Abstract
Drosophila melanogaster has recently emerged as a useful model system in which to study the genetic basis of regulation of fat storage. One of the most frequently used methods for evaluating the levels of stored fat (triglycerides) in flies is a coupled colorimetric assay available as a kit from several manufacturers. This is an aqueous-based enzymatic assay that is normally used for measurement of mammalian serum triglycerides, which are present in soluble lipoprotein complexes. In this short communication, we show that coupled colorimetric assay kits cannot accurately measure stored triglycerides in Drosophila. First, they fail to give accurate readings when tested on insoluble triglyceride mixtures with compositions like that of stored fat, or on fat extracted from flies with organic solvents. This is probably due to an inability of the lipase used in the kits to efficiently cleave off the glycerol head group from fat molecules in insoluble samples. Second, the measured final products of the kits are quinoneimines, which absorb visible light in the same wavelength range as Drosophila eye pigments. Thus, when extracts from crushed flies are assayed, much of the measured signal is actually due to eye pigments. Finally, the lipoprotein lipases used in colorimetric assays also cleave non-fat glycerides. The glycerol backbones liberated from all classes of glycerides are measured through the remaining reactions in the assay. As a consequence, when these assay kits are used to evaluate tissue extracts, the observed signal actually represents the amount of free glycerols together with all types of glycerides. For these reasons, findings obtained through use of coupled colorimetric assays on Drosophila samples must be interpreted with caution. We also show here that using thin-layer chromatography to measure stored triglycerides in flies eliminates all of these problems.
Highlights
Obesity is a serious health problem in developed societies, affecting more than 35% of the adult population
Lard, glycerol, purified fly fat, tri, di, and monoglycerides, samples were prepared for the coupled colorimetric assay using the procedures described by Pospisilik et al [5], in which 10 mg of a given substance was mixed with 1 ml of PBS with 0.1% Triton X-100 (PBT)
To evaluate the ability of coupled colorimetric assay kits to measure stored fat, we first examined whether butterfat or lard, used as examples of insoluble complex triglyceride mixtures whose composition resembles that of stored fat within cells, can be analyzed for triglyceride content using these assays
Summary
Obesity is a serious health problem in developed societies, affecting more than 35% of the adult population. Efforts to identify mutations in humans that account for a major proportion of the obesity phenotype in the general population have not been successful. Remarkable progress has been made in identifying and characterizing genes mutated in rodent models of obesity [1]. These include leptin and the leptin receptor. Forward genetic screens in genetically accessible model organisms such as Drosophila and C. elegans can help to identify new regulators of fat storage and metabolism. Some of the molecular pathways known to be involved in mammalian fat store regulation are important in flies [2], suggesting that Drosophila may represent a useful genetic model for mammalian obesity
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