Oleic Acid Derived Metabolites in Mouse Neuroblastoma N₁₈TG₂ Cells

Abstract

Oleamide is an endogenous sleep-inducing lipid that has been isolated from the cerebrospinal fluid of sleep-deprived mammals. Oleamide is the best-understood member of the primary fatty acid amide family. One key unanswered question regarding oleamide and all other primary acid amides is the pathway by which these molecules are produced. One proposed pathway involves oleoyl-CoA and N-oleoylglycine as intermediates: oleic acid --> oleoyl-CoA --> N-oleoylglycine --> oleamide. The first and third reactions are known reactions, catalyzed by acyl-CoA synthetase and peptidylglycine alpha-amidating monooxygenase (PAM). Oleoyl-CoA formation from oleic acid has been demonstrated in vitro and in vivo while, to date, N-oleoylglycine cleavage to oleamide has been established only in vitro. PAM catalyzes the final step in alpha-amidated peptide biosynthesis, and its proposed role in primary fatty acid amide biosynthesis has been controversial. Mouse neuroblastoma N(18)TG(2) cells are an excellent model system for the study of oleamide biosynthesis because these cells convert [(14)C]-oleic acid to [(14)C]-oleamide and express PAM in a regulated fashion. We report herein that growth of the N(18)TG(2) cells in the presence of [(14)C]-oleic acid under conditions known to stimulate PAM expression generates an increase in [(14)C]-oleamide or in the presence of a PAM inhibitor generates [(14)C]-N-oleoylglycine. This represents the first identification of N-oleoylglycine from a biological source. In addition, N(18)TG(2) cell growth in the presence of N-oleoylglycine yields oleamide. These results strongly indicate that N-oleoylglycine is an intermediate in oleamide biosynthesis and provide further evidence that PAM does have a role in primary fatty acid amide production in vivo.

Biosynthesis, degradation and pharmacological importance of the fatty acid amides

“…Arafat et al [93,106]. A melding of the two proposed pathways could also lead to PFAMs: first the cytochrome c-mediated production of the N-fatty acylglycine followed by PAM oxidation to the corresponding PFAM.…”

“…FAAH will hydrolyze the N-acyltaurines and N-arachidonoylglycine to the corresponding fatty acid and amino acid [33,76], but the other N-acylamino acids are not degraded by FAAH [77]. We have shown that N-acylglycines are biosynthetic precursors to the PFAMs using purified PAM [72] and in PAM-expressing neuroblastoma cells [93][73] [92][E1]. Marnett and co-workers have found that the N-arachidonoylamino acids are substrates for lipoxygenase and cyclooxygenase in vitro [94,95], pointing either to a mechanism for the inactivation of the N-arachidonoylamino acids or for the formation of other bioactive, oxidized amino acid conjugates.…”

“…This reaction is unlikely to occur in vivo because the K M for ammonia was high (65 mM), and the pH optimum for oleamide synthesis was > 9. Mouse neuroblastoma N 18 TG 2 cells secrete [1-14 C]-oleamide when cultured in the presence of [1-14 C]-oleic acid [93,104]; thus, these cells must contain the enzymatic machinery required for oleamide biosynthesis. Oleamide production in the N 18 TG 2 cells increases upon the inhibition of FAAH, providing further evidence against a role for this enzyme in PFAM production in vivo.…”

In vivo evidence that N-oleoylglycine acts independently of its conversion to oleamide

“…Extensive efforts of this and other laboratories have failed to identify the key enzyme for generating N-oleoylglycine. Nevertheless, the production of N-oleoylglycine by a recombinant bile acid conjugating enzyme [22] and by cultured neuroblastoma cells [23] indicate the feasibility for a PAM-based mechanism for the generation of oleamide in vivo.…”

“…The approaches currently available rely upon complex gas chromatographic-mass spectral procedures [16,23,24] which are cumbersome and expensive. To address these limitations we have developed a highly specific enzyme-linked immunosorbant assay (ELISA) for measuring blood levels of oleamide.…”