Colorimetric Measurement of Triglycerides Cannot Provide an Accurate Measure of Stored Fat Content in Drosophila

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.

Modulation of Longevity and Tissue Homeostasis by the Drosophila PGC-1 Homolog

“…In Drosophila , metabolized nutrients are primarily stored as TAG and glycogen in the fat body, the insect equivalent of the mammalian liver and white adipose tissue. Using thin-layer chromatography, the most accurate method to measure stored TAG in Drosophila (Al-Anzi and Zinn, 2010), we observed ~20% reduction in TAG levels in dPGC-1 overexpressing flies (Figure 2D). In contrast, there was a significant increase in both the amount of stored glycogen (Figure 2E) and free glucose levels (Figure 2F) in dPGC-1 overexpressing flies.…”

Methods for studying metabolism in Drosophila

“…The most widely used assay for detecting triglycerides, however, is a coupled colorimetric assay that detects free glycerol levels after cleaving TAG with lipoprotein lipase [32, 34, 35]. Although a few studies have suggested that this approach does not provide an accurate assessment of stored fat in insects [36, 37], both TLC and the colorimetric assay produce similar results [32]. It is, however, worth noting that the colorimetric assay releases glycerol from mono- and diacylglycerides [38] in addition to TAG and, therefore, conclusions regarding any particular form of glycerolipid should be validated by TLC.…”

Hypoxia causes triglyceride accumulation via HIF-1-mediated stimulation of lipin 1 expression

“…The mixture was then sonicated (five 10 sec pulses at maximum intensity), boiled for 5 minutes to facilitate the formation of an even suspension (Al-Anzi and Zinn, 2010) and 20 ml aliquots were tested using a biochemical triglyceride determination kit (Biosys, Athens, Greece). Triglyceride values were normalized to protein measured with Bio-Rad Protein Assay (Bio-Rad, Hercules, CA).…”