Juniperonic Acid Biosynthesis is Essential in Caenorhabditis Elegans Lacking Δ6 Desaturase (fat-3) and Generates New ω-3 Endocannabinoids

Abstract

In eukaryotes, the C20:4 polyunsaturated fatty acid arachidonic acid (AA) plays important roles as a phospholipid component, signaling molecule and precursor of the endocannabinoid-prostanoid axis. Accordingly, the absence of AA causes detrimental effects. Here, compensatory mechanisms involved in AA deficiency in Caenorhabditis elegans were investigated. We show that the ω-3 C20:4 polyunsaturated fatty acid juniperonic acid (JuA) is generated in the C. elegansfat-3(wa22) mutant, which lacks Δ6 desaturase activity and cannot generate AA and ω-3 AA. JuA partially rescued the loss of function of AA in growth and development. Additionally, we observed that supplementation of AA and ω-3 AA modulates lifespan of fat-3(wa22) mutants. We described a feasible biosynthetic pathway that leads to the generation of JuA from α-linoleic acid (ALA) via elongases ELO-1/2 and Δ5 desaturase which is rate-limiting. Employing liquid chromatography mass spectrometry (LC-MS/MS), we identified endocannabinoid-like ethanolamine and glycerol derivatives of JuA and ω-3 AA. Like classical endocannabinoids, these lipids exhibited binding interactions with NPR-32, a G protein coupled receptor (GPCR) shown to act as endocannabinoid receptor in C. elegans. Our study suggests that the eicosatetraenoic acids AA, ω-3 AA and JuA share similar biological functions. This biosynthetic plasticity of eicosatetraenoic acids observed in C. elegans uncovers a possible biological role of JuA and associated ω-3 endocannabinoids in Δ6 desaturase deficiencies, highlighting the importance of ALA.

Keywords: C. elegans; C20:4 polyunsaturated fatty acid; arachidonic acid (AA); biosynthesis; endocannabinoids; juniperonic acid (JuA).

Figure 1

Detection and quantification of FAs in N2 (WT) and fat-3(wa22) C. elegans. (A) Chromatographic resolution in LC-MS/MS showing the presence of C20:4 FAs AA, ω-3 AA in N2 (WT) animals and new C20 FA JuA in fat-3(wa22) mutants. (B) Quantification of AA, ω-3 AA and JuA in wild type N2 and fat-3(wa22) mutants deficient in Δ6 desaturase activity. Scatter plot shows mean ± SD.

Figure 2

Recovery of growth defects with addition of FAs. (A) Recovery of growth delay after 36 h of hatching at 23 °C of fat-3(wa22) mutant with the supplementation of C20:4 FAs (Scale bar = 100 mm). (B) Brood size of fat-3(wa22) C. elegans after addition of different C20:4 FAs. Brood size experiments were carried out with n = 5 animals per condition in at least three independent trials (*** p < 0.0001). Data show mean values ± SD.

Figure 3

FAs supplementation leads to alteration in lifespans. (A) fat-3(wa22) mutants are significantly long-lived than N2 (WT) animals (p < 0.0001). (B) N2 (WT) animal lifespans do not get significantly altered with supplementation of different FAs. (C) Lifespans of fat-3(wa22) mutants are significantly reduced (with supplementation of different FAs (p < 0.0001). All assays were performed at 23 °C with 50 μg/mL FUDR. At least two biological replicate experiments were performed per condition. Statistical analyses were performed in Graphpad Prism 5.0 by using Kaplan–Meier lifespan analysis and p values were calculated using the log-rank test. p < 0.05 was accepted as statistically significant.

Figure 4

Schematic representation of polyunsaturated fatty acid (PUFA) generation in C. elegans. (A) Model of generation of PUFAs under normal conditions in N2 (WT) with the intact enzymatic machinery, where LA and ALA act as precursors for generation of PUFAs (AA and EPA). (B) Model of novel PUFA synthesis in fat-3(wa22) mutants, where ALA acts as the precursor for synthesis of JuA.

Figure 5

Detection of C20:3 and C18:3. (A) Detection of DHGLA, GLA and ALA in N2 (WT) and fat-3(wa22) animals; no ScA was detected in the fat-3(wa22) animals. (B) Corresponding quantification by LC-MS/MS of the mentioned C20:3 and C18:3. Data represent values from independent experiments. Scatter plots show mean ± SD.

Figure 6

ELO-2 and FAT-4 required for synthesis of JuA. (A) shows the effect of RNAi mediated knockdown of elongases and Δ5 desaturase in fat-3(wa22) animals. elo-2 and fat-4 knockdowns show growth delay after 48 h of hatching at 23 °C as compared L4440 controls (Scale bar = 100 mm). (B) A slight decrease in brood size was observed with the elo-2 knockdown. A significant reduction in brood size was observed with the fat-4 know-down (* p < 0.01). Data show mean values ± SD. (C) Significant reduction in the levels of JuA after the knockdown of fat-4 (*** p < 0.0001) and elo-2 (* p < 0.01) in fat-3(wa22) animals. All brood size experiments were carried out with n = 5 animals per condition in three independent trials. Scatter plot shows mean ± SD.

Figure 7

Recovery of brood size of fat-3(wa22) C. elegans RNAi knockdowns. Brood size of fat-3(wa22) C. elegans where RNAi knockdowns of elo-2 and fat-4 were performed showing a significant improvement after supplementation of JuA. (A) Recovery in brood size in elo-2 (** p < 0.001) C. elegans. (B) Recovery in brood size in fat-4 (*** p < 0.0001). All brood size experiments were carried out with n = 5 animals per condition in three independent trials. Data show mean values ± SD.

Figure 8

Detection of new EC-like molecules in N2 (WT) and fat-3(wa22) animals. (A) LC-MS/MS chromatograms of AA, ω-3 AA and JuA derived EC-like molecules in simultaneously bred N2 (WT) animals and fat-3(wa22) mutants. (B) Quantification of the glycerol derived 1/2-AG, ω-3 1/2-AG and 1/2-JG of the ethanolamine-derived AEA, ω-3 AEA and JEA in N2 (WT) and fat-3(wa22) C. elegans by LC-MS/MS. Scatter plots show mean ± SD.

Figure 9

Reduction in the levels of EC like molecules in fat-3(wa22) after knockdown of fat-4 and elo-2. LC-MS/MS quantification shows that significant reduction in the levels of JEA and 1/2 JG occurs with knockdowns of fat-4 and elo-2. (A) Reduction of JEA levels as compared L4440 controls when compared to knockdowns elo-2 (*** p < 0.0001) and fat-4 (*** p < 0.0001). (B) Reduction of 1/2 JG levels as compared L4440 controls when compared to knockdowns elo-2 (** p < 0.001) and fat-4 (*** p < 0.0001). Scatter plots show mean ± SD.

Figure 10

Binding activities of ECs on membrane preparations of N2 (WT) and npr-32(ok2541) mutants. WT (full dots) and npr-32(ok2541) (empty dots) membranes were incubated with 3 nM [³H]CP55,940 in the presence of different concentrations (0.1 to 100 µM) of (A) AEA and 2-AG (B) ω-3 AEA and ω-3 1/2-AG and (C) JEA and 1/2-JG. Data show displacement curves with mean values ± SD of at least three independent experiments each performed in triplicate.