Name: high fat diet feeding and high throughput triacylglyceride assay in drosophila melanogaster
Uploaded: 2017-09-13T12:30:00
Description: Watch this Scientific Journal Video about High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster at JoVE.com

We would like to thank Erika Taylor for editing this manuscript. This work was funded by grants from the National Institutes of Health (P01 HL098053, P01 AG033561 and R01 HL054732) to R.B., a post-doctoral research supplement (R01 HL085481) and fellowship (AAUW) to S.B.D., and grants from the American Heart Association to S.B.D. and R.T.B.

1. HFD Feeding Protocol
<img alt="Table 1" src="/files/ftp_upload/56029/56029table1.jpg" /> 
Table 1. Fly food recipe. 
This table summarizes the different ingredients used to prepare our control food. Once made, 10 mL of the food is poured into vials, cooled and stored at 4 &#176;C for long term storage.
<ol>
	<li>HFD preparation
	<ol>
		<li>To make 1 kg of high fat food, weigh 700 g of pre-made normal fly food (see Table 1) and 3...

<ol>
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Y., Liao, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+diet-induced+obesity+and+insulin+resistance.">A mouse model of diet-induced obesity and insulin resistance.</a> Methods Mol Biol. 821, 421-433 (2012).</li><li>Stocker, H., Gallant, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+started:+an+overview+on+raising+and+handling+Drosophila.">Getting started: an overview on raising and handling Drosophila.</a> Methods Mol Biol. 420, 27-44 (2008).</li><li>Mathews, K. W., 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=Sexual+Dimorphism+of+Body+Size+Is+Controlled+by+Dosage+of+the+X-Chromosomal+Gene+Myc+and+by+the+Sex-Determining+Gene+tra+in+Drosophila.">Sexual Dimorphism of Body Size Is Controlled by Dosage of the X-Chromosomal Gene Myc and by the Sex-Determining Gene tra in Drosophila.</a> Genetics. 205, 1215-1228 (2017).</li><li>Golay, A., Bobbioni, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+dietary+fat+in+obesity.">The role of dietary fat in obesity.</a> Int J Obes Relat Metab Disord. 21, 2-11 (1997).</li><li>Diop, S. B., 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=PGC-1/Spargel+Counteracts+High-Fat-Diet-Induced+Obesity+and+Cardiac+Lipotoxicity+Downstream+of+TOR+and+Brummer+ATGL+Lipase.">PGC-1/Spargel Counteracts High-Fat-Diet-Induced Obesity and Cardiac Lipotoxicity Downstream of TOR and Brummer ATGL Lipase.</a> Cell Rep. 10, 1-13 (2015).</li><li>Chatterjee, D., 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=Control+of+metabolic+adaptation+to+fasting+by+dILP6-induced+insulin+signaling+in+Drosophila+oenocytes.">Control of metabolic adaptation to fasting by dILP6-induced insulin signaling in Drosophila oenocytes.</a> Proc Natl Acad Sci U S A. 111, 17959-17964 (2014).</li><li>Palanker, L., 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=Drosophila+HNF4+regulates+lipid+mobilization+and+beta-oxidation.">Drosophila HNF4 regulates lipid mobilization and beta-oxidation.</a> Cell Metab. 9, 228-239 (2009).</li><li>Heinrichsen, E. 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Obesity induction in mice can take months19,20. In flies, this HFD feeding protocol allows for induction of excess fat accumulation in a matter of days or less, causing increases in fat accumulation only after 18 h (see Figure 2). HFD feeding with the described protocol increases glucose content 12 and decreases Bmm lipase and PGC-1 expression24. This is in contrast to fasting ...

