Name: high-fat feeding paradigm for larval zebrafish: feeding, live imaging, and quantification of food intake
Uploaded: 2016-10-27T15:00:00
Description: Watch this Scientific Journal Video about High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake at JoVE.com

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.

These protocols have been approved by the Carnegie Institution for Science Institutional Animal Care and Use Committee (protocol no. 139).
1. Animal Preparation
<ol>
	<li>Maintain adults and larvae at 28 &#176;C on a 14 hr:10 hr light:dark cycle. Feed adults twice daily with shell free Artemia (decapsulated, non-hatching, starting at 14 dpf) and commercial micropellets.</li>
	<li>These protocols are optimized for the use of 6-7 dpf larvae collected by natural spawning of the AB background. Protocols can...

<ol>
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C., Amsterdam, A., Soroka, C., Boyer, J., Hopkins, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+screen+in+zebrafish+identifies+the+mutants+vps18,+nf2+and+foie+gras+as+models+of+liver+disease.">A genetic screen in zebrafish identifies the mutants vps18, nf2 and foie gras as models of liver disease.</a> Development. 132 (15), 3561-3572 (2005).</li><li>Matthews, R. 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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+Akt1+enhances+adipogenesis+and+leads+to+lipoma+formation+in+zebrafish.">Overexpression of Akt1 enhances adipogenesis and leads to lipoma formation in zebrafish.</a> PLoS One. 7 (5), 36474 (2012).</li><li>Song, Y., Cone, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+a+genetic+model+of+obesity+in+a+teleost.">Creation of a genetic model of obesity in a teleost.</a> FASEB J. 21 (9), 2042-2049 (2007).</li><li>Watts, S. A., Powell, M., D'Abramo, L. 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The techniques described here allow researchers to treat larval zebrafish with a lipid-rich feed, visualize dietary lipid processing in live larvae, and quantify larval food intake. To ensure success, special attention should be given to several critical steps. Commercial chicken eggs vary; to minimize potential variability we perform all assays on organic eggs from cage-free chickens that have not been enriched for omega-3 fatty acids. Lower feeding rates may be observed in fish younger than 6 dpf with remaining endogen...

The mechanisms by which the intestine regulates dietary lipid processing, the liver controls complex lipid synthesis and lipoprotein metabolism, and how these organs work with the central nervous system to control food intake are incompletely understood. It is of biomedical interest to elucidate this biology in light of the current epidemics of obesity, cardiovascular disease, diabetes, and non-alcoholic fatty liver disease. Studies in cell culture and mice have provided the majority of our understanding of the mechanistic relationships between dietary lipids and disease, and zebrafish (Danio rerio) are emerging as an ideal model to complement this work.
Zebrafish have similar gastrointestinal (GI) organs, lipid metabolism, and lipoprotein transport to higher vertebrates 1,2, develop rapidly, and are genetically tractable. The optical clarity of the larval zebrafish facilitates in vivo studies, a particular advantage for study of the GI system as its extracellular milieu (i.e., bile, microbiota, endocrine signaling) is virtually impossible to model ex vivo. In accordance, a body of research combining the genetic tractability and conduciveness to live imaging of zebrafish larvae with a variety of dietary manipulations (high-fat3,4, -cholesterol5, and -carbohydrate diets6,7), and models of cardiovascular disease8, diabetes9,10, hepatic steatosis11-13, and obesity14-16, are emerging to provide a host of metabolic insights.
An essential aspect of transitioning the larval zebrafish into metabolic research is the optimization of techniques developed in other model animals to the zebrafish and the development of novel assays that exploit the unique strengths of the zebrafish. This protocol presents techniques developed and optimized to feed larval zebrafish a lipid-rich meal, visualize dietary lipid processing from whole body to subcellular resolution, and measure food intake. Chicken egg yolk was chosen to compose the lipid-rich meal as it contains high levels of fats and cholesterol (lipids compose ~58% of chicken egg yolk, of which ~5% is cholesterol, 60% are triglycerides, and 35% are phospholipids). Chicken egg yolk provides more fat than typical commercial zebrafish micropellet foods (~15% lipids) and the advantage that it is a standardized feed with known percentages of specific fatty acids species, as zebrafish diets and feeding regiments have not been standardized across labs17. Moreover, fluorescent lipid analogs provided in the egg yolk visualize transport and accumulation of dietary lipids18, image cellular components including lipid droplets by acting both as vital dyes3 and through covalent incorporation into complex lipids, investigate metabolism through thin layer chromatography (TLC)19 and high performance liquid chromatography (HPLC) (S.A.F. unpublished data), and provide a quantitative assay for total food intake20.

