To date most studies focused on metabolic changes induced by modifying the expression of desaturases in C. elegans have used GC-based approaches to profile the total fatty acids present. In this study we extended this analysis to also include aqueous metabolism and intact lipids to demonstrate a profound perturbation in global metabolism induced by perturbing the expression of the desaturases. The perturbations detected across the network of metabolism highlights the usefulness of global profiling tools for functional genomics. The use of LC-MS to profile intact lipids provides greater detail in terms of the fatty acid changes when compared with GC alone, and the profiling of amino acid analysis provides a further insight into core aqueous metabolism, something which is often ignored in many studies of fat metabolism in C.elegans.
The expression profiles of stearoyl-CoA desaturases are regulated by a large number of transcriptional and environmental inputs. In turn, the activities of these enzymes affect a large number of processes in the organism. The direct effects of knocking-out a desaturase enzyme is to modulate the ratio of saturated to unsaturated fatty acids, but this in turns affects membrane fluidity, activates specific signalling pathways, and influences mobility or activity of membrane bound proteins. As part of this, reorganisation may happen through feedback mechanisms involving metabolites as signals, and hence a global metabolomic strategy is needed in order to fully define all of the perturbations induced across the network of metabolism and not only those in fatty acid synthesis.
The higher content of fatty acids in the wild type strain compared with the Δ9 desaturase mutants was demonstrated by both GC-FID and LC-MS analysis, yet it was not detected by Oil Red-O staining. Previously, the fat content of these mutant strains has been measured by coherent anti-Stokes Raman scattering (CARS) , showing a 1.4-fold decrease in the expression level of neutral lipid droplets for the fat-5;fat-6 double mutant. One reason for this discrepancy between the Oil Red-O staining and other methods is that the Oil Red-O method is typically not sensitive enough to detect such a variation. The decrease detected by GC and LC-MS is not only due to a reduction in the actual synthesis of the lipids, but also due to an increase in the catabolism of the fatty acids, with dietary fatty acids representing a larger proportion of the total fatty acid compliment. This is also supported by the increase in succinate, which indicates an increase in acetyl-CoA metabolism via the glyoxylate cycle. A dramatic increase in the expression of genes involved in mitochondrial and peroxisomal β-oxidation has been reported in mutants where the desaturases have been knocked out . In mice, the deletion of SCD-1 leads to a decrease in the content of TAG and cholesterol esters and down-regulates de novo fatty acid synthesis. Furthermore, the deletion reduces lipid content by enhancing oxidation  and thus, an increase in catabolism following reduced desaturase activity appears to be highly conserved. It has also been shown that during fasting the concentration of stearic acid is maintained due to a strong fasting-dependent repression of the fat-7 gene, when peroxisomal and mitochondrial fatty acid β-oxidation are selectively stimulated , suggesting a regulatory interaction between fat-7 and β-oxidation, possibly by sensing the concentration of stearic acid. Moreover, an increase in the fatty-acid-binding proteins lpd-1-lpd-7 has been observed in the desaturase knocked-out mutants : these proteins sequester and transport fatty acids and acyl-CoA esters, facilitating their intracellular utilization, storage and signalling functions. An increase in their expression suggests an increment in the traffic of the fatty acids in the cell and is in agreement with an increase in their catabolism detected in this study.
However, this is not the only reorganization in the lipid content of the mutants. The results show a reduction in some of the phosphocholine derivatives in the mutants, in particular in those containing unsaturated fatty acids, suggesting a reorganisation of the cell membrane. This may be because the cell membranes have a reasonable amount of tolerance to the inclusion of saturated fatty acids, and the conservation of PUFAs for specific roles such as the synthesis of signalling molecules. Indeed, there is an enhancement in the heat-stress tolerance when animals are treated with RNAi for fat-6 and fat-7  arising from the increased incorporation of saturated fatty acids in the membranes. The changes in phosphocholine metabolism could only be followed in this high level of detail by the use of LC-MS. The approach detects the lipids directly and using fragmentation patterns we can deduce how changes in total fatty acid profiles effect the different phospholipids found in C.elegans.
The efficient storage of lipids within lipid droplets appears to be highly dependent on the generation of MUFAs. Unsaturated FAs, but not saturated FAs, induce the formation of lipid droplets or increase the size of the existing lipid droplets. This is confirmed by the fact that when supplemented with vaccenic acid (18:1n7) the concentration of TAGs were increased in mutants with peroxisomal dysfunctions . Furthermore, an impairment in desaturase activity is correlated with a decreased ability to generate and expand lipid droplets to facilitate TAG storage. Under conditions where MUFAs are in short supply to prevent the accumulation of toxic saturated lipids within the cytoplasm mitochondrial and peroxisomal β-oxidation is stimulated , possibly through the action of nhr-49, the C. elegans homologue of PPARα.
The fact that both BFAs and MUFAs decrease in the present study further suggests a general increase in the catabolism of fatty acids, and thus while unsaturated fats may compensate for a lack of BFAs the reverse is not necessarily true. Indeed, when the double mutant fat-6;fat-7 is treated with nhr-64(RNAi) (a gene, whose knock-down suppresses the low fat phenotype of the double mutant) an increase in the concentrations of branched fatty acid was found. This was attributed to a general increase in fatty acids synthesis .
There is an inverse correlation between the concentration of C17iso in TAGs and fat storage levels . C17iso is proposed to act as a chemical/nutritional indicator of the metabolic state of C. elegans. As this fatty acid is the final step of a synthesis that is initiated from the amino acid leucine, the concentration of C17iso may reflect the concentrations of essential dietary amino acids and more generally protein and lipid homeostasis in the organism. Δ9 desaturase-deleted mutants have both reduced content of branched fatty acids and essential amino acids.
Previously, the focus of metabolic studies on these strains has been on the changes in the fatty acid composition. In the present study, in addition to profiling intact lipid metabolites and cleaved fatty acids, also a non-targeted profiling of the aqueous phases was performed, demonstrating that the changes associated with the deletion of the desaturases are not limited to the lipid fractions but extend to the entire metabolome. The increase in the concentration of alanine in the mutants, indicative of increased glycolysis and consumption of amino acids, as well as an increase in succinate suggesting increased metabolism through the citric acid cycle are indicative of a general increase in catabolism. Furthermore, the increased content of branched amino acids in the wild type compared to the mutants seems to confirm this higher catabolic state accompanying the deletions of the Δ9 desaturase genes. These amino acids are the starting point for the synthesis of the branched fatty acids, but both branched chain amino acids and fatty acids decrease in concentration in the mutant, suggesting increased catabolism of both metabolite classes. The higher content of choline, a precursor of membrane phospholipids, in the mutants compared to wild type is another indicator of the lipid reorganization.
There is numerous evidence suggesting that fat-6 and fat-7 have both redundant and non-redundant functions. Our results show differences in the behaviour of both the two single mutants and the two double mutants. The analysis of the amino acid profiles by GC-MS indicates a different influence of the two genes on other metabolic pathways, such as amino acid anabolism and catabolism. The analysis on the intact lipids also demonstrated an increase in absorption of cyclopropane fatty acids in the fat-6 single mutant to compensate the absence of desaturation. This strain lacks the most highly expressed Δ9 desaturase. Other strains, instead, show an increase in the content of saturated fatty acids in the lipids. These results are confirmed also by the analysis of fatty acids by GC-FID: there is an increase in the content of C17:0_Δ in the fat-6 verses wild type model, not present in the fat-7 verses wild type model. However, without LC-MS it would have not been possible to follow this remodelling of the triglycerides.