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 300 g of coconut oil into two separate containers using an analytical balance.</li>
		<li>Heat each container individually in the microwave, until the contents melt completely. Pour the coconut oil into the fly food container. Stir using a whisk until the oil is well mixed into the fly food. 
		NOTE: No chunks of food or oil should be visible. When the fly food is melting in the microwave, stop at intervals of 1 min to mix the food and prevent boiling over.</li>
		<li>Re-weigh the high fat food. If the total weight is less than 1 kg (700 g of food + 300 g of coconut oil), add water (around 10 mL) to compensate for evaporation during heating and to bring back the weight to 1 kg.</li>
		<li>Pour about 10 mL well-mixed high fat food per vial (around 25% of vial volume). Next, cover with cheesecloth to prevent flies&#39; contamination. Cool for 1 h at room temperature then transfer the vials to 4 &#176;C for storage up to 4 weeks. 
		NOTE: The Normal food (NF) is used as a control food. Also pour 10 mL (around 25% of vial volume) of NF per vial. For the HFD food, replace 30% of the NF food with coconut oil. For control and experimental conditions, the flies were given the same amount of NF and HFD (10 mL of food per vial).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56029/56029fig1.jpg" /> 
Figure 1. HFD feeding in Drosophila. 
The scheme shows the different feeding steps for flies on a control food (normal diet without addition of coconut oil-NF) or HFD (with the addition of coconut oil). The entire process takes 10 days after the initial collection of adult flies. <a href="//cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56029/56029fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>HFD feeding of D. melanogaster 
	NOTE: This protocol includes control food and HFD feeding procedures that are compatible with any fly line. Control and experimental flies are put in vials containing the same amount food (NF or HFD).
	<ol>
		<li>Use CO2 to anesthetize the flies. 
		NOTE: For neophytes, please read reference21 for an overview on raising and handling D. melanogaster.</li>
		<li>Collect 0-5 day old flies from vials and transfer (by flipping) the flies into new vials containing NF (previously warmed at room temperature). Let the flies age for 5 additional days at 21 &#176;C.</li>
		<li>At the end of the 5 days, take out the vials containing HFD and NF from 4 &#176;C. 
		NOTE: As cold temperatures can stress living flies, let the vials sit for 30 min at room temperature to warm up before use.</li>
		<li>Using wipes, clean and remove excess fluid from the sides of the vials.</li>
		<li>Insert a small piece of filter paper, about 1 cm x 3 cm, into the high fat food to absorb any surplus liquid. 
		NOTE: This step is critical for preventing the flies from sticking to the HFD food.</li>
		<li>Next, transfer the flies onto the high fat food (by flipping). Lay the vials horizontally on their side, (not standing) at 21-22 &#176;C for 5 days. 
		NOTE: Avoid higher temperatures as this can partially melt the high fat food, causing the flies to stick to the food and die.</li>
		<li>After 5 days, transfer (by flipping) the flies from NF and HFD into new vials without food to allow their guts to empty for 30 min. Next, proceed to weighing the flies. 
		NOTE: One of the critical steps in this HFD feeding protocol is to prepare a well-mixed HFD, free of any chunks of fly food or condensed oil. Homogenous mixing and cooling the HFD to 45-30 &#176;C is advisable, before pouring into vials and storing the food at 4 &#176;C. Proper age matching of control and experimental flies is important as well as controlled nutritional and environmental conditions prior to the HFD feeding. During the feeding phase, control and experimental flies must be given the same amount of NF and HFD. Never collect flies from overcrowded vials or bottles (to avoid transgenerational effects). Always put a maximum of 25 flies per vial during NF and HFD feeding to avoid dietary restriction or other crowding effects. The HFD is composed of 30% coconut oil, thus temperatures above 22 &#176;C might partially melt the HFD, causing the flies to stick on the oily food and die. Before putting the flies on the HFD, clean the vials to remove any residual oil and insert a filter paper to absorb any excess fluid in the food. During the incubation of the flies on HFD, the vials are laid on their sides to provide a fat-free horizontal surface for the flies.</li>
	</ol>
	</li>
	<li>Weighing the flies
	<ol>
