Figure 4

PEGCs and endocannabinoids stimulate cholesterol transport by employing parallel pathways. (a) RNAi of let-767 leads to a significant increase of dauers compared to control plates in daf-7 mutant background (empty vector; from ~20% to ~80%), but has no effect on wild type (N2) dauer formation. Kruskal-Wallis one-way analysis of variance on ranks, p < 0.001, All pairwise multiple comparison procedures (Dunn’s Method), (*) indicates statistically significant difference between daf-7/empty vector and daf-7/let-767 RNAi, p < 0.05. Bars represent mean values and error bars represent standard errors. The number of independent experiments is n = 6 for N2/empty vector, and N2/let-767 RNAi, n = 27 for daf-7/empty vector, n = 33 for daf-7/let-767 RNAi, and n = 6 for daf-7/let-767 RNAi+DA. [DA] = 90 nM. (b) daf-7 on let-767 RNAi at 20 ºC forms ~80% dauers. Supplementation with either of the endocannabinoids 2-AG and AEA, or the addition of methyl-C17iso suppresses dauer formation significantly. Kruskal-Wallis one-way analysis of variance on ranks, p < 0.001. Multiple comparisons versus control group (Dunn’s Method). (*) indicates statistically significant difference with empty vector, p < 0.05. Bars represent mean values from at least three independent experiments and error bars represent standard errors. [2-AG] or [AEA] = 50 µM. [methyl-C17iso] = 100 µM. (c) daf-7 let-767 RNAi worms partly bypass dauer arrest if grown in excess cholesterol (130 µM). (*) indicates statistically significant difference with control plates (13 µM cholesterol). Mann-Whitney rank sum test p = 0.013. Bars represent mean values and error bars represent standard errors. The number of independent experiments is n=26 for daf-7 let-767 RNAi at 13 µM cholesterol, and n = 4 for daf-7 let-767 RNAi at 130 µM cholesterol. (d) Inhibition of the d17iso-glucosylceramide synthesis (and hence PEGCs) by PDMP treatment leads to an increase in dauer formation in daf-7 background, that can be largely suppressed by endocannabinoids, DA and excess cholesterol, but not by methyl-C17iso. One-way analysis of variance, p < 0.001. Multiple comparisons versus control group (Holm-Sidak method). (*) indicates statistically significant difference with PDMP treatment, p < 0.001 for all conditions except for empty vector (p = 0.002). Bars represent mean values and error bars represent standard errors. The number of independent experiments is n = 3 for all conditions. [DA] = 90 nM. [Cholesterol] = 130 µM. [2-AG] or [AEA] = 50 µM. [methyl-C17iso] = 100 µM. (e) mmPEGC-C22 is partly able to suppress dauer formation of daf-7;fat-3 at 20 ºC. One-way analysis of variance, p < 0.001. Multiple comparisons versus control group (Holm-Sidak method). (*) indicates statistically significant difference with solvent control, p < 0.001 for both conditions. Bars represent mean values and error bars represent standard errors. The number of independent experiments is n = 2 for all conditions. [DA] = 1 µM. [mmPEGC-C22] = 0.5 mM. (f) Endocannabinoids and PEGCs act in parallel to promote transport/mobilization of cholesterol from internal pools. Endocannabinoids are synthesized from their precursors DAG and NAPE most probably by DAGL and NAPE-PLD enzymatic activities. The enzyme(s) that produce(s) PEGC from d17iso-GlcCer is(are) unknown but the synthesis is dependent on NCR-1/2. Both endocannabinoids and PEGCs have the ability to mobilize cholesterol. The latter is then delivered to the sites of DAF-9 mediated DA synthesis. After systemic distribution of DA within the organism, it binds and inhibits the dauer-promoting activities of DAF-12. DAG – diacylglycerol; NAPE - N-acyl-phosphatidylethanolamine; DAGL - DAG lipase; NAPE-PLD - NAPE-specific phospholipase D.