• 2-arachidonoylglycerol;
  • anandamide;
  • HRASLS family;
  • N-acyl-phosphatidylethanolamine;
  • phospholipid


  1. Top of page
  2. Abstract
  3. Introduction
  4. Canonical pathways for anandamide and other NAEs
  5. Alternative pathways for anandamide and other NAEs
  6. Metabolism of 2-AG
  7. Conclusions
  8. Acknowledgements
  9. References

Endocannabinoids are endogenous ligands of the cannabinoid receptors CB1 and CB2. Two arachidonic acid derivatives, arachidonoylethanolamide (anandamide) and 2-arachidonoylglycerol, are considered to be physiologically important endocannabinoids. In the known metabolic pathway in mammals, anandamide and other bioactive N-acylethanolamines, such as palmitoylethanolamide and oleoylethanolamide, are biosynthesized from glycerophospholipids by a combination of Ca2+-dependent N-acyltransferase and N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D, and are degraded by fatty acid amide hydrolase. However, recent studies have shown the involvement of other enzymes and pathways, which include the members of the tumor suppressor HRASLS family (the phospholipase A/acyltransferase family) functioning as Ca2+-independent N-acyltransferases, N-acyl-phosphatidylethanolamine-hydrolyzing phospholipaseD-independent multistep pathways via N-acylated lysophospholipid, and N-acylethanolamine-hydrolyzing acid amidase, a lysosomal enzyme that preferentially hydrolyzes palmitoylethanolamide. Although their physiological significance is poorly understood, these new enzymes/pathways may serve as novel targets for the development of therapeutic drugs. For example, selective N-acylethanolamine-hydrolyzing acid amidase inhibitors are expected to be new anti-inflammatory and analgesic drugs. In this minireview, we focus on advances in the understanding of these enzymes/pathways. In addition, recent findings on 2-arachidonoylglycerol metabolism are described.


α/β-hydrolase domain-containing protein








diacylglycerol lipase


fatty acid amide hydrolase


fatty acid amide hydrolase-like anandamide transporter


glycerophosphodiester phosphodiesterase 1




human embryonic kidney


HRAS-like suppressor


lysophosphatidic acid




lecithin retinol acyltransferase




monoacylglycerol lipase


metabotropic glutamate receptor


N-acylethanolamine-hydrolyzing acid amidase






N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D




phosphatidic acid




polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract




phospholipase A/acyltransferase












  1. Top of page
  2. Abstract
  3. Introduction
  4. Canonical pathways for anandamide and other NAEs
  5. Alternative pathways for anandamide and other NAEs
  6. Metabolism of 2-AG
  7. Conclusions
  8. Acknowledgements
  9. References

Endocannabinoids are endogenous ligands of the cannabinoid receptors CB1 and CB2 [1, 2]. Arachidonoylethanolamide (anandamide) [3] and 2-arachidonoylglycerol (2-AG) [4, 5] are considered to be two physiologically important endocannabinoids (Fig. 1). Although both lipid molecules possess an arachidonic acid chain in their structure, the former is an N-acylethanolamine (NAE), and the latter is a monoacylglycerol (MAG). It should be noted that anandamide is a minor component in animal tissues, whereas other NAEs, such as palmitoylethanolamide (PEA), stearoylethanolamide, oleoylethanolamide (OEA), and linoleoylethanolamide, are abundant [6]. PEA exerts anti-inflammatory, analgesic and neuroprotective effects, and functions as an agonist of peroxisome proliferator-activated receptor-α rather than CB1 or CB2 [7]. OEA is noted for its appetite-suppressing effect, and has been reported to be an agonist of not only peroxisome proliferator-activated receptor-α, but also of transient receptor potential vanilloid 1 and the G protein-coupled receptor GPR119 [8]. By analogy, MAGs other than 2-AG may also show cannabinoid receptor-independent activities. In fact, like OEA, 2-oleoylglycerol activates GPR119 and stimulates the release of glucagon-like peptide-1 in vivo [9].


Figure 1. Chemical structures of endocannabinoids and related compounds. 2-OG, 2-oleoylglycerol.

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It is believed that, in response to a variety of cellular stimuli, endocannabinoids and related bioactive lipid molecules are principally generated from membrane phospholipids by specific hydrolases or a combination of acyltransferases and hydrolases. After functioning as receptor ligands, these molecules are degraded by hydrolases. Generally, the synthesizing enzymes for bioactive lipid molecules are tightly regulated and expressed at much lower levels than the degrading enzymes, enabling their generation on demand and quick degradation. Despite their structural similarities, the metabolic pathways of anandamide (Fig. 2) and 2-AG (Fig. 3) in animal tissues are completely different [10-12]. The ‘canonical pathways’ for both endocannabinoids are relatively simple. However, recent studies, including the analysis of gene-deficient mice, have shown that: (a) the pathways are more complicated than expected; and (b) one reaction is often catalyzed by multiple enzymes or isozymes. As specific inhibitors of ‘endocannabinoid hydrolases’, such as fatty acid amide hydrolase (FAAH), MAG lipase (MAGL), and NAE-hydrolyzing acid amidase (NAAA), as well as other related enzymes, are expected to be therapeutic drugs [13], it is crucial to elucidate the complete pathways involved in endocannabinoid metabolism in humans and rodents. In the present article, we will briefly discuss the metabolic pathways for anandamide and other NAEs, with special reference to the ‘alternative pathways’. We will also review the new findings on 2-AG metabolism. Although we focus on mammals, it should be noted that some of these pathways are conserved in other organisms, such as frogs, fishes, worms, Arabidopsis, and Dictyostelium, and play unique roles [14-18].