		<li>For each genotype and gender to be tested, use an ultra-sensitive scale to record the weights of pre-labeled, empty 1.7 mL tubes.</li>
		<li>Using fly anesthetic or CO2, anesthetize and collect 36 flies of the same genotype, gender, and feeding status with a brush into one pre-weighed 1.7 mL tube.</li>
		<li>Weigh the tube containing flies with the same ultra-sensitive scale.</li>
		<li>Determine the average fly weight following this formula: 
		<img alt="Equation 1" src="/files/ftp_upload/56029/56029eq1.jpg" /> 
		NOTE: The number of flies in this protocol is 36.</li>
		<li>Flash freeze the 1.7 mL tubes containing flies by plunging into liquid nitrogen for 2 min. Collect and put the tubes into boxes. Next, transfer the boxes to -80 &#176;C (preferable temperature) for storage until use with the TAG assay. 
		CAUTION: Liquid nitrogen is at an extremely low temperature. Please use appropriate personal protective gear.</li>
	</ol>
	</li>
</ol>
2. TAG Assay
<ol>
	<li>Preparation of the TAG standard concentrations
	<ol>
		<li>Prepare 500 mL of PBT (PBS 1X, 0.05% Triton).</li>
		<li>Using 0.5 mL tubes and the TAG solution (Table of Materials), prepare 100 &#181;L of blank (PBT only) and 100 &#181;L of six standard TAG concentrations (2 &#181;g/&#181;L; 1 &#181;g/&#181;L; 0.5 &#181;g/&#181;L; 0.25 &#181;g/&#181;L; 0.125 &#181;g/&#181;L; 0.0625 &#181;g/&#181;L) with the PBT.</li>
		<li>Place the tubes on ice and proceed with grinding the flies.</li>
	</ol>
	</li>
	<li>Grinding the flies
	<ol>
		<li>Take out the frozen flies from -80 &#176;C (step 1.3). Transfer the tubes containing the flies on ice.</li>
		<li>First, place 2 mm metal grinding balls into a 96-well ball dispenser, use a small paintbrush to remove extra balls.</li>
		<li>Place the 96-well grinding plate upside down on top of the ball dispenser and flip to transfer the metal balls directly into the grinding plate. There should be one ball per well.</li>
		<li>Using a multi-channel pipette, add 600 &#181;L of PBT into each row of the 96-well grinding plate.</li>
		<li>With forceps, add 3 flies per well. Indicate the genotype, gender and food condition of the flies in each row/well of the 96-well grinding plate.</li>
		<li>Tightly place the caps onto the 96-well grinding plate. Tape may be placed on top to ensure that there is no leakage.</li>
		<li>Properly place the 96-well grinding plates into the grinding machine. Set the machine to the highest speed and press &#34;run&#34; to grind the flies for 3 min.</li>
		<li>After 3 min, stop the grinding and proceed with plate centrifugation: centrifuge for 15 min at 4,000 x g, 4 &#176;C. After centrifugation, manage the grinding plate carefully to avoid mixing the supernatant with the pellet.</li>
	</ol>
	</li>
	<li>Sample loading and TAG content determination
	<ol>
		<li>Take a new 96-well spectrophotometer plate. Using a multi-channel pipette, load 200 &#181;L of the TAG reagent into each row of the plate.</li>
		<li>Load 20 &#181;L of PBT into the first well of the first row, to create a blank. Mix by pipetting up and down. Avoid forming bubbles.</li>
		<li>Load 20 &#181;L of each standard dilution (from step 2.1.2) into the remaining wells of the first row and mix by pipetting up and down. Avoid forming bubbles.</li>
		<li>Using a multi-channel pipette, transfer 20 &#181;L 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. Avoid forming bubbles.</li>
		<li>Next, place a gas-permeable foil over the top of the 96-well plate.</li>
		<li>Incubate the plate at 37 &#176;C for 10 min. 
		NOTE: If there are bubbles in the wells, centrifuge the plate for 2 min at 2,000 x g to remove all bubbles. The presence of bubbles will interfere with the absorbance reading.</li>
		<li>Read the absorbance of each well of the plate at 550 nm using a compatible microplate reader. Next, trace the standard curve and determine the concentration [&#181;g/&#181;L] of the unknown samples.</li>
		<li>To determine the amount of TAG content per mean fly weight, use the following formula: 
		<img alt="Equation 2" src="/files/ftp_upload/56029/56029eq2.jpg" /> 
		NOTE: In this protocol, 3 flies were minced in 600 &#181;L of PBT; in consequence, the mean volume per fly is 200 &#181;L.</li>
	</ol>
	</li>
	<li>Bradford assay and normalization of TAG content with protein content
	<ol>
		<li>Prepare seven 100 &#181;L of bovine serum albumin (BSA) standard dilutions (75 &#181;g/mL; 100 &#181;g/mL; 150 &#181;g/mL; 200 &#181;g/mL; 250 &#181;g/mL; 500 &#181;g/mL; 1,000 &#181;g/mL) with the PBT.</li>
		<li>Take a new 96-well plate and load 200 &#181;L of 1X Bradford reagent into each row of the plate.</li>
