Højt fedtindhold Fodring Paradigme for Larver Zebrafisk: Fodring, Levende Imaging, og kvantificering af fødeindtagelse
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 mekanismer, som tarmen regulerer kosten lipid behandling, leveren styrer komplekse lipid syntese og lipoprotein metabolisme, og hvordan disse organer arbejder med det centrale nervesystem til at styre fødeindtagelsen er ufuldstændigt forstået. Det er for biomedicinsk interesse at belyse denne biologi i lyset af de nuværende epidemier af fedme, hjertekarsygdomme, diabetes og ikke-alkoholiske fedtlever sygdom. Studier i cellekultur og mus har givet de fleste af vores forståelse af de mekanistiske relationer mellem kosten lipider og sygdomme, og zebrafisk (Danio rerio) dukker op som en ideel model til at supplere dette arbejde.
Zebrafisk har lignende gastrointestinale (GI) organer, lipid metabolisme, og lipoprotein transport til højere hvirveldyr 1,2, udvikler sig hurtigt, og er genetisk medgørlig. Den optiske klarhed af larvestadiet zebrafisk letter in vivo-undersøgelser, en particular fordel for studiet af GI-systemet som sin ekstracellulære miljø (dvs. galde, mikrobiota, endokrine signalering) er næsten umuligt at modellere ex vivo. I overensstemmelse, en mængde forskning kombinerer den genetiske sporbarhed og fremmende at leve billeddannelse af zebrafisk larver med en bred vifte af kosten manipulationer (højt fedtindhold 3,4, kolesterol 5 og -carbohydrate kost 6,7), og modeller af hjertekarsygdomme 8, diabetes 9,10, leversteatose 11-13, og fedme 14-16, dukker til at give et væld af metaboliske indsigter.
Et væsentligt aspekt af overgangen larve zebrafisk i metabolisk forskning er optimering af teknikker udviklet i andre dyremodeller til zebrafisk og udvikling af nye analyser, der udnytter de unikke styrker i zebrafisk. Denne protokol præsenterer teknikker udviklet og optimeret til at fodre larver zebrafisk en Lipid-rige måltid, visualisere kosten lipid behandling fra hele kroppen til subcellulær opløsning, og måle fødeindtagelse. Kylling æggeblomme blev valgt til at komponere lipid-rige måltid som det indeholder høje niveauer af fedt og kolesterol (lipider komponere ~ 58% af kylling æggeblomme, hvoraf ~ 5% er cholesterol, 60% er triglycerider, og 35% er phospholipider ). Kylling æggeblomme giver mere fedt end typiske kommercielle zebrafisk mikropellet fødevarer (~ 15% lipider) og den fordel, at det er en standardiseret foder med kendte procentdele af specifikke fedtsyrer arter, som zebrafisk kost og fodring regimenter ikke er standardiseret på tværs labs 17. Desuden fluorescerende lipid-analoger tilvejebragt i æggeblommen visualisere transport og akkumulering af ernæringsmæssige lipider 18, billed- cellulære komponenter, herunder lipiddråber Ved at handle både som vitale farvestoffer 3 og gennem kovalent inkorporering i komplekse lipider, undersøge metabolisme gennem tyndtlagskromatografi (TLC) 19 </sup> Og højtydende væskekromatografi (HPLC) (SAF upublicerede data), og tilvejebringe en kvantitativ analyse for samlede fødeindtagelse 20.
Protocol
Representative Results
Discussion
De her beskrevne teknikker gør det muligt for forskerne at behandle larvernes zebrafisk med en lipid-rige foder, visualisere kosten lipid forarbejdning i levende larver, og kvantificere larvernes fødeindtagelse. For at sikre succes, bør man være særlig opmærksom på flere kritiske trin. Kommercielle hønseæg varierer; at minimere eventuel variabilitet vi udføre alle assays på økologiske æg fra bur-fri kyllinger, som ikke er beriget med omega-3 fedtsyrer. Lavere fodring priserne kan observeres i fisk yngre end…
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 |
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