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.