		<li>In the first well of the first row, add 10 &#181;L of PBT to create a blank. Mix by pipetting up and down. Avoid forming bubbles.</li>
		<li>Add 10 &#181;L of each standard dilution into the remaining wells of the first row and mix by pipetting up and down. Avoid forming bubbles.</li>
		<li>Using a multi-channel pipette, transfer 10 &#181;L of the fly supernatant from each row of the grinding plate into the corresponding row in the 96-well plate. Pipette up and down to mix well. Avoid forming bubbles.</li>
		<li>Place a gas-permeable foil over the top of the 96-well plate.</li>
		<li>Incubate the plate at room temperature for 5 min. If there are bubbles in the wells, centrifuge the plate for 2 min at 2,000 x g to remove them. 
		NOTE: The presence of bubbles will interfere with the absorbance reading.</li>
		<li>Use a spectrophotometer to read the absorbance of the blank, standards and unknown samples at 595 nm. Use the standard curve to determine the concentrations [&#181;g/&#181;L] of the samples in each row.</li>
		<li>Normalize the TAG content with protein level using the following formula: 
		<img alt="Equation 3" src="/files/ftp_upload/56029/56029eq3.jpg" /></li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 of adult Drosophila that causes a fast decrease in both fat and glucose contents25,26 and increased Bmm expression24. Also, elevating Bmm or PGC-1 levels protects against HFD-induced obesity14,24. While 30% HFD has been used in this protocol, feeding flies with 3%, 7%, or 15% fat diet induced obesity in a dose-dependent fashion12, as does increasing the duration of HFD feeding (Figure 3). We also found that feeding single fatty acids of the main components of coconut oil, i.e., 14% lauric acid or 5% myristic acid, causes significantly elevated fat accumulation12.
The accelerated metabolic response with these flies on a HFD regimen is correlated with a reduced lifespan27 as observed in mammals28, but &#62; 80% of the flies survive past 20 days on HFD27. The rapid induction of fat accumulation mediated by conserved cellular and molecular processes controlling lipid and glucose metabolism is advantageous for many obesity-related studies, such as diabetic or lipotoxic cardiomyopathy10,12,13,14,15,16. Key findings in the consequences of metabolic imbalances and related cardiac dysfunctions will likely allow comparative translational studies in humans.
The HFD feeding protocol is compatible for use with other Drosophila species and insects models that share similar diets with D. melanogaster. This HFD feeding protocols could be appropriately adapted for organism like C. elegans or also other insects that require different &#39;normal&#39; food media. The 30% HFD is not suitable for use in experimentation requiring high temperatures; however, reduced concentrations of coconut oil or single fatty acids could be adequate alternatives12.
This TAG protocol is based on PBS, a non-toxic buffer that allows easy handling and experimentation without a fume hood, in contrast to previously used organic solvents (methanol/chloroform or diethyl ether) in lipid extraction and quantification15,29,30. Another advantage of this buffer is that it can normalize the TAG levels to protein content based on the same initial homogenate, making the fat determination in small tissue samples easily achievable and offering new opportunities to study organ cross-talk in organismal metabolic homeostasis. Also, the same initial fly homogenate obtained after grinding, can be used to perform parallel glucose and glycogen assays, allowing the study of changes in both the lipid and glucose metabolism from the same biological samples. This TAG protocol is less laborious or time-consuming than previous protocols15,29,30, and is rather high throughput, using a 96-well format that allows for rapid testing of close to 100 samples within an hour. This protocol could also be used to quantify fat content under other dietary conditions, including high sugar diet13,31, dietary restriction and starvation, as well as for aging studies to understand age-associated changes in fat metabolism.
Obesity, metabolic syndrome and related pathologies are at an all-time high with disastrous consequences on human health1,2. The combined HFD protocol and the high throughput TAG assay offer a unique platform to use model organisms, such as Drosophila, to rapidly induce obesity and screen for genetic factors, or natural and synthetic chemical compounds, to better understand metabolic imbalance. Ultimately this might greatly contribute to the advancement of scientific discoveries for developing new treatments or cures for metabolic diseases.