Figure 2. Metabolic pathways of anandamide and other NAEs. Asterisks indicate the enzymes included in the canonical pathway. Ca-NAT, Ca2+-dependent N-acyltransferase; PTPN22, protein tyrosine phosphatase, nonreceptor type 22; SHIP1, Src homology 2 domain-containing inositol-5-phosphatase 1; sPLA2, secretory PLA2.

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Figure 3. Metabolic pathways of 2-AG.

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Canonical pathways for anandamide and other NAEs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Canonical pathways for anandamide and other NAEs
  5. Alternative pathways for anandamide and other NAEs
  6. Metabolism of 2-AG
  7. Conclusions
  8. Acknowledgements
  9. References

Schmid and his group delineated the metabolic pathways of NAE on the basis of their biochemical studies with animal tissues such as heart and brain [19]. The classic biosynthetic route, composed of two enzyme reactions, was called ‘the transacylation–phosphodiesterase pathway’. The intermediate metabolite, N-acylated ethanolamine phospholipid, is an unusual glycerophospholipid molecule in which an extra acyl chain is covalently linked to the amino group of ethanolamine phospholipid [10, 20, 21]. N-acylated ethanolamine phospholipid species comprise the diacyl type [N-acyl-phosphatidylethanolamine (NAPE)], the alkenylacyl type [plasmalogen-type, N-acyl-plasmenylethanolamine (pNAPE)], and the alkylacyl type (N-acyl-plasmanylethanolamine), which have an acyl chain, an alk-1-enyl chain, or an alkyl chain, respectively, at the sn-1 position (Fig. 4). Alternatively, NAPE may be used as a generic term for all three types of N-acylated ethanolamine phospholipid. In addition to serving as a precursor of various NAEs, NAPE seems to function in membrane stabilization [22]. NAPE was also reported to be an anorexic hormone [23]. However, this finding has been questioned, because a decrease in food intake was also observed after intraperitoneal injection of the negative control phosphatidylethanolamine (PtdEtn) into mice [24]. Regarding the degradation of NAEs, hydrolysis to free fatty acid and ethanolamine is a major pathway [25, 26]. Oxygenation of the arachidonic acid moiety of anandamide appears to be another degradative pathway [27, 28].


Figure 4. Chemical structures of N-acylated ethanolamine phospholipids.

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Ca2+-dependent N-acyltransferase

The first reaction of NAE biosynthesis is N-acylation of diacyl-type (PtdEtn), alkenylacyl-type (plasmalogen) and alkylacyl-type ethanolamine phospholipids, leading to the formation of N-acylated ethanolamine phospholipids such as NAPE (Fig. 2). The enzyme responsible, ‘N-acyltransferase’, has several unique properties [29-31]. First, the enzyme uses phosphatidylcholine (PtdCho), 1-acyl-lyso-PtdCho, PtdEtn and cardiolipin as donor substrates, and extracts an acyl group selectively from the sn-1 position of these donor phospholipids. Thus, 2-acyl-lysophospholipid, a lysophospholipid that is typically produced by phospholipase (PL)A1, is another product in this reaction. Acyl-CoA is not utilized, although this molecule serves as a donor substrate for many other acyltransferases. Interestingly, NAPE synthase in plants catalyzes direct incorporation of free fatty acid into PtdEtn [18]. Second, the enzyme is associated with membranes, and can be solubilized with the nonionic detergent Nonidet P-40 [31]. Third, the activity of the enzyme is stimulated by Ca2+. It is well known that NAPE accumulates in ischemic heart and brain, or after toxic insult to tissues or cells [29, 32]. Recently, Janfelt et al. showed, with desorption ESI imaging MS, that, during ischemia–reperfusion in the brain of neonatal rats, the levels of many NAPE species increased in the whole injured area, where the cells seemed to be dead [33]. These findings suggest that a remarkable increase in intracellular Ca2+ levels activates N-acyltransferase to generate NAPE. However, it is unclear whether a modest increase in Ca2+ levels caused by physiological stimuli potentiates the enzymatic activity. Despite efforts over many years this Ca2+-dependent N-acyltransferase remains molecularly uncharacterized.

NAPE-hydrolyzing PLD (NAPE-PLD)

The second step in the canonical pathway is the release of NAE from NAPE by a PLD-type enzyme known as NAPE-PLD [34]. cDNA cloning revealed that NAPE-PLD belongs to the metallo-β-lactamase family and is molecularly distinguished from the known PLD isoforms, which hydrolyze common glycerophospholipids such as PtdCho to produce phosphatidic acid (PA), an intracellular signal molecule [35]. NAPE-PLD appears to contain catalytically essential zinc, and the purified recombinant enzyme is specific for NAPE, being almost inactive with major glycerophospholipids such as PtdCho and PtdEtn [36]. At this time, NAPE-PLD is the sole enzyme in animal tissues known to directly release NAE from NAPE. This is in marked contrast to plants, which lack NAPE-PLD, but express multiple PLD isoforms, such as PLDβ and PLDγ, that hydrolyze different glycerophospholipids, including NAPE [16]. NAPE-PLD is tightly bound to membranes. The soluble enzyme prepared by treatment with detergent can be stimulated by millimolar concentrations of divalent cations, including Ca2+ and Mg2+ [37], and 10–100 μm PtdEtn [38]. However, the physiological regulators of NAPE-PLD activity are unknown. Concerning transcriptional regulation, lipopolysaccharide (LPS) downregulates the expression of NAPE-PLD mRNA in RAW264.7 macrophage cells [39]. LPS altered the acetylation state of histone proteins bound to the NAPE-PLD promoter and suppressed the transcription of NAPE-PLD mRNA [40]. The transcription factor Sp1 was involved in the regulation of baseline NAPE-PLD expression, but not in the suppression by LPS. In addition, the expression of NAPE-PLD in rodent brains is age-dependently upregulated at the mRNA and protein levels [41], in agreement with the increase in NAPE-PLD activity [41, 42]. In contrast, N-acyltransferase activity decreases during development [42]. The opposite changes of these two enzymes probably explain why the NAPE level in the ischemic brain of young rodents is much higher than that in adult rodents.

