Abstract
Like all animals, Drosophila shows robust fat (triglyceride) turnover, i.e., they synthesize, store and utilize triglyceride for their daily metabolic needs. The protocol describes a simple assay to measure this turnover of triglycerides in Drosophila.
Background
Almost all animals store energy reserves in the form of glycogen and triglycerides. Many physiological, pathological and environmental conditions cause changes in the total level of these energy reserves, especially triglycerides. However, it’s not always clear whether the resulting changes in triglycerides are due to reduced breakdown, increased synthesis or vice versa. With this protocol, it is possible to determine both the rate of synthesis and degradation of the newly synthesized triglycerides in flies.
Materials and Reagents
Equipment
Procedure
Data analysis
The amount of 14C present in 0 h sample denotes the de novo synthesis of TG and is expressed as CPM/mg fly weight. One experiment containing 5-6 independent samples of 15-25 flies is used to obtain mean ± SEM values. Additional experimental repeats should be carried out to confirm the observed results. Based on the design of the study, one can use Student’s t-test or ANOVA for obtaining the significance. Here, two different diets have been used, and the results (Figure 3) suggest that female flies on low yeast (LY) diet show increased de novo synthesis and a faster breakdown of TG when compared to the flies fed on high yeast (HY) diet. Note: It’s best to use independent bottles to obtain flies for replicates in one experiment. Figure 3. Fat turnover in adult female flies fed on high yeast (HY) and low yeast (LY) diets. The amount of 14C incorporation in 0 h sample indicates the de novo synthesis of TGs. The 60 h sample indicates the breakdown. Student’s t-test was used to measure statistical significance, and error bars denote SEM of five independent preparations (*indicates P < 0.05 and **indicates P < 0.001)
Recipes
Acknowledgments
SDK acknowledges support from American Federation of Aging Research and Larry L. Hillblom Foundation grants.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.