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.

<table><tbody><tr><td>Talboys Ball dropper/bead Dispenser</td><td>Talboys</td><td>#: 930150</td><td></td></tr><tr><td>Talboys High Throughput Homogenizer</td><td>Talboys</td><td>#: 930145</td><td></td></tr><tr><td>Grinding Balls, Stainless Steel&amp;nbsp;</td><td>OPS Diagnostics, LLC</td><td># GBSS 156-5000-01</td><td>5000 balls</td></tr><tr><td>Masterblock 96 Well deep Microplates</td><td>Greiner Bio-One</td><td># T-3058-1</td><td>case of 80 plates</td></tr><tr><td>Greiner&amp;nbsp; 96 well microplate flat bottom</td><td>Sigma Aldrich</td><td># M4436</td><td>40 plates</td></tr><tr><td>Greiner CapMat for sealing multiwell plates</td><td>Sigma Aldrich</td><td># C3606</td><td>50 sealing plates</td></tr><tr><td>Reagent Reservoirs&amp;nbsp;</td><td>Thomas Scientific</td><td># 1192T71</td><td>12/PK</td></tr><tr><td>Thermo Scientific Finnpipette 4661040</td><td>Thermo Scientific</td><td># 4661040</td><td>1-10 ul multipipette</td></tr><tr><td>Thermo Scientific Finnpipette 4661070</td><td>Thermo Scientific</td><td># 4661070</td><td>30-300ul multipipette</td></tr><tr><td>Thermo Scientific Finnpipette 4661020</td><td>Thermo Scientific</td><td>#4661020</td><td>10-100ul multipipette</td></tr><tr><td>Multichannel tips</td><td>Denville Scientific Inc</td><td># P3131-S</td><td>for 10 uL pipette</td></tr><tr><td>Multichannel tips</td><td>Denville Scientific Inc</td><td># P3133-S</td><td>for 200 uL pipette</td></tr><tr><td>Multichannel tips</td><td>Denville Scientific Inc</td><td>#P1125</td><td>for 100 uL pipette</td></tr><tr><td>Forceps&amp;nbsp;</td><td>Roboz Surgical</td><td># 5 Dumonts</td><td>Super fine forceps</td></tr><tr><td>Mettler Toledo Excellence XS Analytical Balance Mfr# XS64</td><td>Cole-Parmer scientific experts</td><td># EW-11333-00</td><td></td></tr><tr><td>Metler Toledo Excellence XS Toploading Balance</td><td>Cole-Parmer scientific experts</td><td># EW-11333-49</td><td></td></tr><tr><td>96-Well microplate Centrifuge</td><td>Hettich Zentrifugen</td><td># Rotina 420R</td><td></td></tr><tr><td>Microplate Reader</td><td>Molecular devices</td><td># SpectraMax 190</td><td></td></tr><tr><td>Lab-Line Bench Top Orbit Environ Shaker Incubator</td><td>Biostad</td><td># 3527</td><td></td></tr><tr><td>Infinity Triglycerides reagent</td><td>Thermo Scientific</td><td># TR22421</td><td></td></tr><tr><td>Triglyceride Standard</td><td>Stanbio</td><td>#2103 - 030</td><td></td></tr><tr><td>Quick Start Bradford Protein Assay</td><td>Bio-RAD</td><td># 500-0205</td><td>1x dye Reagent</td></tr><tr><td>Coconut oil</td><td>Nutiva</td><td># 692752200014</td><td>15 0z jar</td></tr><tr><td>PBS 10X</td><td>Thermo Scientific</td><td># AM9625</td><td>500 ml</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td># 9002-93-1</td><td></td></tr><tr><td>Gas-permeable Foil</td><td>Macherey-Nagel</td><td># 740675</td><td>50 pieces</td></tr><tr><td>filter Paper</td><td>VWR</td><td># 28317-241</td><td>Pack of 100</td></tr><tr><td>Drosophila vials</td><td>Genesee Scientific</td><td>Cat #: 32-116SB</td><td></td></tr><tr><td>Quick Start Bovine Serum Albumin Standard</td><td>Bio-Rad</td><td># 5000206</td><td></td></tr><tr><td>FlyNap Anesthetic</td><td>Carolina</td><td># 173025</td><td>100 mL</td></tr><tr><td>Kimwipes Low-Lint</td><td>Uline</td><td># S-8115</td><td>1-Ply, 4.4 x 8.4&quot;</td></tr></tbody></table>