We are in a time where obesity, and its associated economic burdens, is a worldwide problem1. Two out of every three Americans are overweight or obese with related heart pathologies, the primary cause of death within the adult population2. New efficient methods are needed to adequately investigate the genetic and molecular components implicated in the regulation of metabolic syndrome using model organisms. For this reason, we choose the fruit fly Drosophila model because it shares the most basic biological processes with mammals, including mice and humans3,4,5,6. Drosophila&#39;s genome is highly conserved during evolution but overall much smaller with less gene duplication and metabolic complexity, making it ideal for understanding the fundamental mechanisms implicated in many human diseases4,7,8. Also, characteristic processes carried out by adipose tissue, the gut and pancreas are represented in the fly and mediate regulatory functions in glucose and lipid metabolism, for example, that are similar to humans9,10,11. Moreover, the basic molecular pathways involved in the control of obesity, insulin resistance and diabetes in humans are functionally conserved in Drosophila melanogaster12,13,14,15,16. Like higher organisms, Drosophila has a beating heart that is formed during development by similar processes to that of the mammalian heart3,17. Thus, the development of a reliable HFD feeding protocol and high throughput TAG assay, adapted for efficient screening purposes using the genetic tool box of Drosophila, provide an important means to study and understand the fundamental genetic basis underlying complex metabolic diseases.
The HFD food itself is made from a standard laboratory fly food supplemented with coconut oil, which is composed mostly of saturated fatty acids known to be associated with metabolic syndrome18. While inducing obesity in mammalian models, such as rodents, can take months19,20, our optimized HFD feeding protocol in Drosophila effectively and reproducibly increases organismal fat content in a matter of days12,14. This protocol, in conjunction with a high throughput TAG assay, allows efficient mass screening for the effects of genetic factors, environmental influences and drug candidates to discover new modulators of fat metabolism. In consequence, these protocols are likely relevant to understand and/or combat obesity and obesity-associated human pathologies.
The feeding protocol is versatile and may be applied to study the metabolic and functional effects of single saturated or unsaturated fatty acid. The use of this high throughput TAG assay is not limited to D. melanogaster, but may be adapted to a variety of small model organisms with cuticle or tough extracellular matrices (e.g., other Drosophila species, C. elegans and other emerging invertebrate model organisms) to measure fat content under different environmental, genetic or physiological conditions, at any stage of development, adulthood or phase of metabolic disease. The TAG assay is based on a colorimetric measurement of a series of enzymatic reactions that degrade the TAGs into free fatty acids, glycerol, Glycerol 3-phosphate and finally H2O2 that reacts with 4-aminoantipyrine (4-AAP) and 3,5-dichloro-2-hydroxybenzene sulfonate (3,5 DHBS) to produce a red colored product that is measured using a 96-well spectrophotometer.

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			<td>1-Ply, 4.4 x 8.4&#34;</td>
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high fat diet feeding and high throughput triacylglyceride assay in drosophila melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

In D. melanogaster, as is the case with other species, there is sexual dimorphism between males and females22. It is well known that females are larger, with more fat in their abdomens, than males22. To test the effectiveness of our protocol, we performed TAG assays to determine the differences in TAG content between males and females of standard laboratory wildtype (w1118) flies. The data show that females have ...

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High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

