幼虫のゼブラフィッシュのためのパラダイムを摂食高脂肪：摂食、ライブイメージング、および食物摂取の定量化
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
腸は食物脂質の処理を調節する機構は、肝臓は、複合脂質の合成およびリポタンパク質代謝を制御し、これらの器官は、食物摂取量を制御するために、中枢神経系でどのように機能するか完全には理解されています。これは、肥満、心臓血管疾患、糖尿病、および非アルコール性脂肪肝疾患の現在の流行に照らして、この生物学を解明するために、生物医学的関心です。細胞培養およびマウスにおける研究食物中の脂肪と病気の間の機械的な関係の理解の大部分を提供している、およびゼブラフィッシュ（ ゼブラフィッシュ）は、この作業を補完する理想的なモデルとして浮上しています。
ゼブラフィッシュは、急速に発展、高等脊椎動物1,2と同様の胃腸（GI）器官、脂質代謝、およびリポタンパク質の輸送があり、遺伝的に扱いやすいです。幼虫のゼブラフィッシュの光学的透明性は、in vivo試験で particulaを容易にGI系の研究のためのRの利点その細胞外環境（ すなわち 、胆汁、微生物、内分泌シグナリングは）ex vivoでのモデル化することは事実上不可能であるように。とゼブラフィッシュ幼生のイメージングを生きるために、遺伝的扱いやすさとconducivenessを組み合わせた研究によれば、体食物の操作（高脂肪3,4、 -コレステロール5、および-炭水化物ダイエット6,7）、および心血管疾患8の様々なモデル、糖尿病9,10、脂肪肝11-13、および肥満14-16、代謝洞察のホストを提供するために浮上しています。
代謝研究に幼虫のゼブラフィッシュの移行の重要な側面は、ゼブラフィッシュとゼブラフィッシュの独自の強みを活用した新規アッセイの開発に他のモデル動物で開発された技術の最適化です。このプロトコルは、技術がLIPI幼虫のゼブラフィッシュを養うために開発され、最適化された提示しますDが豊富な食事は、細胞内の解像度に全身から食餌脂質処理を可視化し、食物摂取量を測定します。それが脂肪やコレステロールの高レベルが含まれているとして、鶏の卵黄を〜（脂質が構成する脂質の豊富な食事を構成するために〜5％がコレステロールであるそのうちの鶏の卵黄の58％を、選ばれた、60％はトリグリセリドであり、35％がリン脂質であります）。ゼブラフィッシュダイエットと摂食連隊は、ラボ17全体で標準化されていないとして、鶏の卵黄は、典型的な市販のゼブラフィッシュマイクロペレット食品（〜15％の脂質）、それは特定の脂肪酸種の既知の割合で標準化された飼料であるという利点よりも多くの脂肪を提供します。また、卵黄に設けられた蛍光脂質類似食物脂質18の輸送および蓄積を可視化、脂質滴を含む画像の細胞成分複合脂質に両方としてバイタル色素3および共有結合の取り込みを介して作用することにより、薄層クロマトグラフィー（TLC）を介して代謝を調査19 </sup>高速液体クロマトグラフィー（HPLC）（SAF未発表データ）、および総食物摂取量20についての定量的アッセイを提供します。
Protocol
Representative Results
Discussion
ここに記載された技術は、研究者は、脂質の豊富な飼料と幼虫のゼブラフィッシュを扱うライブ幼虫に食餌脂質処理を可視化し、幼虫の食物摂取量を定量化することができます。成功を確実にするために、特別な注意は、いくつかの重要なステップに与えられるべきです。コマーシャル鶏の卵は異なります。潜在的な変動を最小限に抑えるために、我々は、オメガ3脂肪酸について濃縮されて…
Divulgations
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 |
References
- Carten, J. D., Farber, S. A. A new model system swims into focus: using the zebrafish to visualize intestinal metabolism in vivo. Clin Lipidol. 4 (4), 501 (2009).
- Babin, P. J., Vernier, J. M. Plasma lipoproteins in fish. J Lipid Res. 30 (4), 467-489 (1989).
- Carten, J. D., Bradford, M. K., Farber, S. A. Visualizing digestive organ morphology and function using differential fatty acid metabolism in live zebrafish. Dev Biol. 360 (2), 276-285 (2011).
