Fettrik Utfodring Paradigm för Larvernas Zebrafish: Feeding, Live avbildning och kvantifiering av matkonsumtionen
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 genom vilka tarmen reglerar dietary lipid bearbetning, kontrollerar levern komplexa lipid syntes och lipoprotein metabolism, och hur dessa organ arbetar med det centrala nervsystemet för att kontrollera matintag är ofullständigt känd. Det är av biomedicinska intresse att belysa denna biologi i ljuset av de nuvarande epidemier av övervikt, hjärt-kärlsjukdomar, diabetes och alkoholfri fettlever. Studier i cellodling och möss har gett majoriteten av vår förståelse av de mekaniska relationer mellan kost lipider och sjukdom, och zebrafisk (Danio rerio) växer fram som en idealisk modell för att komplettera detta arbete.
Zebrafisk har liknande gastrointestinala (GI) organ, lipidmetabolism och lipoprotein transport till högre ryggradsdjur 1,2, utvecklas snabbt, och är genetiskt lätthanterlig. Den optiska klarhet larver zebrafisk underlättar in vivo-studier, en particular fördel för studier av GI-systemet som dess extracellulära miljön (dvs, galla mikrobiota endokrin signalering) är praktiskt taget omöjligt att modellera ex vivo. Enligt en kropp av forskning som kombinerar den genetiska spårbarhet och gynnsamma villkor för att leva avbildning av zebrafisk larver med en mängd olika kost manipulationer (fettrik 3,4, -kolesterol 5 och -carbohydrate dieter 6,7), och modeller av hjärt-kärlsjukdom 8, diabetes 9,10, leversteatos 11-13, och fetma 14-16, dyker upp för att ge en mängd metaboliska insikter.
En viktig aspekt av övergången larver zebrafisk i metabolisk forskning är optimering av tekniker som utvecklats i andra djurmodeller till zebrafisk och utveckling av nya analyser som utnyttjar de unika styrkor zebrafisk. Detta protokoll presenterar tekniker utvecklas och optimeras för att mata larver zebrafisk en Lipid-rik måltid, visualisera kosten lipid behandling från hela kroppen till subcellulär upplösning och mäta födointaget. Kyckling äggula valdes för att komponera lipid-rika måltid eftersom det innehåller höga halter av fett och kolesterol (lipider komponera ~ 58% av kyckling äggula, varav ~ 5% är kolesterol, 60% är triglycerider, och 35% är fosfolipider ). Kyckling äggula ger mer fett än typiska kommersiella zebrafisk micropellet livsmedel (~ 15% lipider) och den fördelen att det är ett standardiserat foder med kända procenthalter av specifika fettsyror arter, som zebrafisk dieter och utfodring regementen inte har standardiserats tvärs labs 17. Dessutom fluorescerande lipid-analoger som i äggulan visualisera transport och ackumulation av dietlipider 18, bildcellkomponenter inklusive lipiddropparna genom att agera både som vitala färgämnen 3 och genom kovalent inkorporering i komplexa lipider, undersöka metabolism genom tunnskiktskromatografi (TLC) 19 </sup> Och högupplösande vätskekromatografi (HPLC) (SAF opublicerade data), och ger en kvantitativ analys för total födointaget 20.
Protocol
Representative Results
Discussion
De tekniker som beskrivs här tillåter forskare att behandla larver zebrafisk med en lipid rikt foder, visualisera kosten lipid bearbetning i levande larver, och kvantifiera larv födointag. För att säkerställa framgång, bör särskild uppmärksamhet ägnas åt flera viktiga steg. Kommersiella hönsägg varierar; att minimera potentiella variabilitet vi utföra alla analyser på ekologiska ägg från buren fria kycklingar som inte har berikats för omega-3-fettsyror. Lägre matningshastigheter kan observeras i fisk…
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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