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 more whole body fat than their male counterparts (Figure 2A, B). The data also showed that the assay is stable, with no variation in TAG quantification over time (up to 50 min after incubation) and no variation depending on biological sample size (3 or 5 flies).
HFD consumption has been shown to cause obesity in human and mouse19,20,23. To test the efficacy of our feeding protocol, we performed TAG assays with our HFD and control fed female flies. We found that consumption of HFD in Drosophila causes increased fat content that progressively accumulates over time (Figure 3A-C). Another important finding is that after only 18 h of HFD feeding (Figure 3C), we were able to induce a significant increase of fat content in these flies. These findings suggest that this genetic model system is an ideal tool for accelerated research into finding novel regulators of HFD-induced obesity.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56029/56029fig2.jpg" /> 
Figure 2. TAG assays in male and female flies. 
2-week old flies on a normal diet were collected, grouped by sex (males and females), weighed and ground up (3 or 5 flies per well) for TAG analysis. TAG assays were performed following the procedures described in this paper. The absorbance of each sample at 550 nm was read at different time-points (0 min, 5 min, 20 min and 50 min) to determine eventual fluctuations in TAG quantification over time, fat content variation in different population sizes (3 and 5 flies) of w1118 flies, and differences between male and female TAG contents. The results showed that females accumulate more fat than their male counterparts (A-B), TAG measurements do not fluctuate up to 50 min after the reaction incubation at 37 &#176;C (A-B). Also, the mean TAG levels remain unchanged between TAG assays using 3 or 5 flies (A-B). The data are presented as Mean &#177; SEM. Statistics: no significant difference. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56029/56029fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56029/56029fig3.jpg" /> 
Figure 3. Effects of HFD on fat accumulation. 
A-B: 2-week old female flies raised on a normal or HFD for 5 days were collected and the TAG assays were performed to determine levels of fat. TAG content was normalized either with weight (A) or protein levels (B). The data showed that HFD consumption leads to increased fat content with both methods of normalization. C: 2-week old female flies on normal/control food (NF) and 18 h, 1 day or 2 days on HFD are assayed for TAG content. The results show a significant and progressive increase of TAG levels from 18 h to 2 days of exposure to HFD. The data are presented as Mean &#177; SEM. Statistics: student t-test. *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56029/56029fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster at JoVE.com