- Marza, E., et al. Developmental expression and nutritional regulation of a zebrafish gene homologous to mammalian microsomal triglyceride transfer protein large subunit. Dev Dyn. 232 (2), 506-518 (2005).
- Stoletov, K., et al. Vascular lipid accumulation, lipoprotein oxidation, and macrophage lipid uptake in hypercholesterolemic zebrafish. Circ Res. 104 (8), 952-960 (2009).
- Fang, L., et al. Programming effects of high-carbohydrate feeding of larvae on adult glucose metabolism in zebrafish, Danio rerio. Br J Nutr. 111 (5), 808-818 (2014).
- Wang, Z., Mao, Y., Cui, T., Tang, D., Wang, X. L. Impact of a combined high cholesterol diet and high glucose environment on vasculature. PLoS One. 8 (12), 81485 (2013).
- Fang, L., et al. In vivo visualization and attenuation of oxidized lipid accumulation in hypercholesterolemic zebrafish. J Clin Invest. 121 (12), 4861-4869 (2011).
- Curado, S., et al. Conditional targeted cell ablation in zebrafish: a new tool for regeneration studies. Dev Dyn. 236 (4), 1025-1035 (2007).
- Pisharath, H., Rhee, J. M., Swanson, M. A., Leach, S. D., Parsons, M. J. Targeted ablation of beta cells in the embryonic zebrafish pancreas using E. coli nitroreductase. Mech Dev. 124 (3), 218-229 (2007).
- Passeri, M. J., Cinaroglu, A., Gao, C., Sadler, K. C. Hepatic steatosis in response to acute alcohol exposure in zebrafish requires sterol regulatory element binding protein activation. Hepatology. 49 (2), 443-452 (2009).
- Sadler, K. C., Amsterdam, A., Soroka, C., Boyer, J., Hopkins, N. A genetic screen in zebrafish identifies the mutants vps18, nf2 and foie gras as models of liver disease. Development. 132 (15), 3561-3572 (2005).
- Matthews, R. P., et al. TNFalpha-dependent hepatic steatosis and liver degeneration caused by mutation of zebrafish S-adenosylhomocysteine hydrolase. Development. 136 (5), 865-875 (2009).
- Oka, T., et al. Diet-induced obesity in zebrafish shares common pathophysiological pathways with mammalian obesity. BMC Physiol. 10, 21 (2010).
- Chu, C. Y., et al. Overexpression of Akt1 enhances adipogenesis and leads to lipoma formation in zebrafish. PLoS One. 7 (5), 36474 (2012).
- Song, Y., Cone, R. D. Creation of a genetic model of obesity in a teleost. FASEB J. 21 (9), 2042-2049 (2007).
- Watts, S. A., Powell, M., D’Abramo, L. R. Fundamental approaches to the study of zebrafish nutrition. ILAR J. 53 (2), 144-160 (2012).
- Farber, S. A., et al. Genetic analysis of digestive physiology using fluorescent phospholipid reporters. Science. 292 (5520), 1385-1388 (2001).
- Miyares, R. L., de Rezende, V. B., Farber, S. A. Zebrafish yolk lipid processing: a tractable tool for the study of vertebrate lipid transport and metabolism. Dis Model Mech. 7 (7), 915-927 (2014).
- Otis, J. P., et al. Zebrafish as a model for apolipoprotein biology: comprehensive expression analysis and a role for ApoA-IV in regulating food intake. Dis Model Mech. 8 (3), 295-309 (2015).
- Bligh, E., Dyer, W. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-918 (1959).
- Otis, J. P., Farber, S. A. Imaging vertebrate digestive function and lipid metabolism. Drug Discov Today Dis Models. 10 (1), (2013).
- Andre, M., et al. Intestinal fatty acid binding protein gene expression reveals the cephalocaudal patterning during zebrafish gut morphogenesis. Int J Dev Biol. 44 (2), 249-252 (2000).
- Shimada, Y., Hirano, M., Nishimura, Y., Tanaka, T. A high-throughput fluorescence-based assay system for appetite-regulating gene and drug screening. PLoS One. 7 (12), 52549 (2012).
