Haute teneur en graisses Alimentation Paradigm pour larvaire Zebrafish: Alimentation, imagerie en direct, et la quantification de la prise alimentaire
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
Les mécanismes par lesquels l'intestin réglemente le traitement des lipides alimentaires, le foie contrôle la synthèse des lipides et des lipoprotéines de métabolisme complexe, et comment ces organes fonctionnent avec le système nerveux central pour contrôler la prise alimentaire sont encore mal compris. Il est d'intérêt biomédical pour élucider cette biologie à la lumière des épidémies actuelles de l'obésité, les maladies cardiovasculaires, le diabète, et non alcoolisées maladie du foie gras. Des études en culture cellulaire et les souris ont fourni la majorité de notre compréhension des relations mécanistes entre les lipides et les maladies alimentaires, et le poisson zèbre (Danio rerio) sont en train de devenir un modèle idéal pour compléter ce travail.
Zebrafish ont gastrointestinal similaire (GI) organes, le métabolisme des lipides, et le transport des lipoprotéines de vertébrés supérieurs 1,2, se développent rapidement, et sont génétiquement traitable. La clarté optique du poisson zèbre larvaire facilite les études in vivo, une particular avantage pour l' étude du système gastro – intestinal comme son milieu extracellulaire (ie, la bile, le microbiote, la signalisation endocrine) est pratiquement impossible de modéliser ex vivo. Conformément, un organisme de recherche combinant la traçabilité génétique et favorables à l' imagerie active des larves de poisson zèbre avec une variété de manipulations diététiques (riches en matières grasses 3,4, -cholestérol 5, et les régimes alimentaires 6,7), hydrate de carbone et des modèles de maladies cardio – vasculaires 8, le diabète 9,10, stéatose hépatique 11-13, 14-16 et de l' obésité, émergent de fournir une foule d'idées métaboliques.
Un aspect essentiel de la transition du poisson zèbre larvaire dans la recherche métabolique est l'optimisation des techniques développées dans d'autres modèles animaux au poisson zèbre et le développement de nouveaux tests qui exploitent les atouts uniques du poisson zèbre. Ce protocole présente les techniques développées et optimisées pour nourrir le poisson zèbre larvaire un lipid-repas riche, visualisent alimentaire traitement lipidique du corps entier à la résolution subcellulaire, et de mesurer l'apport alimentaire. jaune d'oeufs de poules a été choisi pour composer le repas riche en lipides, car il contient des niveaux élevés de graisses et de cholestérol (lipides composent ~ 58% de jaune d'oeuf de poulet, dont ~ 5% est le taux de cholestérol, 60% sont des triglycérides, et 35% sont des phospholipides ). Jaune poulet oeuf fournit plus de matières grasses que les aliments typiques commerciaux de poisson zèbre de micropellets (~ 15% de lipides) et l'avantage qu'il est un aliment normalisé avec des pourcentages connus d'espèces d'acides gras spécifiques, comme les régimes de poisson zèbre et régiments d'alimentation ne sont pas normalisées dans les laboratoires 17. En outre, les analogues de lipides fluorescents prévus dans le jaune d'oeuf visualiser le transport et l' accumulation de lipides alimentaires 18, les composants cellulaires d'image , y compris gouttelettes lipidiques en agissant les deux colorants vitaux 3 et par liaison covalente incorporation dans des lipides complexes, l' étude du métabolisme par chromatographie sur couche mince (CCM) 19 </sup> Et la chromatographie à haute performance liquide (HPLC) (SAF données non publiées), et de fournir une analyse quantitative pour l' apport alimentaire totale 20.
Protocol
Representative Results
Discussion
Les techniques décrites ici permettent aux chercheurs de traiter des larves de poisson zèbre avec une alimentation riche en lipides, visualiser le traitement des lipides alimentaires dans des larves vivantes, et de quantifier l'apport alimentaire des larves. Pour assurer le succès, une attention particulière devrait être accordée à plusieurs étapes critiques. œufs de poule commerciales varient; pour réduire au minimum la variabilité potentielle que nous effectuons tous les tests sur les oeufs organiques d…
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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