NAPE-PLD−/− mice were born at the expected Mendelian frequency, were viable, and were apparently healthy [43, 44]. The accumulation of NAPEs in the brains of NAPE-PLD−/− mice demonstrated an important role of NAPE-PLD in the degradation of NAPE in this tissue. However, the detection of unaltered or moderately reduced NAE levels strongly suggested the existence of other NAE-forming enzyme(s) or route(s) in NAPE-PLD−/− mice. Apart from this gene-modified mouse model, a recent cohort study suggested a physiological role of NAPE-PLD. In this study, a common haplotype in NAPE-PLD was reported to be protective against severe obesity in a Norwegian population [45].

NAEs with different N-acyl chains appear to share the transacylation–phosphodiesterase pathway for their biosynthesis. In fact, N-acyl species were not distinguished in the reactions catalyzed by N-acyltransferase [46] and NAPE-PLD [36]. The N-acyl chain originates from the sn-1 position of the donor phospholipid, and, in general, polyunsaturated fatty acyl chains such as the arachidonoyl chain are mostly linked to the sn-2 rather than the sn-1 position. Most likely, this is the main reason why the tissue levels of anandamide are usually much lower than those of saturated or monounsaturated NAEs. Apart from this pathway, anandamide may be formed by the condensation of free arachidonic acid with ethanolamine in the reverse reaction of FAAH (Fig. 2) [47, 48]. This reverse reaction can occur if both arachidonic acid and ethanolamine are present in sufficient amounts [49]. Anandamide was actually formed through this route in vivo after partial hepatectomy of mice [50]. Moreover, anandamide could be spontaneously formed from arachidonoyl-CoA and ethanolamine [51].


A membrane-associated amidohydrolase that hydrolyzes anandamide and other NAEs to their corresponding fatty acids and ethanolamine has been extensively studied [25, 26]. When its cDNA cloning was reported, the enzyme was named FAAH [52]. FAAH is a serine hydrolase belonging to the amidase signature family. The catalytic triad is composed of Lys142, Ser217, and Ser241 (the catalytic nucleophile) [53]. Analysis of FAAH−/− mice demonstrated the central role of FAAH in the degradation of not only anandamide but also other NAEs, including PEA and OEA [54, 55]. FAAH also hydrolyzes other fatty acid amides, such as oleamide (primary amide of oleic acid) [52] and N-acyltaurine (taurine-conjugated fatty acid) [56]. Such broad reactivity with cannabinoid receptor-insensitive bioactive fatty acid amides should be considered when the molecular mechanisms for the phenotype of FAAH−/− mice are examined. Although FAAH hydrolyzes 2-AG at a high rate [57], its contribution to 2-AG degradation in the brain was estimated to be minor [58, 59]. Considering the physiological importance of FAAH, naturally occurring single-nucleotide polymorphisms were examined in the human FAAH gene. Interestingly, the cytosine 385 to adenine missense mutation was found to be strongly associated with street drug use and problem drug/alcohol use [60]. This mutation results in the substitution of a threonine for Pro129, and the P129T variant showed enhanced sensitivity to proteolytic degradation. Thus, the functional abnormality of FAAH could be linked to drug abuse and dependence in humans.

As FAAH has been suggested to be involved in a variety of symptoms, including anxiety, depression, and neuropathic pain, FAAH inhibitors are expected to be therapeutic drugs [13]. A large number of specific FAAH inhibitors have been developed, and include URB597 [61], OL-135 [62], PF-3845 [63], and PF-04457845 [64]. Furthermore, the full spectrum of cannabimimetic activities was expected with dual inhibition of FAAH and MAGL, the principal enzyme for the degradation of 2-AG. In fact, treatment with fluorophosphonate compounds as the dual inhibitors increased the brain levels of both anandamide and 2-AG more than 10-fold, resulting in the complete appearance of the ‘tetrad of cannabinoid’ (analgesia, hypomotility, hypothermia, and catalepsy) [65]. JZL195, which inhibits both FAAH and MAGL, also mimicked the pharmacological activities of the CB1 receptor agonist in vivo [66].

An isozyme of FAAH with ~ 20% sequence identity at the amino acid level is expressed in humans, but not in rodents [67]. The original FAAH and this newly discovered isozyme were designated FAAH-1 and FAAH-2, respectively. Interestingly, unlike FAAH-1, which was localized in endoplasmic reticulum, FAAH-2 was localized on lipid droplets, and its N-terminal hydrophobic region was identified as a lipid droplet localization sequence, suggesting that the role of FAAH-2 is different from that of FAAH-1 [68]. Recently, a catalytically inactive, truncated variant of FAAH-1 [FAAH-like anandamide transporter (FLAT)] was reported to have the ability to bind to anandamide and drive anandamide transport in neurons [69]. However, this finding has been questioned, for several reasons, including the lack of endogenous FLAT in brain and neural cells, and the remaining FAAH-like catalytic activity of recombinant FLAT [70].

Similarly to the oxygenation of free arachidonic acid to various bioactive eicosanoids, the arachidonic acid chain of anandamide can be oxygenated (Fig. 2) [27, 28]. These metabolites include hydroxyl derivatives of anandamide produced by cytochrome P450 [71], hydroperoxy derivatives produced by lipoxygenases [72, 73], and prostaglandin-like ethanolamides (called prostamides) produced by cyclooxygenase (COX)-2 [74, 75]. Despite their reported biological activities, the physiological significance of these compounds is poorly understood.