<table border="0" cellpadding="0" cellspacing="0" width="832">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">Tricaine (ethyl 3-aminobenzoate methanesulofnate salt)</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:127px;">A5040-25G</td>
			<td style="width:335px;">Anesthesia for larval zebrafish</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Chicken eggs</td>
			<td style="width:143px;">N/A</td>
			<td style="width:127px;">N/A</td>
			<td>Organic, cage-free eggs, not enriched for omege-3 fatty acids</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Ultrasonic processor 3000 sonicator</td>
			<td style="width:143px;">Misonix, Inc.</td>
			<td style="width:127px;">S-3000</td>
			<td>To make egg yolk liposomes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Sonabox acoustic enclosure</td>
			<td style="width:143px;">Misonix, Inc.</td>
			<td style="width:127px;">432B</td>
			<td>To make egg yolk liposomes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">1/8&#8221; tapered microtip</td>
			<td style="width:143px;">Misonix, Inc.</td>
			<td style="width:127px;">419</td>
			<td>To make egg yolk liposomes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Amber vials (4 ml, glass)</td>
			<td style="width:143px;">National Scientific</td>
			<td style="width:127px;">13-425</td>
			<td>Lipid storage; includes vials, open-top caps, and cap septa</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Incu-Shaker Mini&#160;</td>
			<td style="width:143px;">Benchmark</td>
			<td style="width:127px;">1222U12</td>
			<td>Incubated shaker for feeds</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">BODIPY FL C16&#160;</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">D3821</td>
			<td>Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">BODIPY FL C12&#160;</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">D3822</td>
			<td>Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">BODIPY FL C5&#160;</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">D3834</td>
			<td>Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Pentanoic Acid)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">BODIPY FL C5</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">D2183</td>
			<td>Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Propionic Acid)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">TopFluor cholesterol&#160;</td>
			<td style="width:143px;">Avanti Polar Lipids Inc.</td>
			<td style="width:127px;">810255</td>
			<td>Fluorescent lipid analog; 23-(dipyrrometheneboron difluoride)-24-norcholesterol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Fatty acid-free BSA</td>
			<td>Sigma-Aldrich</td>
			<td style="width:127px;">A0281-1G</td>
			<td>For TopFluor cholesterol solubilization</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Methyl cellulose</td>
			<td>Sigma-Aldrich</td>
			<td style="width:127px;">M0387</td>
			<td>Mounting media for live larval imaging; 75 x 25 x 1 mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">Low melt agarose</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">BP165-25</td>
			<td>Mounting media for live larval imaging; 22 x 30</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">VWR microscope slides&#160;</td>
			<td style="width:143px;">VWR&#160;</td>
			<td style="width:127px;">16004-422</td>
			<td>Mounting larvae for live imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Coverslips&#160;</td>
			<td style="width:143px;">Cover Glass</td>
			<td style="width:127px;">12-544A</td>
			<td>Mounting larvae for live imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Super glue</td>
			<td style="width:143px;">Loctite</td>
			<td style="width:127px;">LOC01-30379</td>
			<td>Mounting larvae for live imaging</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">FluoroDish (glass bottom dish)</td>
			<td style="width:143px;">World Precision Instruments, Inc.&#160;</td>
			<td style="width:127px;">FD35-100</td>
			<td>Mounting larvae for live imaging; 35 mm dish, 23 mm glass, 0.17 mm glass thickness&#160;&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Confocal microscope</td>
			<td>Leica Microsytems</td>
			<td style="width:127px;">SP-2, SP-5</td>
			<td>Microscope for high magnification live imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Stereoscope</td>
			<td style="width:143px;">Nikon</td>
			<td style="width:127px;">SM21500</td>
			<td>Microscope for low magnification live imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Glass culture tubes&#160;</td>
			<td style="width:143px;">Kimble</td>
			<td style="width:127px;">73500-13100</td>
			<td>Lipid extraction; (13 x 100 mm; 13 ml)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Savant SpeedVac Plus&#160;</td>
			<td style="width:143px;">ThermoQuest</td>
			<td style="width:127px;">SC210A</td>
			<td>Lipid extraction</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Channeled TLC plates</td>
			<td style="width:143px;">Whatman Scientific</td>
			<td style="width:127px;">WC4855-821</td>
			<td>Food intake assay; LK5D Silica Gel 150 A, 20 x 20 cm, 250 um thick; Discontinued</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Channeled TLC plates</td>
			<td style="width:143px;">Analtech, Inc.</td>
			<td style="width:127px;">66911</td>
			<td>Food intake assay; Direct replacement for Whatman Scientific TLC plates</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Typhoon 9410 Variable Mode Imager</td>
			<td style="width:143px;">GE Healthcare</td>
			<td style="width:127px;">9410</td>
			<td>Fluorescent plate reader for food intake assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">ImageQuant software</td>
			<td style="width:143px;">GE Healthcare</td>
			<td>29000605</td>
			<td>Analysis of food intake assay</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">5 3/4&#8217; Wide bore, borosilicate disposable pasteur pipets&#160;&#160;&#160;</td>
			<td style="width:143px;">Kimble</td>
			<td style="width:127px;">63A53WT</td>
			<td>Transfering larvae</td>
		</tr>
	</tbody>
</table>

high-fat feeding paradigm for larval zebrafish: feeding, live imaging, and quantification of food intake

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.

When fed on a rocker at 29-31 &#176;C, the majority of healthy larvae (&#8805;95%) will eat within 1 hr. Upon consuming the egg yolk emulsion, the larval intestine darkens in color. Very dark intestines can be observed at 2 hr (Figure 1). If larvae are unfed or fail to feed, the intestine remains clear. Larvae fed egg white exhibit a distended intestinal lumen that does not darken in color.
<img alt="Figure 1" src="/files/ftp_upload/54735/54735...

Watch this Scientific Journal Video about High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake at JoVE.com

High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake

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.

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich ...

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Biology

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In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

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