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 ...

high-fat-diet-feeding-high-throughput-triacylglyceride-assay

Nanyang Technological University

Research

JoVE Journal

Genetics

11.2K Views.  Sanford Burnham Prebys Medical Discovery Institute. Dit is een hoog vet dieet voeding protocol om zwaarlijvigheid in Drosophila, een model voor begrip fundamentele moleculaire mechanismen betrokken bij lipotoxicity. Het biedt ook een hoge doorvoersnelheid triglyceride assay voor het meten van vet accumulatie in Drosophila en mogelijk andere (insect) modellen onder verschillende voeding, milieu, genetische of fysiologische omstandigheden.

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

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and lowered activity levels. These multifactorial diseases arise from a combination of genetic, environmental, and behavioral factors. One such complex disease is Metabolic Syndrome (MetS), which is a cluster of metabolic disorders, including hypertension, hyperglycemia, and abdominal obesity. Exercise and dietary intervention are the primary treatments recommended by doctors to mitigate obesity and its subsequent metabolic diseases. Exercise intervention, in particular aerobic interval training, stimulates favorable changes in the common risk factors for Type 2 Diabetes Mellitus (T2DM), Cardiovascular Disease (CVD), and other conditions. With the influx of evidence describing the therapeutic effect exercise has on metabolic health, establishing a system that models exercise in a controlled setting provides a valuable tool for assessing the effects of exercise in an experimental context. Drosophila melanogaster is a great tool for investigating the physiological and molecular changes that result from exercise intervention. The flies have short lifespans and similar mechanisms of metabolizing nutrients when compared to humans. To induce exercise in Drosophila, we developed a machine called the TreadWheel, which utilizes the fly&#39;s innate, negative geotaxis tendency to gently induce climbing. This enables researchers to perform experiments on large cohorts of genetically diverse flies to better understand the genotype-by-environment interactions underlying the effects of exercise on metabolic health.

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The TreadWheel uses rotational motion to gently induce exercise in adult Drosophila melanogaster by exploiting flies&#39; innate, negative geotaxis. It allows for analysis of the interactions between exercise and factors, such as genotype, sex, and diet, and their effect on physiological and molecular assays to assess metabolic health.

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and ...

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, and issues commands to start or stop feeding. Deciphering the processes underlying the decision-making of timely and moderate feeding carries major implications in our understanding of physiological and psychological disorders related to feeding control. Simple, quantitative, and robust methods are required to measure the food ingestion of animals after experimental manipulation, such as forcibly increasing the activities of certain target neurons. Here, we introduced dye-labeling-based feeding assays to facilitate the neurogenetic study of feeding control in adult fruit flies. We review available feeding assays, and then describe our methods step-by-step from setup to analysis, which combine thermogenetic and optogenetic manipulation of neurons controlling feeding motivation with dye-labeled food intake assay. We also discuss the advantages and limitations of our methods, compared with other feeding assays, to help readers choose an appropriate assay.

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Quantitative food-intake assays with dyed food provide a robust and high-throughput means to evaluate feeding motivation. Combining the food consumption assay with thermogenetic and optogenetic screens is a powerful approach to investigate the neural circuits underlying appetite in adult Drosophila melanogaster.

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, ...