Alternative pathways for anandamide and other NAEs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Canonical pathways for anandamide and other NAEs
  5. Alternative pathways for anandamide and other NAEs
  6. Metabolism of 2-AG
  7. Conclusions
  8. Acknowledgements
  9. References

These newly discovered pathways for NAE metabolism include: (a) members of the PLA/acyltransferase (PLA/AT) family; (b) multistep pathways via N-acylated lysophospholipid; and (c) NAAA. These enzymes/pathways were found in animal tissues as possible alternatives to Ca2+-dependent N-acyltransferase, NAPE-PLD, and FAAH, respectively.

PLA/AT family proteins as Ca2+-independent N-acyltransferases

In an attempt to identify Ca2+-dependent N-acyltransferase, we noticed that the reaction catalyzed by N-acyltransferase is similar to that catalyzed by lecithin retinol acyltransferase (LRAT). LRAT transfers an acyl chain from the sn-1 position of lecithin (PtdCho) to retinol (vitamin A alcohol), resulting in the formation of retinyl ester (the storage form of vitamin A). We speculated that the primary structure of N-acyltransferase might be analogous to that of LRAT. LRAT is a member of the NlpC/P60 thiol protease superfamily [76], and has considerable sequence homology with five members (HRASLS1–5) of the HRAS-like suppressor (HRASLS) family (Fig. 5) [77]. HRASLS3 (also referred to as H-rev107) is a representative molecule of this family, and has been analyzed as a class II tumor suppressor that negatively regulates the activity of the oncoprotein Ras [78]. Later, HRASLS1 (A-C1) [79], HRASLS2 [80], and HRASLS4 [tazarotene-induced protein 3 (TIG3)] [81] were also reported to have tumor-suppressing activity. As catalytically important residues, including cysteine and histidine as the putative catalytic dyad, are highly conserved among LRAT and HRASLS family members, HRASLS3 was earlier suggested to be an acyltransferase [76]. However, the enzymatic properties of the family members were not examined until we reported that HRASLS5 (H-rev107-like protein 5) has an N-acyltransferase activity, forming NAPE by transferring an acyl group from PtdCho to PtdEtn [31, 82]. HRASLS5 was present mainly in the cytosolic fraction, and its N-acyltransferase activity was only slightly stimulated by Ca2+. Interestingly, the enzyme removed a fatty acyl group from both the sn-1 and sn-2 positions of the acyl donor PtdCho. These results strongly suggested that HRASLS5 is different from the known membrane-associated Ca2+-dependent N-acyltransferase. Our studies revealed that all other members of the HRASLS family (HRASLS1–4) also have N-acyltransferase activity [77, 83, 84]. Very recently, Golczak et al. also reported that purified HRASLS2, HRASLS3 and HRASLS4 generate NAPE from PtdCho and PtdEtn [85].


Figure 5. Primary structures of human LRAT and five members of the HRASLS (PLA/AT) family. Closed and shaded boxes indicate identity in all six and any four or five polypeptides, respectively. Dashes denote deletion of amino acids as compared with the other sequences. Asterisks indicate amino acids forming the catalytic triad.

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Importantly, HRASLS1–5 showed not only N-acyltransferase activity, but also PLA1 and PLA2 (PLA1/2) activity, hydrolyzing the sn-1 and sn-2 ester bonds of PtdCho or PtdEtn, and O-acyltransferase activity, transferring an acyl group from PtdCho to the hydroxyl group of lyso-PtdCho. We thus proposed calling HRASLS1–5 PLA/AT-1–5 [77]. PLA/AT-1, PLA/AT-2 and PLA/AT-5 showed relatively high N-acyltransferase activity relative to PLA1/2 activity, whereas PLA/AT-3 and PLA/AT-4 showed lower N-acyltransferase activity [77, 86]. When the homogenate of human embryonic kidney (HEK)293 cells, stably expressing PLA/AT-2, was examined, both the soluble and particulate fractions showed N-acyltransferase activity, with 1.4-fold higher activity in the soluble fraction. The N-acyltransferase activity was not stimulated by Ca2+ in either fraction. Thus, PLA/AT-2 was also presumed to be different from the known Ca2+-dependent N-acyltransferase.

Although PLA/AT proteins show N-acyltransferase activity in vitro, it remained unclear whether these molecules generate NAPE in living cells. When we metabolically labeled COS-7 cells transiently expressing recombinant PLA/AT-1–5 with [14C]ethanolamine, we found that PLA/AT-1, PLA/AT-2, PLA/AT-4 and PLA/AT-5 generated significant amounts of [14C]NAPE and [14C]NAE [86]. Liquid chromatography–tandem MS demonstrated that the stable expression of PLA/AT-2 in HEK293 cells greatly increased endogenous levels of NAPEs and NAEs. Furthermore, additional expression of NAPE-PLD in the PLA/AT-2-expressing cells led to efficient conversion of the increased NAPE to NAE. RT-PCR revealed that human HeLa cells expressed endogenous PLA/AT-2, and the knockdown of this protein by small interfering RNA lowered the endogenous level of NAPE. Taken together, these results suggested that the PLA/AT proteins produce NAPE, which serves as a precursor of NAE in living cells. In the cells overexpressing recombinant PLA/AT proteins, the generation of NAPE proceeded without any cellular stimuli. However, we could not rule out the possibility that the N-acyltransferase activity of native PLA/AT proteins is regulated by intracellular Ca2+ or other signaling molecules.

