A Buoyancy-based Method of Determining Fat Levels in Drosophila
Summary
Here we present a method to measure organismal fat levels in the third instar (L3) larval stage of Drosophila melanogaster. This method exploits the comparatively low density of fat tissue to differentiate between larvae with altered fat stores. Buoyancy-based analysis is a valuable tool for rapid, reproducible, and economical screening.
Abstract
Drosophila melanogaster is a key experimental system in the study of fat regulation. Numerous techniques currently exist to measure levels of stored fat in Drosophila, but most are expensive and/or laborious and have clear limitations. Here, we present a method to quickly and cheaply determine organismal fat levels in L3 Drosophila larvae. The technique relies on the differences in density between fat and lean tissues and allows for rapid detection of fat and lean phenotypes. We have verified the accuracy of this method by comparison to body fat percentage as determined by neutral lipid extraction and gas chromatography coupled with mass spectrometry (GCMS). We furthermore outline detailed protocols for the collection and synchronization of larvae as well as relevant experimental recipes. The technique presented below overcomes the major shortcomings in the most widely used lipid quantitation methods and provides a powerful way to quickly and sensitively screen L3 larvae for fat regulation phenotypes while maintaining the integrity of the larvae. This assay has wide applications for the study of metabolism and fat regulation using Drosophila.
Introduction
Drosophila melanogaster has been used for over a century in the study of genetics and other basic biological questions. In the last few decades, it has become clear that Drosophila is a powerful tool in the study of many human diseases. As 70 – 80% of genes associated with human diseases have an identified fly ortholog1–4, Drosophila provides a simplified yet translatable system in which to study complex diseases. Metabolism in particular has benefited from such study. Not only are the genetics of metabolism well conserved between flies and humans, but the relevant organs and cell types are also very similar2,5. For example, the fat body of the fly stores both triacylglycerides (TAG) and glycogen, functions analogous to those performed in mammalian liver and white adipose tissue6. Using Drosophila as a model for human obesity has vastly improved our understanding of lipid metabolism and the genetics of obesity7. The larval stage of development is particularly useful for studying the segregation of nutrients to storage or utilization as it is dedicated to feeding and the storage of energy to be used during pupariation.
Currently, there are many different quantitative methods of determining fat storage levels in Drosophila. The most widely used method is the coupled colorimetric assay (CCA)8,9. CCA was developed for determining TAG levels in human serum and operates on the premise that glycerol liberated from the backbone of triglycerides will undergo several reactions, ultimately resulting in a redox-coupled reaction generating a colored product. Absorbance of specific wavelengths of light is then measured to determine the initial amount of glycerol present. However, glycerol can also be liberated from mono- and diacylglycerides in addition to TAG and therefore may not be an accurate measure of stored body fat9. Furthermore, eye pigment of crushed adult flies can interfere with some absorbance readings and complicate results9,10. Therefore, CCA must be accompanied and validated by thin layer chromatography (TLC), which allows for the separation of most lipid families that can be quantitated by densitometry10,11. However, some lipid classes like sterols cannot be analyzed and must be quantified a different way12. Mass spectrometry (MS) is an accurate way to quantitate all classes of major cellular lipids12,13. However, the lipid extraction procedures necessary to analyze by MS are both time consuming (most taking nearly a full day) and costly. Here we present an alternative method to quickly and cheaply determine organismal fat levels in the L3 larvae of Drosophila melanogaster.
The method presented below exploits the difference in density between fat tissue and lean tissue. Mammalian fat tissue has a density of approximately 0.9 g/ml14 while skeletal muscle as a density of 1.06 g/ml15. This difference means that animals with higher stores of fat will have lower density than animals with lower stores of fat, which will allow them to float better in a solution of fixed density. This property allows for extremely quick screening of a large number of animals while being both inexpensive and non-invasive. Buoyancy-based analysis has been used both to confirm the phenotypes of altering known regulators of fat levels as well as to identify new genetic and neurological regulators of obesity16,17.
Protocol
Representative Results
Discussion
There are a multitude of techniques that have been developed to measure lipid levels8–10. However, each of these methods comes with some major drawbacks that are addressed by the buoyancy-based assay outlined above. First, this assay is extremely quick. Testing a full concentration gradient takes no more than 30 – 60 min. This is a huge improvement on most of the techniques currently in use. For example, lipid quantitation by MS takes 7 – 9 hr to isolate the lipids and another several hou…
Disclosures
The authors have nothing to disclose.
Acknowledgements
K.E.H. was supported by the Training Grant in Molecular Biology NIH-T32-GM08730. This work was supported by NIH, NIDDK Grant 5K01DK095932 and AHA Award 12BGIA11930014 to T.R.
Materials
| Bacto Agar | VWR | 214030 | |
| Welch's Original 100% Grape Juice | |||
| Sucrose | Fisher | S512 | |
| Tegosept | Genesee Scientific | 20-259 | |
| Ethanol | 200 proof | ||
| Baker's yeast | Fleichmann's | ||
| Yellow Corn Meal | Quaker | Enriched degerminated | |
| Drosophila Agar Type II | Apex | 66-104 | |
| Soy Flour | ADM Specialty Ingredients | 062-100 | |
| Malt Extract | Breiss | 5748 | |
| Light Corn Syrup | Karo | ||
| Propionic Acid | VWR | U330-09 | |
| Sodium chloride | Fisher | 50147491 | |
| Potassium phosphate monobasic | Sigma | P5655-100G | |
| Sodium phosphate anhydrous | Fisher | S3933 | |
| Potassium chloride | Fisher | P330-500 | |
| 35 x 10mm petri plate | Fisher | 08-757-100A | |
| 50mL Conical | Fisher | 50-869-569 |
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