像细胞成分包括既作为活体染料3和通过共价掺入复合脂脂滴，通过薄层色谱法调查代谢（TLC）19 </sup>和高效液相色谱（HPLC）（SAF未发表数据），并提供总食物摄入量20的定量测定。
Protocol
Representative Results
Discussion
此处所描述的技术允许研究治疗幼虫斑马鱼用富脂质的饲料，在活幼虫可视膳食脂质加工，并量化幼虫的食物摄取。为确保成功，应特别注意考虑到几个关键步骤。商业鸡蛋不同;尽量减少潜在的可变性，我们执行的有机鸡蛋所有检测从没有被富集的Omega-3脂肪酸免费笼鸡。较低的投饲率可能在鱼类观察不到6岁DPF剩余卵黄内生的能源供应，不健康的鱼或鱼妨碍食物的摄入量（ 即下巴或肠畸形…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank Meng-Chieh Shen for images, Jennifer Anderson for providing helpful comments on the manuscript, and members of the Farber laboratory for their contributions in developing these techniques. This study was funded by NIDDK-NIH award RO1DK093399 (S.A.F.), RO1GM63904 (The Zebrafish Functional Genomics Consortium: PI Stephen Ekker and Co-PI S.A.F), and F32DK096786 (J.P.O.). This content is solely the responsibility of the authors and does not necessarily represent the official views of NIH. Additional support was provided by the G. Harold and Leila Y. Mathers Charitable Foundation to the laboratory of S.A.F and the Carnegie Institution for Science endowment.
Materials
| Tricaine (ethyl 3-aminobenzoate methanesulofnate salt) | Sigma-Aldrich | A5040-25G | Anesthesia for larval zebrafish |
| Chicken eggs | N/A | N/A | Organic, cage-free eggs, not enriched for omege-3 fatty acids |
| Ultrasonic processor 3000 sonicator | Misonix, Inc. | S-3000 | To make egg yolk liposomes |
| Sonabox acoustic enclosure | Misonix, Inc. | 432B | To make egg yolk liposomes |
| 1/8” tapered microtip | Misonix, Inc. | 419 | To make egg yolk liposomes |
| Amber vials (4 ml, glass) | National Scientific | 13-425 | Lipid storage; includes vials, open-top caps, and cap septa |
| Incu-Shaker Mini | Benchmark | 1222U12 | Incubated shaker for feeds |
| BODIPY FL C16 | Thermo Fisher Scientific | D3821 | Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid) |
| BODIPY FL C12 | Thermo Fisher Scientific | D3822 | Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid) |
| BODIPY FL C5 | Thermo Fisher Scientific | D3834 | Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Pentanoic Acid) |
| BODIPY FL C5 | Thermo Fisher Scientific | D2183 | Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Propionic Acid) |
| TopFluor cholesterol | Avanti Polar Lipids Inc. | 810255 | Fluorescent lipid analog; 23-(dipyrrometheneboron difluoride)-24-norcholesterol |
| Fatty acid-free BSA | Sigma-Aldrich | A0281-1G | For TopFluor cholesterol solubilization |
| Methyl cellulose | Sigma-Aldrich | M0387 | Mounting media for live larval imaging; 75 x 25 x 1 mm |
| Low melt agarose | Thermo Fisher Scientific | BP165-25 | Mounting media for live larval imaging; 22 x 30 |
| VWR microscope slides | VWR | 16004-422 | Mounting larvae for live imaging |
| Coverslips | Cover Glass | 12-544A | Mounting larvae for live imaging |
| Super glue | Loctite | LOC01-30379 | Mounting larvae for live imaging |
| FluoroDish (glass bottom dish) | World Precision Instruments, Inc. | FD35-100 | Mounting larvae for live imaging; 35 mm dish, 23 mm glass, 0.17 mm glass thickness |
| Confocal microscope | Leica Microsytems | SP-2, SP-5 | Microscope for high magnification live imaging |
| Stereoscope | Nikon | SM21500 | Microscope for low magnification live imaging |
| Glass culture tubes | Kimble | 73500-13100 | Lipid extraction; (13 x 100 mm; 13 ml) |
| Savant SpeedVac Plus | ThermoQuest | SC210A | Lipid extraction |
| Channeled TLC plates | Whatman Scientific | WC4855-821 | Food intake assay; LK5D Silica Gel 150 A, 20 x 20 cm, 250 um thick; Discontinued |
| Channeled TLC plates | Analtech, Inc. | 66911 | Food intake assay; Direct replacement for Whatman Scientific TLC plates |
| Typhoon 9410 Variable Mode Imager | GE Healthcare | 9410 | Fluorescent plate reader for food intake assay |
| ImageQuant software | GE Healthcare | 29000605 | Analysis of food intake assay |
| 5 3/4’ Wide bore, borosilicate disposable pasteur pipets | Kimble | 63A53WT | Transfering larvae |
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