We reported that PLA/AT-3 (H-rev107) functions mainly as PLA1/2, with low N-acyltransferase activity [83, 84]. However, Duncan et al. termed this molecule adipose-specific PLA2, and classified it as group XVI PLA2 (PLA2G16) [87]. Recently, two groups demonstrated that PLA/AT-3 has PLA1 activity that is as potent as its PLA2 activity [85, 88]. PLA/AT-3 attracted attention because of its abundant expression in adipose tissue, its induction during adipose differentiation, and its ability to suppress lipolysis [89, 90]. We also showed that the overexpression of PLA/AT-3 in HEK293 cells caused dysfunction of peroxisomes and a remarkable decrease in ether-type triglycerides and plasmalogens [91]. It remains to be determined whether the N-acyltransferase activity of PLA/AT proteins is related to their activities regarding tumor suppression, obesity, and dysfunction of peroxisomes. Recently, the N-terminal catalytic domain of PLA/AT-3 was characterized by solution NMR structure analysis [92] and X-ray crystallography [85, 88]. Together with site-directed mutagenesis studies, these studies increased our knowledge of the structure and function of PLA/AT-3, including the following: (a) the catalytic triad in the active site is composed of His23, His35, and Cys113 (His35 is replaced by asparagine in PLA/AT-1); (b) the acylation site in the acyl–protein complex is Cys113; and (c) the C-terminal transmembrane domain is required for the interfacial catalysis.

Multistep pathways via N-acylated lysophospholipid

Whereas NAPE-PLD directly releases NAE from NAPE, multistep pathways for the biosynthesis of NAE from NAPE via N-acylated lysophospholipid were also suggested in the 1980s (Fig. 2) [93]. We showed that the secretory PLA2 isoforms of groups IB, IIA and V hydrolyzed NAPE to 1-acyl-lyso-NAPE, which was further converted to NAE by a membrane-associated lyso-PLD-like enzyme existing in brain and other tissues of rat [94]. Although this ‘lyso-PLD’ was distinguished from NAPE-PLD by its catalytic properties, its further characterization has not been carried out. The presence of NAEs in the tissues of NAPE-PLD−/− mice clarified that NAE can be formed by an alternative pathway(s) in vivo [43]. The double O-deacylation of NAPE via lyso-NAPE and further hydrolysis of the resultant metabolite glycerophospho-NAE (GP-NAE) to NAE and glycerol 3-phosphate were proposed as one alternative pathway (Fig. 2) [95]. GP-NAE was actually detected in mouse brain tissue [96]. In the analysis of NAPE-PLD−/− mice, we found a remarkable increase in endogenous brain levels of lyso-NAPE and GP-NAE as well as of NAPE [44]. These results suggested that NAPE accumulates in the brain because of the deficiency of NAPE-PLD, and is degraded in the alternative pathway via lyso-NAPE and GP-NAE. By the functional proteomic isolation method with fluorophosphonate-biotin probe, α/β-hydrolase 4 [α/β-hydrolase domain-containing protein (ABHD)4] was demonstrated to be responsible for the double O-deacylation that generates GP-NAE from NAPE via lyso-NAPE [95]. ABHD4 thus hydrolyzes both NAPE and lyso-NAPE by PLA1/2 (or PLB) and lysophospholipase activities. ABHD4 preferentially hydrolyzed lyso-NAPE among various lysophospholipids, including lyso-PtdEtn, lyso-PtdCho, and lysophosphatidylserine. Regarding the N-acyl species of lyso-NAPEs, ABHD4 did not distinguish between saturated and polyunsaturated acyl chains. Ser146, in the consensus sequence GXSXG, was presumed to be the catalytic nucleophile. It was recently reported that knockdown of ABHD4 inhibits anoikis (cell death in response to loss of cell–cell and cell–matrix interactions) in prostate epithelial cells [97]. Its relationship to NAE metabolism is unclear.

Glycerophosphodiester phosphodiesterase 1 (GDE1) was shown to hydrolyze GP-NAE to NAE and glycerol 3-phosphate [96]. On the basis of tissue distribution and catalytic properties, including Mg2+ requirement, the brain enzyme activity hydrolyzing GP-NAE to NAE was attributed to GDE1. Thus, the combination of ABHD4 and GDE1 was considered to form a NAPE-PLD-independent pathway. Initially, this pathway was expected to be responsible for anandamide formation, as the brain level of anandamide was not altered in NAPE-PLD−/− mice, in contrast to the decrease in the levels of saturated NAEs [43]. However, neither ABHD4 nor GDE1 showed a preference for N-arachidonoyl species of lyso-NAPE and GP-NAE (precursors of anandamide). It remains unclear whether or not the ABHD4–GDE1 pathway is involved in the selective formation of anandamide. Alternatively, in our analysis of NAPE-PLD−/− mice, the brain level of anandamide was significantly lower, together with the levels of other NAEs [44].

GDE1 is an integral membrane glycoprotein, and was originally discovered as MIR16, a protein interacting with RGS16 (a regulator of G protein signaling) [98]. Later GDE1 was shown to have a phosphodiesterase activity, preferentially hydrolyzing glycerophosphoinositol [99]. GDE1−/− mice were born at the expected Mendelian frequency, were viable and healthy, and showed no abnormal cage behavior [100]. Expectedly, the formation of NAE from GP-NAE or lyso-NAPE was hardly detected in the brain homogenates. However, endogenous brain levels of NAEs were not significantly different between the homozygous and heterozygous mice. Furthermore, no significant difference was seen between NAPE-PLD−/− mice and mice with double knockout of GDE1 and NAPE-PLD. These results suggest that enzymes or pathways other than NAPE-PLD and the ABHD4–GDE1 pathway are involved in NAE formation. Further analysis of GDE1−/− mice demonstrated that brain levels of glycerophosphoinositol, glycerophosphoserine and glycerophosphoglycerate were highly elevated [101]. In agreement with these findings, the brain level of free serine, which should be released from glycerophosphoserine by GDE1, was significantly reduced.