Behavior

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental factors, and the toxicological and pharmacological effects of numerous substances. Few methods currently implemented are amenable to high throughput measurement. The Microplate Feeder Assay (MFA) was developed for quantifying the consumption of liquid food for individual flies using absorbance. In this assay, flies consume liquid food medium from select wells of a 1536-well microplate. By incorporating a dilute tracer dye into the liquid food medium and loading a known volume into each well, absorbance measurements of the well acquired before and after consumption reflect the resulting change in volume (i.e., volume consumed). To enable high throughput analysis with this method, a 3D-printed coupler was designed that allows flies to be sorted individually into 96-well microplates. This device precisely orients 96- and 1536-well microplates to give each fly access to up to 4 wells for consumption, thus enabling food preference quantification in addition to regular consumption. Furthermore, the device has barrier strips that toggle between open and closed positions to allow for controlled containment and release of a column of samples at a time. This method enables high throughput measurements of consumption of aqueous solutions by many flies simultaneously. It also has the potential to be adapted to other insects and to screen consumption of nutrients, toxins, or pharmaceuticals.

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

The microplate feeder assay offers an economical, high throughput method for quantifying liquid food consumption in Drosophila. A 3D-printed device connects a 96-well microplate in which flies are housed to a 1536-well microplate from which flies consume a feeding solution with a tracer dye. The solution volume decline is measured spectrophotometrically.

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

Neuroscience

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Biology

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating the neuronal regulation of these behaviors is essential for the understanding of the physiological and molecular mechanisms underlying nutritional homeostasis. The use of genetically tractable animal models such as worms, flies, and fish greatly facilitates these types of studies. In the last decade, the fruit fly Drosophila melanogaster has been used as a powerful animal model by neurobiologists investigating the neuronal control of feeding and foraging behaviors. While undoubtedly valuable, most studies examine adult flies. Here, we describe a protocol that takes advantage of the simpler larval nervous system to investigate neuronal substrates controlling feeding behaviors when larvae are exposed to diets differing in their protein and carbohydrates content. Our methods are based on a quantitative colorimetric no-choice feeding assay, performed in the context of a neuronal thermogenetic-activation screen. As a read-out, the amount of food eaten by larvae over a 1 h interval was used when exposed to one of the three dye-labeled diets that differ in their protein to carbohydrates (P:C) ratios. The efficacy of this protocol is demonstrated in the context of a neurogenetic screen in larval Drosophila, by identifying candidate neuronal populations regulating the amount of food eaten in diets of different macronutrient quality. We were also able to classify and group the genotypes tested into phenotypic classes. Besides a brief review of the currently available methods in the literature, the advantages and limitations of these methods are discussed and, also, some suggestions are provided about how this protocol might be adapted to other specific experiments.

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Described here is a protocol that enables the colorimetric quantification of the amount of food eaten within a defined interval of time by Drosophila melanogaster larvae exposed to diets of different macronutrient quality. These assays are conducted in the context of a neuronal thermogenetic screen.

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating ...

A Rapid Food-Preference Assay in Drosophila

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate their food environment. The fruit fly, Drosophila melanogaster, is a genetically tractable model organism that is widely used to decipher the molecular, cellular, and neural underpinnings of food preference. To analyze fly food preference, a robust feeding method is needed. Described here is a two-choice feeding assay, which is rigorous, cost-saving, and fast. The assay is Petri-dish-based and involves the addition of two different foods supplemented with blue or red dye to the two halves of the dish. Then, ~70 prestarved, 2-4-day-old flies are placed in the dish and&#160;allowed to choose between blue and red foods in the dark for about 90 min. Examination of the abdomen of each fly is followed by the calculation of the preference index. In contrast to multiwell plates, each Petri dish takes only ~20 s to fill and saves time and effort. This feeding assay can be employed to quickly determine whether flies like or dislike a particular food.

A Rapid Food-Preference Assay in Drosophila

We present a protocol for a two-choice feeding assay for flies. This feeding assay is fast and easy to run and is suitable not only for small-scale laboratory research, but also for high-throughput behavioral screens in flies.

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate ...

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

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