Alto contenuto di grassi alimentazione paradigma per larvale Zebrafish: Alimentazione, l'imaging dal vivo, e la quantificazione del cibo
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
I meccanismi mediante i quali l'intestino regola l'elaborazione lipidico dietetico, fegato controlla complesso metabolismo sintesi dei lipidi e lipoproteine, e come questi organi funzionano con il sistema nervoso centrale per controllare l'assunzione di cibo non sono completamente compresi. È di interesse biomedico per chiarire questo biologia alla luce delle epidemie attuali di obesità, malattie cardiovascolari, diabete e malattie del fegato grasso non-alcolica. Studi in colture cellulari e topi hanno fornito la maggior parte della nostra comprensione delle relazioni meccanicistiche tra lipidi alimentari e malattie, e zebrafish (Danio rerio) stanno emergendo come un modello ideale per completare questo lavoro.
Zebrafish hanno gastrointestinale simile (GI) gli organi, il metabolismo dei lipidi, e il trasporto delle lipoproteine a vertebrati superiori a 1,2, si sviluppano rapidamente, e sono geneticamente trattabili. La chiarezza ottica del pesce zebra larvale facilita studi in vivo, una particulaR vantaggio per lo studio del sistema gastrointestinale come il suo ambiente extracellulare (vale a dire, la bile, microbiota, endocrino segnalazione) è praticamente impossibile per modellare ex vivo. In accordo, un corpo di ricerca che unisce la trattabilità genetica e conduciveness di vivere l'imaging di larve di zebrafish con una varietà di manipolazioni dietetiche (alto contenuto di grassi, 3,4 -cholesterol 5, e diete -carbohydrate 6,7), e modelli di malattia cardiovascolare 8, diabete 9,10, steatosi epatica 11-13, 14-16 e l'obesità, stanno emergendo per fornire una serie di approfondimenti metabolici.
Un aspetto essenziale della transizione del pesce zebra larvale nella ricerca metabolica è l'ottimizzazione delle tecniche sviluppate in altri animali modello per il pesce zebra e lo sviluppo di nuovi test che sfruttano i punti di forza del pesce zebra. Questo protocollo presenta le tecniche sviluppati e ottimizzati per alimentare zebrafish larvale un Lipiricco d-pasto, visualizzare trasformazione alimentare di lipidi da tutto il corpo per la risoluzione subcellulare, e misurare l'assunzione di cibo. tuorlo d'uovo di gallina è stato scelto per comporre il pasto ricco di lipidi in quanto contiene livelli elevati di grassi e colesterolo (lipidi compongono ~ 58% di tuorlo d'uovo di gallina, di cui ~ 5% è il colesterolo, il 60% sono i trigliceridi, e il 35% sono fosfolipidi ). Tuorlo d'uovo di gallina fornisce più grasso di prodotti tipici commerciali zebrafish micropellet (~ 15% di lipidi) e il vantaggio che si tratta di un feed standardizzato con percentuali note di specifiche specie di acidi grassi, come le diete zebrafish e reggimenti di alimentazione non sono stati standardizzati attraverso laboratori 17. Inoltre, analoghi lipidici fluorescenti fornite nel tuorlo d'uovo visualizzare trasporto e accumulo di lipidi alimentari 18, componenti cellulari di immagine tra goccioline lipidiche agendo sia coloranti vitali 3 e attraverso covalente incorporazione in lipidi complessi, indagare metabolismo attraverso cromatografia su strato sottile (TLC) 19 </sup> E la cromatografia liquida ad alte prestazioni (HPLC) (SAF dati non pubblicati), e di fornire un metodo quantitativo per una totale assunzione di cibo 20.
Protocol
Representative Results
Discussion
Le tecniche qui descritte permettono ai ricercatori di trattare zebrafish larvale con mangimi ricchi di lipidi, visualizzare trasformazione dei lipidi nella dieta in larve vive, e quantificare l'assunzione di cibo larvale. Per garantire il successo, particolare attenzione deve essere data a diversi passaggi critici. uova di gallina commerciali variano; Per minimizzare la variabilità potenziale eseguiamo tutti i saggi su uova biologiche da polli privi di gabbia che non sono stati arricchiti per gli acidi grassi omeg…
Declarações
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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