The fact that the brain contains plasmalogen-type ethanolamine phospholipid (plasmenylethanolamine) in abundance [102] suggests that pNAPE also exists in the same tissue and serves as a precursor of NAE (Fig. 6). In rat brain, 65% of N-arachidonoylethanolamine phospholipids were of the plasmalogen type [103]. In agreement with an earlier report using rat heart microsomes [104], recombinant NAPE-PLD hydrolyzed N-palmitoyl-plasmenylethanolamine to PEA at 70% of the rate of N-palmitoyl-PtdEtn hydrolysis [44]. The brain levels of pNAPE and its lyso form (lyso-pNAPE) in NAPE-PLD−/− mice were much higher than those in wild-type mice, as shown by liquid chromatography–tandem MS. Furthermore, the brain homogenate of NAPE-PLD−/− mice converted pNAPE to NAE, and the homogenate also released NAE from lyso-pNAPE. As lyso-pNAPE has a vinyl ether bond rather than an ester bond at the sn-1 position, lysophospholipases such as ABHD4 should be inactive with this lysophospholipid. Therefore, it was likely that a lyso-PLD-type phosphodiesterase directly releases NAE from lyso-pNAPE. The lyso-PLD-type enzyme found in the brain was active at neutral pH, and converted N-palmitoyl-lysoplasmenylethanolamine, N-oleoyl-lysoplasmenylethanolamine and N-arachidonoyl-lysoplasmenylethanolamine to their corresponding NAEs at similar rates. The activity was stimulated by 2 mm Mg2+ and inhibited by 0.1% Triton X-100. We found that recombinant GDE1 showed weak lyso-PLD activity in hydrolyzing N-palmitoyl-lysoplasmenylethanolamine in addition to the aforementioned GP-NAE-hydrolyzing activity. As GDE1 is expressed in brain, GDE1 may be, at least in part, responsible for the brain lyso-PLD activity.


Figure 6. Biosynthetic pathways of NAE from pNAPE.

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PLC-mediated hydrolysis of NAPE to NAE phosphate and dephosphorylation to generate NAE is another multistep route for the conversion of NAPE to NAE that does not go through lyso-NAPE (Fig. 2). Treatment of RAW264.7 mouse macrophages with LPS potently enhanced anandamide levels, despite the downregulation of NAPE-PLD [39, 105]. Protein tyrosine phosphatase, nonreceptor type 22 was identified as one of the gene products that increase the anandamide level, and exhibited a phosphatase activity that generates anandamide from anandamide phosphate. Src homology 2 domain-containing inositol-5-phosphatase 1 showed the same phosphatase activity. Although the PLC–phosphatase pathway was suggested to function in the brain of NAPE-PLD−/− mice [106], the PLC-like enzyme responsible remains uncharacterized.


We found a lysosomal enzyme hydrolyzing NAEs, first in CMK human megakaryoblastic cells [107], and later in the lung and other tissues of rats [108]. cDNA cloning of this enzyme, referred to as NAAA, showed it to be a cysteine hydrolase belonging to the N-terminal nucleophile hydrolase superfamily (Fig. 7) [109-111]. No sequence homology was seen between NAAA and FAAH. Prior to our identification, NAAA was recognized as acid ceramidase-like protein from sequence homology [112]. Acid ceramidase is a lysosomal enzyme that hydrolyzes ceramide to sphingosine and fatty acid. In agreement with its localization in lysosomes, NAAA is active only at acidic pH, and hydrolyzes various NAEs, with a preference for PEA in vitro. Human NAAA is a glycoprotein with four N-glycosylation sites [113, 114]. Like acid ceramidase [115], recombinant NAAA is produced as an inactive proenzyme, and is converted by autocatalytic cleavage between Phe125 and Cys126 to a catalytically active heterodimer composed of α and β subunits. The N-terminal 28 amino acids form a signal peptide, which is not contained in the α subunit [114]. Although the β subunit was purified from rat lung [108], native αβ heterodimer has not been isolated. It remains unclear whether native NAAA stably exists as the heterodimer. On the basis of the sequence homology among the family members, the N-terminal cysteine of the β subunit (Cys126 in human NAAA) has been presumed to be the catalytic nucleophile. This was recently demonstrated by showing that β-lactones, which inhibit NAAA, acylate Cys126 [116, 117].


Figure 7. The primary structures of human NAAA and acid ceramidase. Asterisks and dots denote identity and similarity of the two sequences, respectively. Dashes indicate deletion of amino acids as compared with the other sequence. Diamonds and the circle indicate N-glycosylation sites and the catalytic nucleophile of NAAA, respectively. The arrow and the arrowhead denote a site of cleavage by signal peptidase (NAAA) and that between the α and β subunits (both NAAA and acid ceramidase), respectively.

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NAAA is expressed in various human and rodent tissues, with predominant expression in macrophages [118, 119]. The expression of NAAA was highest in the prostate among various human tissues [120]. Nonionic detergents (Triton X-100 and Nonidet P-40) and the thiol-reducing agent dithiothreitol have been used as NAAA stimulators in vitro. As endogenous stimulators, choline-containing or ethanolamine-containing phospholipids (PtdCho, PtdEtn, and sphingomyelin) and dihydrolipoic acid (the reduced form of α-lipoic acid) could substitute for Nonidet P-40 and dithiothreitol, respectively [121]. These endogenous molecules may function by keeping NAAA active in lysosomes. As it is likely that FAAH and NAAA contribute to the degradation of NAEs in vivo, we investigated whether or not compensatory induction of NAAA mRNA occurs in the tissues of FAAH−/− mice. However, the expression levels in various tissues were not significantly different from those in wild-type mice [121].

