The endocannabinoid anandamide is a precursor for the signaling lipid N-arachidonoyl glycine by two distinct pathways

Abstract

Background: N-arachidonoyl glycine (NAGly) is an endogenous signaling lipid with a wide variety of biological activity whose biosynthesis is poorly understood. Two primary biosynthetic pathways have been proposed. One suggests that NAGly is formed via an enzymatically regulated conjugation of arachidonic acid (AA) and glycine. The other suggests that NAGly is an oxidative metabolite of the endogenous cannabinoid, anandamide (AEA), through an alcohol dehydrogenase. Here using both in vitro and in vivo assays measuring metabolites with LC/MS/MS we test the hypothesis that both pathways are present in mammalian cells.

Results: The metabolic products of deuterium-labeled AEA, D4AEA (deuterium on ethanolamine), indicated that NAGly is formed by the oxidation of the ethanolamine creating a D2NAGly product in both RAW 264.7 and C6 glioma cells. Significantly, D4AEA produced a D0NAGly product only in C6 glioma cells suggesting that the hydrolysis of AEA yielded AA that was used preferentially in a conjugation reaction. Addition of the fatty acid amide (FAAH) inhibitor URB 597 blocked the production of D0NAGly in these cells. Incubation with D8AA in C6 glioma cells likewise produced D8NAGly; however, with significantly less efficacy leading to the hypothesis that FAAH-initiated AEA-released AA conjugation with glycine predominates in these cells. Furthermore, the levels of AEA in the brain were significantly increased, whereas those of NAGly were significantly decreased after systemic injection of URB 597 in rats and in FAAH KO mice further supporting a role for FAAH in endogenous NAGly biosynthesis. Incubations of NAGly and recombinant FAAH demonstrated that NAGly is a significantly less efficacious substrate for FAAH with only ~50% hydrolysis at 30 minutes compared to 100% hydrolysis of AEA. Co-incubations of AEA and glycine with recombinant FAAH did not, however, produce NAGly.

Conclusion: These data support the hypothesis that the signaling lipid NAGly is a metabolic product of AEA by both oxidative metabolism of the AEA ethanolamine moiety and through the conjugation of glycine to AA that is released during AEA hydrolysis by FAAH.

Figure 1

Structures of AEA and NAGly. A) the endocannabinoid, N-arachidonoyl ethanolamine (anandamide; AEA) and B) the related signaling lipid, N-arachidonoyl glycine (NAGly); C) deuterium-labeled AEA with eight deuteriums on the arachidonic acid moiety; D) deuterium-labeled NAGly with eight deuteriums on the arachidonic acid moiety; E) deuterium-labeled AEA with four deuteriums on the ethanolamine moiety; F) deuterium-labeled NAGly with 2 deuteriums on the glycine moiety.

Figure 2

Chromatograms of the tandem mass spectrometric (MS) method for deuterium-labeled NAGly (D₈NAGly) in which the parent mass in negative mode [368.3]^- is paired fragment mass of glycine [74.0]^-. A) Chromatogram of an MS scan for the 368.3/74 pair from RAW 267.4 cell extracts that were incubated with deuterium-labeled anandamide. B) Chromatogram of a scan for the 368.3/74 pair with the synthesized D₈NAGly standard (std). C) Overlay of the two independent scans.

Figure 3

Chromatograms of the tandem mass spectrometric (MS) method for deuterium-labeled N-arachidonoyl glycine (D₂NAGly) in which the parent mass in negative mode [362.3]^- is paired fragment mass of deuterium-labeled glycine [76.0]^- or the non-deuterium-labeled N-arachidonoyl glycine (D₀NAGly) that has a parent mass in negative ion mode of [360.3]^- and a paired fragment mass of glycine [74.0]^-. A) Chromatogram of an MS scan for the 362.3/76 pair from RAW 267.4 cell extracts that were incubated with deuterium-labeled anandamide (D₄AEA). B) Chromatogram of the MS scan for the 360.3/74 pair with the synthesized D₀NAGly standard (std). C) Overlay of the two independent scans.

Figure 4

Mass spectrometric product ion scans of synthesized standards and RAW 264.7 lipid extracts. The numbers above the peaks are the calculated centroid mass. A) Positive ion mode product ion scan of the mass [348.3]⁺ of the synthesized standard of non-deuterium labeled N-arachidonoyl ethanolamine (D₀AEA). B) Positive ion mode product ion scans of the mass [352.3]⁺ of the synthesized standard of deuterium-labeled AEA (D₄AEA). C) Positive ion mode product ion scan of the mass [362.3]⁺ of the synthesized standard of non-deuterium labeled NAGly (D₀NAGly). D) Positive ion mode product ion scan of the mass [364.3]⁺ of the RAW 264.7 cell extract that was incubated with D₄AEA.

Figure 5

Chromatograms of lipid extracts of C6 Glioma of the tandem mass spectrometric (MS) methods for both deuterium-labeled NAGly (D₂NAGly) in which the parent mass in negative mode [362.3]^- is paired fragment mass of deuterium-labeled glycine [76.0]^- and the non-deuterium-labeled N-arachidonoyl glycine (D₀NAGly) that has a parent mass in negative ion mode of [360.3]^- and a paired fragment mass of glycine [74.0]^-. A) Chromatogram of MS scans for D₂NAGly and D₀NAGly from the lipid extract of C6 Glioma cells that were incubated with DMSO followed by D₄AEA. B) Chromatogram of MS scans for D₂NAGly and D₀NAGly from the lipid extract of C6 Glioma cells that were incubated with 1 μM URB 597 followed by D₄AEA.

Figure 6

Comparison of NAGly production in C6 glioma cells after incubation with deuterium-labeled arachidonic acid (D₈AA) or ethanolamine moiety deuterium-labeled AEA (D₄AEA). The products measured were arachidonoyl chain deuterium-labeled NAGly (D₈NG); non-deuterium-labeled NAGly (D₀NG); and glycine moiety deuterium-labeled NAGly (D₂NG). + denotes an addition of the compound to the cell media for 1 hour before lipid extraction. -denotes compounds that were not present during the incubation. # p ≤ 0.05 compared to levels of D₈NG in D₈AA treatment group; * p ≤ 0.05 compared to levels of D₂NG in D₄AEA+ URB 597 treatment group; n = 6–8 per group.

Figure 7

Production of N-arachidonoyl ethanolamine (AEA) and N-arachidonoyl glycine (NAGly) in whole brain. A) Levels of AEA and NAGly in rat whole brain two hours after vehicle (white bar) or 3 μg/kg URB 597 (black bar). * p ≤ 0.05 B) Levels of AEA and NAGly in mouse whole brain of WT (white bar) or FAAH KO (black bar). * p ≤ 0.05.

Figure 8

Comparison of hydrolysis rates of N-arachidonoyl ethanolamine (AEA) and N-arachidonoyl glycine (NAGly) via recombinant FAAH. n = 4–6 per time point. Time points (minutes): 1, 3, 5, 9, 15, 30.

Figure 9

Two pathways for N-arachidonoyl glycine (NAGly) biosynthesis from N-arachidonoyl ethanolamine (AEA).