C. elegans fat stores: Misled by Nile red

Lipidomics Gateway (25 November 2009) [doi:10.1038/lipidmaps.2009.33]

Dyes commonly used to assess fat storage in Caenorhabditis elegans do not stain major triglyceride stores or provide an accurate readout for lipid metabolism in genomic screens.

C. elegans

C. elegans is a useful model for the genetic analysis of energy homeostasis and fat storage pathways in a whole organism. Lipophilic dyes are commonly used in live nematodes to assess regulators of fat mass, and consequently to indicate a role in fat regulation for particular genes. The lipophilic Nile red stain has so far implicated more than 400 genes in lipid regulation, but recent biochemical analyses of lipid content have called this approach into question. Now Gary Ruvken and colleagues demonstrate that neither Nile red nor fluorescently labeled fatty acids stain the major C. elegans fat storage compartment. They report in Cell Metabolism that these vital stains seem to be treated by the nematode as foreign, and are directed to a degradative compartment. They propose the use of fixative staining with oil red O as the most accurate method to reveal C. elegans fat mass in genomic screens.

In cell culture and in vitro, Nile red concentrates in hydrophobic environments, with a concomitant increase in yellow-gold fluorescence. Fatty acids labeled with the fluorescent compound BODIPY behave similarly, and both stain living nematodes when ingested with their food, Escherichia coli. The main C. elegans fat storage organ, the intestine, contains lysosome-related organelles, which are acidic vesicular compartments thought to be a major site of lipid storage because of their staining with Nile red. Nevertheless, some tissues known to be high in fat content are not stained.

The authors compared several C. elegans mutants with wild-type worms by examining Nile red staining and total triglyceride/ phospholipid content as measured by gas chromatography-mass spectrometry. The lipid biochemistry results did not tally with the Nile red staining; for example, mutations to daf-2, encoding the C. elegans insulin/IGF receptor, caused a decrease in Nile red staining but a 130% increase in triglyceride mass. Other mutations caused increased Nile red staining without significant alteration to triglyceride mass. Food deprivation also produced contrasting results; as expected, triglyceride levels fell with fasting, but Nile red signal intensity increased

The use of other stains confirmed that Nile red and BODIPY-labeled fatty acids do not stain the major fat storage compartments, as they do not colocalize with a neutral lipid dye applied post-fixation, and they do colocalize with a lysosome-specific dye. These data indicate that in live worms, Nile red and BODIPY-fatty acids are partitioned to degradative lysosomal compartments, and do not accumulate in the fat stores.

As an alternative, Ruvkun and colleagues tested four fixative-based neutral lipid dyes. Of these, oil red O provided a robust stain, with preservation of worm anatomy and low inter-sample variability. The whole-body oil red O signal of several metabolic mutants correlated with the biochemical measurements of triglyceride mass, and the signal decreased with fasting.

Until a vital dye alternative is available, this study shows that if C. elegans is to remain a useful model, energy balance pathways should be investigated with fixative staining by oil red O or by quantitative lipid biochemistry.

Emma Leah