Høy fett Fôring Paradigm for Larve Sebrafisk: Fôring, Live Imaging, og Kvantifisering av matinntak
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 regulerer tarmen kost lipid behandling, styrer leveren komplekse lipid syntese og lipoproteinmetabolismen, og hvordan disse organene arbeider med sentralnervesystemet for å kontrollere matinntak er ufullstendig forstått. Det er av biomedisinsk interesse å belyse dette biologi i lys av dagens epidemier av fedme, hjerte- og karsykdommer, diabetes og alkoholfrie fatty leversykdom. Studier i cellekultur og mus har gitt mesteparten av vår forståelse av de mekanistiske relasjoner mellom lipider og sykdom, og sebrafisk (Danio rerio) er fremstår som en ideell modell for å utfylle dette arbeidet.
Sebrafisk har lignende gastrointestinal (GI) organer, lipidmetabolismen, og lipoprotein transport til høyere virveldyr 1,2, utvikle seg raskt, og er genetisk medgjørlig. Den optiske klarhet i larvesebrafisk letter in vivo studier, en particular fordel for studier av GI-systemet som sin ekstracellulære miljø (dvs. galle, microbiota, endokrine signalering) er nesten umulig å modellere ex vivo. I samsvar, en mengde forskning kombinere genetiske tractability og conduciveness å leve avbildning av sebrafisk larver med en rekke kosttilskudd manipulasjoner (fettrik 3,4, -kolesterol 5 og -karbohydrat dietter 6,7), og modeller av hjerte-og karsykdommer 8, diabetes 9,10, leversteatose 11-13, og fedme 14-16, dukker opp for å gi en rekke metabolske innsikt.
En viktig del av overgangen larvesebrafisk til metabolsk forskning er optimalisering av teknikker utviklet i modell for andre dyr til sebrafisk og utvikling av nye analyser som utnytter de unike styrkene til sebrafisk. Denne protokollen presenterer teknikker utviklet og optimalisert for å mate larvesebrafisk en Lipid rikt måltid, visualisere kosten lipid behandling fra hele kroppen til subcellulære oppløsning, og måle matinntaket. Kylling eggeplomme ble valgt for å komponere lipidrike måltid som den inneholder høye nivåer av fett og kolesterol (lipider komponere ~ 58% av kylling eggeplomme, hvorav ~ 5% cholesterol, 60% er triglycerider, og 35% er fosfolipider ). Kylling egg gir mer fett enn typiske kommersielle sebrafisk micropellet matvarer (~ 15% lipider) og den fordelen at det er en standardisert feed med kjente prosenter av spesifikke fettsyrer arter, som sebrafisk dietter og fôring regiments ikke har blitt standardisert på tvers av laboratorier 17. Videre fluorescerende lipid analogs anordnet i eggeplommen visualisere transport og akkumulering av lipider 18, bilde cellulære komponenter omfattende lipid-dråper ved å virke både som vitale fargestoffer 3 og gjennom kovalent inkorporering i komplekse lipider, undersøke metabolismen ved tynnsjiktskromatografi (TLC) 19 </sup> Og høy ytelse væskekromatografi (HPLC) (SAF upubliserte data), og tilveiebringe en kvantitativ analyse for total matinntaket 20.
Protocol
Representative Results
Discussion
Teknikkene som beskrives her tillate forskere å behandle larvesebrafisk med en lipid-rik feed, visualisere kosten lipid behandling i levende larver, og kvantifisere larve matinntak. For å sikre suksess, bør spesiell oppmerksomhet gis til flere kritiske trinn. Kommersielle kylling egg variere; for å minimere potensielle variasjonen vi utføre alle analyser på økologiske egg fra bur-free kyllinger som ikke har blitt beriket for omega-3 fettsyrer. Lavere fôring prisene kan observeres i fisk yngre enn 6 dpf med gjenv…
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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