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.
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.
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.
1. Collect Eggs from Flies with Genotypes of Interest
NOTE: Ideal crosses consist of 150 virgin flies and at least 75 males. Stock collections should consist of at least 200 flies.
2. Transfer Larvae to Experimental Food
3. Determine Fat Levels in Larvae
Figure 1 represents an example of how the buoyancy-based assay can detect both higher and lower fat stores in genetically manipulated larvae. Figure 1A shows that excitation or silencing of a certain subset of neurons (E347) in the larval brain induces lower and higher fat stores respectively. The same genetic background control was used in both cases. Figure 1B provides an example of how this phenotype obtained by the density assay is corroborated by neutral lipid extraction and quantification by GCMS.
Figure 1. Neuronal Function in Specific Regions of the Larval Brain is Necessary and Sufficient for Body Fat Regulation. Neuronal activity in the indicated E347-GAL4 line was silenced by overexpression of ShiDN, or activated by overexpression of NB, as indicated in the panels, and effects on body fat were assessed. (A) Percent of floating larvae at equilibrium in different density solutions. Fifty larvae were examined per biological replicate, n= 7 – 9 biological replicates per genotype. (B) Percent body fat (total neutral lipids including triacylglycerides and phospholipids divided by body weight) as measured by GCMS17,20 (n= 8). Blue lines or bars represent GAL4-only (no UAS) animals crossed to w1118 to control for effects of genetic background. P values represent results from two-tailed t tests. **** P < 0.0001, *** P = 0.0001 to 0.001, ** P = 0.001 to 0.01, * P = 0.01 to 0.05. Error bars show SEM. This figure has been modified16. Please click here to view a larger version of this figure.
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 hours to analyze them. This is a strong deterrent when performing a screen on a large number of animals. By measuring a quick buoyancy phenotype, one can quickly focus on the genotypes that have the phenotype of interest. Second, the density assay requires only common reagents such as salts (for PBS) and sucrose. This makes it much easier and cheaper to screen a large number of larvae or quickly test an interesting genotype. Finally, this protocol is non-invasive and the larvae are still alive at the end. This allows further experimentation to be performed on the animals such as specific lipid quantitation or transcript or protein analysis. Additionally, recovered larvae may be allowed to continue growth to adulthood.
An additional benefit of this technique over others is increased sensitivity to small changes in fat levels in a population. By performing a whole concentration gradient on a population of 50 larvae, slight shifts in the population fat levels will be identified by a change in the intermediate concentrations, although the concentration at which the larvae begin floating and are totally floated may not be different than controls. This small shift in the population may not always be identified by other fat quantitation methods as they require a large group of larvae to analyze. The density assay on the other hand interrogates individuals within the population and can consequently identify these small changes or shifts in the overall population distribution.
As this technique relies on the density of the solution to produce reliable results, it is extremely important that the PBS and sucrose solutions are made consistently. We have found that different PBS recipes produce differences in solution density, leading to varying results. We use the recipe outlined above for consistent results. Furthermore, making the 20% sucrose from the same batch of 1x PBS is important as it results in the same background solution density. If the two solutions were made separately, slight variations in the PBS density could bring in additional density variable beyond the addition of sucrose to the experimental conical vial. Lastly, evaporation can be a problem, because over time the solutions will become more concentrated with the evaporation of water. It is therefore important to cap the bottles tightly and to remake the solution every week so that the experimental solutions remain consistent.
Among the steps outlined above, there are several critical steps that if altered, could result in inaccurate results by the buoyancy assay. First, it is important that the larvae transferred into the experimental food vial be the same age. Transferring larvae that vary in size will result in larvae at different developmental stages at the time the buoyancy assay is performed. This protocol has been developed to determine changes in fat levels in wandering L3 larvae and will not work accurately for early L3 or pre-pupae or later. Early third instar larvae have a tendency to float independently of fat levels while pre-pupae and pupae sink even at the highest concentrations used. For this reason, the age of the L1 larvae transferred is critical. Similarly, the point at which the vial is taken to perform the buoyancy assay is important. For the same developmental timing reasons stated above, the vials should be taken only when 20 – 40 larvae are wandering to ensure accurate density measurements. Additionally, the number of L1 transferred can affect results as well. The wide vials used in this protocol allow 50 larvae to eat without competition during development. A much larger number of larvae transferred than this will result in competition for food and may affect the amount of fat stored independent of genotype.
There may be genotypes with such extremely high fat levels that most or all of the larvae will float at the first concentration. In this case, the protocol may be modified by decreasing the concentration of the starting solution in order to obtain a full spectrum of the population density distribution. On the other hand, a specific genotype may be so lean that no larva floats in the initial sucrose. In this case, the initial steps may be omitted and a higher starting concentration used. This assay is very flexible and can be modified to better fit a wide range of fat levels. We advise the use of controls such as appropriate wild type animals (controlled for genetic background), positive controls (established fat mutants, such as adp or Sir217), and negative controls (published lean mutants such as lsd219). Appropriate use of this protocol will allow future screening of collections to determine new regulators of metabolism and obesity predisposition. Furthermore, this protocol provides an easy way for researchers in any field to test whether their genetic manipulation of interest causes a change in fat levels, without the need for complex protocols or expensive equipment. This protocol will help to elucidate the complex relationship between genetics and obesity.
The authors have nothing to disclose.
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.
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 |