Considering that NAAA preferentially hydrolyzes PEA over other NAEs, selective NAAA inhibitors that may increase local levels of endogenous PEA were expected to be anti-inflammatory and analgesic drugs [122]. To date, many compounds have been reported to selectively inhibit NAAA (Fig. 8) [117, 123-130]. The most potent NAAA inhibitors thus far reported are lactone derivatives such as (S)-N-(2-oxo-3-oxetanyl)-3-phenylpropionamide [(S)-OOPP], (S)-N-(2-oxo-3-oxetanyl)biphenyl-4-carboxamide, and (2S,3R)-2-methyl-4-oxo-3-oxetanylcarbamic acid 5-phenylpentyl ester (URB913/ARN077), whose IC50 values were 420, 115 and 127 nm, respectively [125, 126, 128]. (S)-OOPP normalized the decreased PEA levels in carrageenan-stimulated leukocytes and LPS-treated RAW264.7 macrophage cells, and led to a reduction in neutrophil migration and inhibition of carrageenan-induced plasma extravasation [125]. Recently, we reported that lipophilic amines such as pentadecylamine and tridecyl 2-aminoacetate inhibited NAAA with IC50 values of 5.7 and 11.8 μm, respectively, and showed much weaker effects on FAAH [129]. These simple structures may provide a scaffold for further improvement.


Figure 8. Chemical structures of specific NAAA inhibitors. (A) Lactone derivatives. (B) Other compounds. IC50 values are shown in parentheses.

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Metabolism of 2-AG

  1. Top of page
  2. Abstract
  3. Introduction
  4. Canonical pathways for anandamide and other NAEs
  5. Alternative pathways for anandamide and other NAEs
  6. Metabolism of 2-AG
  7. Conclusions
  8. Acknowledgements
  9. References

Biosynthesis of 2-AG

Earlier, the PLC–diacylglycerol lipase (DAGL) pathway was considered to play a role in the release of arachidonic acid from phospholipid [131]. This pathway is now known to be the most important route in the biosynthesis of 2-AG (Fig. 3) [11, 132]. It is generally accepted that 2-AG is formed through this pathway in postsynaptic neurons in response to depolarization and stimulation of Gq/11-coupled receptors. The resultant 2-AG is released, and mediates retrograde synaptic suppression through CB1 receptors at presynaptic terminals [133]. In this pathway, PLC hydrolyzes 2-arachidonoyl-phosphatidylinositol (PtdIns) to arachidonic acid-containing diacylglycerol (DAG), which is subsequently hydrolyzed to 2-AG by DAGL. The most abundant PtdIns molecule in mammalian tissues is 1-stearoyl-2-arachidonoyl-PtdIns, enabling the effective generation of 2-AG over other MAGs. Among the β, γ, δ, ε, ζ, and η subtypes of PLC, the β subtype is characterized by the stimulation by Gq/11-coupled receptors [134], which suggests that it has a central role in neurotransmitter-dependent 2-AG formation mediated by Gq/11-coupled receptors in postsynaptic neurons. In fact, each isoform (β1–β4) of PLCβ, which is expressed regiospecifically in different brain areas, is stimulated by different Gq/11-coupled receptors, including the group I metabotropic glutamate receptors (mGluRs) mGluR1 and mGluR5, and the muscarinic acetylcholine receptor M1 [133]. PA [135] and PtdCho [136] may also be converted to 2-arachidonoyl-DAG, which, in turn, serves as a precursor of 2-AG. Another pathway initiated from PtdIns leads to the formation of lyso-PtdIns by PLA1, followed by the release of 2-AG by lyso-PtdIns-specific PLC (Fig. 3) [137, 138]. Furthermore, 2-AG can be formed from 2-arachidonoyl-lysophosphatidic acid (LPA) (Fig. 3) [139].

DAGL, a membrane-associated enzyme hydrolyzing DAG preferentially at the sn-1 position, was first reported in 1981 [140, 141]. cDNA cloning revealed that human DAGL has two closely related genes, α and β, a lipase-3 motif, a serine lipase motif, and four putative transmembrane domains [142]. DAGLα was widely distributed in human and mouse tissues, with high expression levels in the nervous system (human and mouse) and pancreas (human). DAGLα was colocalized with Gq/11-coupled receptors, Gq/11α and PLCβ at particular synaptic and neuronal surfaces [133]. The knockdown of DAGLα by RNA interference suggested its involvement in the mGluR-dependent formation of 2-AG in neuroblastoma cells [143]. Both DAGLα-deficient and DAGLβ-deficient mice were viable, and their general appearance was normal [144, 145]. DAGLα-deficient mice showed a remarkable reduction in brain 2-AG levels, and failed in Ca2+-dependent and Gq/11-coupled receptor-driven retrograde synaptic suppression. These results strongly suggested the involvement of DAGLα in the generation of 2-AG, which is responsible for CB1 receptor-dependent retrograde signaling. In contrast, the brain 2-AG levels of DAGLβ-deficient mice were normal [144] or reduced by 50% [145]. The classical DAGL inhibitors, RHC80267 and tetrahydrolipstatin, inhibited both DAGL isoforms, and lowered endogenous 2-AG levels [146]. More selective DAGL inhibitors include O-3841, OMDM-188, and O-5596 [147-149].

