Alto teor de gordura Alimentação Paradigma para larval Zebrafish: Alimentação, Imagens ao vivo, e quantificação da ingestão de alimentos
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
Os mecanismos pelos quais o intestino regula o processamento de lípidos na dieta, o fígado controla complexo metabolismo síntese de lípidos e lipoproteínas, e como estes órgãos trabalhar com o sistema nervoso central para controlar a ingestão de alimentos está compreendida de forma incompleta. É de interesse biomédico para elucidar este biologia à luz dos actuais epidemias de obesidade, doenças cardiovasculares, diabetes e doença hepática gordurosa não-alcoólica. Estudos em cultura de células e camundongos forneceram a maioria da nossa compreensão das relações mecanicistas entre lipídios na dieta e doenças, e peixe-zebra (Danio rerio) estão emergindo como um modelo ideal para complementar este trabalho.
Peixe-zebra tem gastrointestinal semelhante (GI) órgãos, metabolismo lipídico e de transporte de lipoproteínas de vertebrados superiores 1,2, desenvolver-se rapidamente, e são geneticamente tratável. A limpidez óptica do peixe-zebra larval facilita estudos in vivo, uma particulavantagem r para o estudo do sistema de GI como seu meio extracelular (ou seja, bile, microbiota, endócrino sinalização) é praticamente impossível modelo ex vivo. Em conformidade, um corpo de pesquisa que combina a rastreabilidade genética e conducividade a viver imagiologia de larvas do peixe com uma variedade de manipulações dietéticas (elevado teor de gordura, 3,4 5 -colesterol, e dietas -carbohydrate 6,7), e os modelos de doença cardiovascular 8, diabetes 9,10, esteatose hepática 11-13, 14-16 e obesidade, estão surgindo para fornecer uma série de insights metabólicas.
Um aspecto essencial da transição peixe-zebra larval em pesquisa metabólica é a otimização de técnicas desenvolvidas em outros animais modelo para o peixe-zebra e do desenvolvimento de novos ensaios que exploram os pontos fortes únicas do peixe-zebra. Este protocolo apresenta técnicas desenvolvidas e otimizadas para alimentar peixe-zebra larval um Lipi-D refeição rica, visualizar processamento de lipídios na dieta de corpo inteiro para subcelular resolução, e medir a ingestão de alimentos. gema de ovo de galinha foi escolhido para compor a refeição rica em lipídios, já que contém altos níveis de gorduras e colesterol (lipídios compor ~ 58% de gema de ovo de galinha, dos quais ~ 5% é o colesterol, 60% são triglicéridos, e 35% são fosfolipídios ). Gema de ovo de galinha fornece mais gordura do que os alimentos típicos comerciais de peixe-zebra de micropastilha (~ 15% de lipídios) e a vantagem de que é uma alimentação padronizada com percentagens conhecidas de espécies de ácidos graxos específicos, como dietas de peixe-zebra e regimentos de alimentação não foram padronizadas em laboratórios 17. Além disso, os análogos de lipídios fluorescentes fornecidas na gema de ovo visualizar transporte e acumulação de lípidos na dieta 18, componentes celulares de imagem, incluindo gotículas lipídicas, agindo ambos os corantes como vitais 3 e através da incorporação covalente em lípidos complexos, investigar o metabolismo através de cromatografia em camada delgada (TLC) 19 </sup> E cromatografia líquida de alta eficiência (HPLC) (SAF dados não publicados), e fornecer um ensaio quantitativo para a ingestão total de alimentos 20.
Protocol
Representative Results
Discussion
As técnicas descritas aqui permitem aos pesquisadores para tratar peixe-zebra larval com uma alimentação rica em lipídios, visualizar o processamento de lípidos na dieta em larvas vivas, e quantificar a ingestão de alimentos larval. Para garantir o sucesso, deve ser dada especial atenção a vários passos críticos. ovos de galinha comerciais variam; para minimizar a variabilidade potencial realizamos todos os ensaios em ovos orgânicos de galinhas livres de gaiolas que não tenham sido enriquecido para ácidos g…
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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