Vetrijke Feeding Paradigm voor larvale zebravis: Voeden, Live Imaging, en kwantificering van voedselinname
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
De mechanismen waarmee de darm reguleert dieet lipide verwerking, de lever stuurt complex lipidesynthese en lipoproteïne metabolisme, en hoe deze organen met het centrale zenuwstelsel voedselinname zijn onvolledig begrepen. Het is van biomedische belang deze biologie verhelderen in het licht van de huidige epidemieën van zwaarlijvigheid, hart- en vaatziekten, diabetes en niet-alcoholische steatohepatitis. Studies in celcultuur en muizen hebben op voorwaarde dat de meerderheid van onze kennis van de mechanistische relaties tussen de voeding vetten en de ziekte, en de zebravis (Danio rerio) zijn in opkomst als een ideaal model om dit werk aan te vullen.
Zebravis soortgelijke gastrointestinale (GI) organen, lipidemetabolisme en lipoproteïne transport hogere vertebraten 1,2, snel ontwikkelen en genetisch handelbaar. De optische helderheid van de larvale zebravis vergemakkelijkt in vivo studies, een particular voordeel voor de studie van het GI-systeem als zijn extracellulaire milieu (dwz, gal, microbiota, endocriene signalering) is het vrijwel onmogelijk om te modelleren ex vivo. Volgens een lichaam van het onderzoek combineren van de genetische tractability en bevorderlijk zijn beeldvorming van de zebravis larven leven diverse manipulaties dieet (vetrijk 3,4, 5 -cholesterol en -carbohydrate diëten 6,7) en modellen van hart- en vaatziekten 8, diabetes 9,10, hepatische steatose 11-13, 14-16 en obesitas, ontstaan van een groot aantal metabole inzichten.
Essentieel overgang de larvale zebravis in de metabole activiteit is het optimaliseren van technieken die in andere modeldieren de zebravis en de ontwikkeling van nieuwe assays die de unieke sterktes van de zebravis benutten. Dit protocol presenteert technieken ontwikkeld en geoptimaliseerd om larvale zebravis diervoeders een Lipid-rijke maaltijd, visualiseren dieet lipide de verwerking van het hele lichaam naar subcellulaire resolutie, en meet voedselinname. Kip eidooier werd gekozen om de vetrijke maaltijd samenstellen aangezien het een hoog gehalte aan vetten en cholesterol (lipiden samen ~ 58% kip eigeel, waarvan ~ 5% cholesterol, 60% triglyceriden en 35% fosfolipiden ). Kip eidooier bevat meer vet dan typische commerciële zebravis micropellet voedingsmiddelen (~ 15% lipiden) en het voordeel dat het een gestandaardiseerde feed met bekende percentages van specifieke vetzuren species, zoals zebravis dieet en voeding regimenten niet in laboratoria 17 gestandaardiseerd. Bovendien fluorescerende lipide analogen die in de eidooier visualiseren transport en de accumulatie van voedingslipiden 18, stock celcomponenten inclusief vetdruppels door zowel fungeert als vitale kleurstoffen 3 en door covalente opname in complexe lipiden onderzoeken metabolisme met dunnelaagchromatografie (TLC) 19 </sup> En hogedrukvloeistofchromatografie (HPLC) (SAF ongepubliceerde gegevens), en een kwantitatieve assay voor totale voedselinname 20.
Protocol
Representative Results
Discussion
De hier beschreven technieken kunnen onderzoekers larvale zebravis behandelen met een lipide-rijke voeding, visualiseren dieet lipide verwerking in levende larven, en te kwantificeren larvale voedselinname. Om succes te garanderen, moet speciale aandacht worden besteed aan een aantal kritische stappen. Commerciële kippeneieren variëren; mogelijke variabiliteit te minimaliseren voeren wij alle assays biologische eieren van kooi-kippen die niet zijn verrijkt voor omega-3 vetzuren. Lagere voeden tarieven kunnen worden wa…
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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