La alimentación de las larvas de pez cebra paradigma para la alta en grasas: Alimentación, imágenes en vivo, y Cuantificación de la ingesta de alimentos
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
Los mecanismos por los que el intestino regula el procesamiento de lípidos de la dieta, el hígado controles complejos síntesis de lípidos y metabolismo de las lipoproteínas, y cómo estos órganos trabajan con el sistema nervioso central para controlar la ingesta de alimentos se conocen por completo. Es de interés biomédico para dilucidar esta biología a la luz de la actual epidemia de la obesidad, las enfermedades cardiovasculares, la diabetes y la enfermedad de hígado graso no alcohólica. Los estudios en cultivos celulares y en ratones han proporcionado la mayor parte de nuestra comprensión de las relaciones mecánicas entre lípidos de la dieta y la enfermedad, y el pez cebra (Danio rerio) están emergiendo como un modelo ideal para complementar este trabajo.
El pez cebra tiene gastrointestinal similar (GI) los órganos, el metabolismo de los lípidos, y el transporte de lipoproteínas de vertebrados superiores a 1,2, se desarrollan rápidamente y son genéticamente manejable. La claridad óptica de la larvas de pez cebra facilita los estudios in vivo, una particulaventaja r para el estudio del sistema GI como su medio extracelular (es decir, la bilis, microbiota, la señalización endocrina) es prácticamente imposible de modelo ex vivo. De acuerdo, un cuerpo de investigación combinando la tratabilidad genética y condiciones favorables para imágenes en directo de las larvas de pez cebra con una variedad de manipulaciones de la dieta (alto contenido de grasa, 3,4 -colesterol 5 y dietas -carbohydrate 6,7), y modelos de enfermedad cardiovascular 8, la diabetes 9,10, esteatosis hepática 11-13, 14-16 y la obesidad, están emergiendo para proporcionar una gran cantidad de puntos de vista metabólico.
Un aspecto esencial de la transición de la larvas de pez cebra en la investigación metabólica es la optimización de las técnicas desarrolladas en otros animales modelo al pez cebra y el desarrollo de nuevos ensayos que explotan las ventajas únicas de la pez cebra. Este protocolo presenta técnicas desarrollados y optimizados para alimentar larvas de pez cebra un lipid-rica comida, visualizar el procesamiento de lípidos de la dieta de todo el cuerpo de la resolución subcelular, y miden la ingesta de alimentos. yema de huevo de pollo fue elegido para componer la comida rica en lípidos, ya que contiene altos niveles de grasas y colesterol (lípidos componen ~ 58% de yema de huevo de gallina, de los cuales ~ 5% es el colesterol, 60% son triglicéridos, y 35% son fosfolípidos ). Yema de huevo de pollo proporciona más grasa que los alimentos típicos comerciales de pez cebra de microgránulos (~ 15% de lípidos) y la ventaja de que es un alimento estandarizado con porcentajes conocidos de especies ácidos grasos específicos, como las dietas de pez cebra y regimientos de alimentación no se han normalizado a través de los laboratorios 17. Por otra parte, los análogos de lípidos fluorescentes previstos en la yema de huevo visualizar el transporte y la acumulación de lípidos de la dieta 18, los componentes celulares de imagen incluyendo gotitas de lípidos, actuando ambos colorantes vitales 3 y a través de la incorporación covalente en lípidos complejos, investigar el metabolismo a través de cromatografía en capa fina (TLC) 19 </sup> Y cromatografía líquida de alta resolución (HPLC) (SAF datos no publicados), y proporcionar un ensayo cuantitativo para la ingesta total de alimentos 20.
Protocol
Representative Results
Discussion
Las técnicas descritas aquí permiten a los investigadores tratan larvas de pez cebra con una alimentación rica en lípidos, visualizar el procesamiento de los lípidos de la dieta en larvas vivas, y cuantificar la ingesta de alimentos de las larvas. Para asegurar el éxito, se debe prestar especial atención a varios pasos críticos. huevos de gallina comerciales varían; para minimizar la variabilidad potencial llevamos a cabo todos los ensayos en los huevos orgánicos de gallinas libres de jaulas que no han sido en…
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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