Degradation of 2-AG

The major degradative pathway of 2-AG is its hydrolysis to arachidonic acid and glycerol [11, 150] (Fig. 3). The hydrolysis of 2-AG in brain is mainly catalyzed by MAGL, and < 15% of the activity is attributed to other hydrolases, such as ABHD6, ABHD12, and FAAH [59]. Mammalian MAGL was purified in 1976 [151] and cloned in 1997 [152]. The enzyme hydrolyzes not only 2-AG but also other 2-MAGs and 1-MAGs, and it is a soluble protein that associates with membranes in a peripheral manner [153]. A serine hydrolase belonging to the α/β-hydrolase superfamily, MAGL has a catalytic triad composed of Ser122, Asp239, and His269. MAGL crystallized as a dimer, and its three-dimensional structure has been analyzed in detail [154, 155]. MAGL is expressed in many tissues, and plays an important role in the degradation of 2-AG that is responsible for endocannabinoid signaling in the brain [152, 156]. In the cerebellum, the expression of MAGL was high within parallel fiber terminals, weak in Bergman glia, and absent in other synaptic terminals. However, 2-AG was broadly degraded in a synapse-type-independent manner by MAGL [157].

MAGL inhibitors have attracted much attention as promising therapeutic drugs and pharmacological probes, owing to their ability to enhance 2-AG signaling [13, 150, 153, 158]. A large number of sulfhydryl blockers and serine hydrolase inhibitors have been reported to be nonspecific or specific MAGL inhibitors. An O-aryl carbamate, JZL184, is one of the most potent inhibitors selective for MAGL. In JZL184-treated mice, the brain level of 2-AG was increased, and CB1-dependent analgesia, hypothermia and hypomotility appeared [159]. KML29, a recently developed derivative of JZL184, was more selective for MAGL, and did not show detectable crossreactivity with FAAH [160]. In MAGL-deficient mice, the brain 2-AG-hydrolyzing activity dramatically decreased and the brain 2-AG level was highly elevated [161-163]. Notably, similarly to mice chronically treated with JZL184, MAGL-deficient mice showed tolerance to CB1 agonists. Downregulation and desensitization of brain CB1 were also observed. Recent studies have revealed roles for MAGL other than 2-AG degradation. For example, MAGL deficiency impaired lipolysis and attenuated diet-induced insulin resistance [163]. Arachidonic acid formation as a result of MAGL generates prostaglandins that promote neuroinflammation in the brain [164]. Moreover, MAGL was highly expressed in aggressive human cancer cells and primary tumors. Its overexpression in nonaggressive cancer cells increased their pathogenicity phenotypes [165].

ABHD6 and ABHD12 are additional MAG hydrolases belonging to the α/β-hydrolase superfamily, with the postulated catalytic triad serine–aspartic acid–histidine [166, 167]. ABHD6 was notable for its expression in the mouse microglial cell line BV-2, in which MAGL was not expressed [168]. In adult mouse cortex, ABHD6 was located postsynaptically in neurons, and it was suggested to constitute a rate-limiting step in 2-AG signaling. UCM710 inhibited FAAH and ABHD6, but not MAGL [169]. Mutations in the ABHD12 gene are causally linked to a neurodegenerative disease termed polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract (PHARC), suggesting essential functions of ABHD12 in the nervous system and eye [170].

As with anandamide, the arachidonoyl moiety of 2-AG can be oxygenated by COX-2 and lipoxygenases, resulting in the formation of glyceryl prostaglandins or hydroperoxy derivatives of 2-AG, respectively (Fig. 3) [171, 172]. Some biological activities of glyceryl prostaglandins have been reported [172]. Interestingly, R-enantiomers of ibuprofen, naproxen and flurbiprofen selectively inhibited the endocannabinoid oxygenation catalyzed by COX-2, but were inactive with free arachidonic acid as a COX-2 substrate. Thus, these inhibitors were considered to be substrate-selective inhibitors of endocannabinoid oxygenation [173].


  1. Top of page
  2. Abstract
  3. Introduction
  4. Canonical pathways for anandamide and other NAEs
  5. Alternative pathways for anandamide and other NAEs
  6. Metabolism of 2-AG
  7. Conclusions
  8. Acknowledgements
  9. References

We have discussed new enzymes and pathways that may be involved in the metabolism of endocannabinoids and related NAEs. The new enzymes/pathways in the metabolism of anandamide and other bioactive NAEs included: (a) the members of the HRASLS family or PLA/AT family, which function as Ca2+-independent N-acyltransferases; (b) NAPE-PLD-independent multistep pathways via N-acylated lysophospholipid; and (c) NAAA, a lysosomal enzyme that preferentially hydrolyzes PEA. Although their physiological significance is poorly understood, these new enzymes/pathways may serve as novel targets for the development of therapeutic drugs. For example, selective NAAA inhibitors may be promising as novel anti-inflammatory and analgesic drugs. Ca2+-dependent N-acyltransferase in the canonical pathway remains molecularly uncharacterized. The identification of the gene is eagerly anticipated. Recent studies enabled the analysis of genetic and pharmacological deficiency of 2-AG-metabolizing enzymes, and confirmed the essential role of 2-AG in the mediation of retrograde synaptic suppression. Elucidation of the physiological functions of ABHD6 and ABHD12 is also expected. After the submission of this manuscript, Blankman et al. reported that ABHD12 is a principal lysophosphatidylserine lipase in the mammalian brain [174]. The mutations in ABHD12 caused the human neurodegenerative disorder PHARC, and the phenotypes of ABHD12−/− mice resembled those of human PHARC patients.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Canonical pathways for anandamide and other NAEs
  5. Alternative pathways for anandamide and other NAEs
  6. Metabolism of 2-AG
  7. Conclusions
  8. Acknowledgements
  9. References

We are grateful to D. G. Deutsch (Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA) for critical reading of this manuscript.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Canonical pathways for anandamide and other NAEs
  5. Alternative pathways for anandamide and other NAEs
  6. Metabolism of 2-AG
  7. Conclusions
  8. Acknowledgements
  9. References
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