The overall goal of this high fat feeding protocol and high throughput triacylglyceride assay is to induce obesity in a model organism such as drosophila melanogaster and monitor the associated changes in fat content under a variety of conditions. This method can help answer key questions in the metabolism field, including organismal obesity and organ specific lipotoxicity relevant to diabetes a metabolic syndrome, liver and cardiac steatosis. The main advantage of this technique is that excess fat accumulation in drosophila can be induced in a matter of days, and fat content determined simultaneously in about an hour.The implications of this technique extends towards diagnosis of metabolic diseases and does their therapy because high throughput protocol allows for accelerated drug discovery studies using this model system. To begin the experiment, prepare one kilogram of high fat food, weigh 700 grams of pre made fly food, and 300 grams of coconut oil into two separate containers using an analytical balance. Heat each container individually in the microwave until the contents melt completely.Pour the melted coconut oil into the fly food container, and stir the contents using a whisk until the oil is mixed into the fly food. Reweigh the high fat food. If the total weight is less than one kilogram, add about 10 milliliters of water to bring back the weight to one kilogram.Next, pour 10 milliliters of the well-mixed high fat food into each vial. Cover the food with the cheesecloth to prevent contamination and allow the food to cool for one hour at room temperature. After cooling, transfer the vials to four degrees Celsius for storage up to four weeks.Use CO2 to anesthetize the flies. Collect zero to five day old flies from vials, and flip the flies into new vials containing previously warmed normal food or NF.Let the flies age for an additional five days at 21 degrees Celsius. At the end of the five days, take out the vials containing the high fat diet, or HFD and NF from four degrees Celsius.Clean and remove excess fluid from the sides of the vials using wipes to prevent flies from sticking to the HFD food. Insert a small piece of filter paper about one centimeter by three centimeters, into the high fat food to absorb any surplus liquid. Next, flip the flies onto the high fat food.Lay the vials horizontally on their side, at 21 to 22 degrees Celsius for five days. After five days, flip the flies from the NF and HFD into new vials without food to allow their intestine to empty for 30 minutes. Proceed to weighing the flies.For each genotype and gender to be tested, use an ultra sensitive scale to record the weights of pre-labeled empty 1.7 milliliter tubes. Anesthetize and collect 36 flies of the same genotype, gender, and feeding status with a brush into one pre-weighed 1.7 milliliter tube. Then, weigh the tube containing flies with the same ultra-sensitive scale.Flash freeze the 1.7 milliliter tubes containing flies by plunging them into liquid nitrogen for two minutes. Collect the tubes, and place them into boxes. Next, transfer the boxes to a negative 80 degrees Celsius freezer for storage until use with the TAG assay.First, remove the frozen flies from the negative 80 degrees Celsius freezer, and place the tubes on ice. Place two millimeters of metal grinding balls into a 96 well ball dispenser, and use a small paintbrush to remove extra balls. Next, place the 96 well grinding plate upside down on top of the ball dispenser, and flip it to transfer the metal balls directly into the grinding plate.Ensure there is one ball per well. Then, using a multi-channel pipette, add 600 microliters of PBT into each row of the 96 well grinding plate. With forceps, add three flies per well.Indicate the genotype, gender, and food condition of the flies in each row or well of the 96 well grinding plate. Tightly place the caps onto the 96 well grinding plate. Place tape on top to ensure there is no leakage.Properly place the 96 well grinding plates into the grinding machine. Set the machine to the highest speed, and press run to grind the flies for three minutes. After three minutes, stop the grinding and proceed with with centrifugalization.After centrifugalization, manage the grinding plate carefully to avoid mixing the supernatant with the pellet. Using a multi-channel pipette, load 200 microliters of the TAG reagent into each row of a new 96 well spectrophotometer plate. Load 20 microliters of PBT into the first well of the first row to create a blank.Mix by pipetting up and down. Load 20 microliters of each standard dilution into the remaining wells of the first row, and mix by pipetting up and down. Using a multi-channel pipette, transfer 20 microliters of supernatant from each row of the grinding plate to the corresponding row in the 96 well spectrophotometer plate.Pipette up and down to mix well. Next, place a gas permeable foil over the top of the 96 well plate. Incubate the plate at 37 degrees celsius for ten minutes.Read the absorbance of each well of the plate at 550 nanometers using a compatible microplate reader. Tag assays of standard laboratory wild type flies show that females have more whole body fat than their male counterparts. The data also showed that the assay is stable with no variation in tag quantification over time and no variation depending on biological sample size.Tag assays of high fat diet, or HFD, and control-fed female flies revealed the consumption of HFD in drosophila causes increased fat content that progressively accumulates over time. After only 18 hours of HFD feeding, a significant increase of fat content was induced in flies. While attempting this procedure, it is important to remember to use well-cleaned vials for high fat diet feeding.Store the flies at 22 degrees or below, also do not mix supernatant with debris while pipetting during the tag assay. After its development, this technique paved the way for researchers studying metabolism and associated diseases to explore the effect of nutrient excess and the genetic basis of fat accumulation and associated organ disfunction such as lipotoxic cardiomyopathy in drosophila.

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