[Vitamins & Hormones] Vitamins and Hormones Volume 81 || Chapter 8 Novel Endogenous N‐Acyl...

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Novel Endogenous N-Acyl Glycines:Identification and Characterization

Heather B. Bradshaw,* Neta Rimmerman,* Sherry S.-J. Hu,*

Sumner Burstein,† and J. Michael Walker*

Contents

I.

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istorical View of Lipid Signaling Discoveries

ormones, Volume 81 # 200

29, DOI: 10.1016/S0083-6729(09)81008-X All r

of Psychological and Brain Sciences, Indiana University, Bloomington, Indiana,of Biochemistry and Molecular Pharmacology, University of Massachusetts MedMassachusetts, USA

1

9 Elsevie

ights rese

USAical Scho

92

II.
T he Identification of Endogenous Signaling
Lipids with Cannabimimetic Activity
192
III.
Id entification of Additional N-Acyl Amides 1 93
IV.
N -Arachidonoyl Glycine Biological Activity 1 94
V.
N -Arachidonoyl Glycine Biosynthesis 1 94
VI.
N -Palmitoyl Glycine Biological Activity 1 95
VII.
N -Palmitoyl Glycine Biosynthesis 1 97
VIII.
P alGly Metabolism 1 97
IX.
Id entification and Characterization of Additional Members
of the N-Acyl Glycines
198
X.
B iological Activity of Novel N-Acyl Glycines 2 00
XI.
C onclusions 2 01
Refe
r ences 2 03
Abstract

Discovery of the endogenous cannabinoid and N-acyl amide, anandamide

(N-arachidonoyl ethanolamine), paved the way for lipidomics discoveries in

the growing family of N-acyl amides. Lipidomics is a field that is broadening

our view of the molecular world to include a wide variety of endogenous lipid

signaling molecules. Many of these lipids will undoubtedly provide new

insights into old questions while others will provide broad platforms for new

questions. J Michael Walker’s last 8 years were dedicated to this search and

he lived long enough to see 54 novel lipids isolated from biological tissues in

his laboratory. Here, we summarize the biosynthesis, metabolism and biological

activity of two of the family of N-acyl glycines, N-arachidonoyl glycine and

r Inc.

rved.

ol,

191

192 Heather B. Bradshaw et al.

N-palmitoyl glycine, and introduce four additional members: N-stearoyl glycine,

N-linoleoyl glycine, N-oleoyl glycine, and N-docosahexaenoyl glycine. Each of

these compounds is found throughout the body at differing levels suggesting

region-specific functionality and at least four of the N-acyl glycines are regu-

lated by the enzyme fatty acid amide hydrolase. The family of N-acyl glycines

presented here is merely a sampling of what is to come in the continuing

discovery of novel endogenous lipids.

I. Historical View of Lipid Signaling Discoveries

The use of natural products for a variety of ailments in folklore medi-cine attracted scientists to explore their mechanisms of bioactivity (Caterinaet al., 1997; Mechoulam and Gaoni, 1965; Naef et al., 2003; Urca et al.,1977). The finding that plant-derived active ingredients such as the opium-derived morphine and cannabis-derived D9-hydrotetracannabinol (D9-THC; cannabinoids) that had a wide range of biological activity promotedthe identification of novel endogenous cannabinoid-like signalingmoleculesand pathways in the mammalian nervous system that would likewise drivethese actions. The discovery of these signaling molecules has opened newavenues into research that continue to expand our knowledge of basicbiochemical functioning.

II. The Identification of Endogenous Signaling

Lipids with Cannabimimetic Activity

The first mammalian counterpart of the analgesic phytocannabinoidD9-THC was identified in porcine brain and named anandamide after theSanskrit word for bliss, ananda (also, N-arachidonoyl ethanolamine—AEA;Fig. 8.1A) [5]. Both exogenous and endogenous compounds were shown toactivate the G protein-coupled receptor (GPCR) cannabinoid receptor1 (CB1) and induced several physiological and behavioral outcomes includinghypothermia, analgesia, hypoactivity, and catalepsy (Devane et al., 1992;Mechoulam and Gaoni, 1965). Additional characteristics of AEA includedbinding to the GPCR cannabinoid receptor 2 (CB2) and activating thetransient receptor potential vanilloid type-1 channel (Felder et al., 1995;Ross, 2003). Furthermore, unique retrograde signaling properties affectingrelease of classical neurotransmitters were revealed (Elphick and Egertova,2001; Ueda et al., 2005). A second endogenous cannabinoid 2-arachidonoylglycerol (2-AG) binding to both cannabinoid receptors was later identifiedin rat brain and canine gut (Mechoulam et al., 1995; Sugiura et al., 1995).

NH

O

OH

N-arachidonoyl ethanolamine(anandamide; AEA)

A

O

NH

OH

ON-arachidonoyl glycine

(NAGly)

B

NH

O

OHD

DD

D

D4-AEA

C

NH

O DDOH

OD2-NAGly

D

Figure 8.1 Molecular structures of N-arachidonoyl ethanolamine (AEA; A) andN-arachidonoyl glycine (NAGly; B). (C) Deuterium-labeled AEA in which the deu-teriums are located on the ethanolamine moiety. (D) Deuterium-labeled NAGly inwhich the deuteriums are on the glycine moiety from the theoretical conversion of AEAto NAGly via the initial step of alcohol dehydrogenase.

Novel Endogenous N-Acyl Glycines 193

Together these two endogenous lipids paved the way for the identificationof a family of lipids often referred to as endocannabinoids.

III. Identification of Additional N-Acyl Amides

Burstein et al. (1997) suggested that N-arachidonoyl glycine (NAGly;Fig. 8.1B) was a putative endogenous compound in 1997. The methodol-ogies used in the isolation and measurements of AEA in biological samples(lipid extractions and HPLC/MS/MS; Walker et al., 1999) enabled Walkerand colleagues to search for other N-acyl amides of similar structure, whichwere hypothesized to have similar function. Huang et al. (2001) were able toisolate three novel N-acyl amide molecules in the brain and periphery:NAGly, N-arachidonoyl GABA, and N-arachidonoyl alanine. Further


work from theWalker laboratory identifiedN-oleoyl dopamine,N-stearoyldopamine, N-palmitoyl dopamine, and N-arachidonoyl dopamine (Chuet al., 2003; Huang et al., 2002). Other research groups identified andcharacterized N-arachidonoyl serine (Milman et al., 2006) and the N-acyltaurines (McKinney and Cravatt, 2006) adding support to the hypothesisthat there is a potentially large family of N-acyl amides that are putativesignaling molecules with a wide range of biological activity (for review,see Bradshaw and Walker, 2005).

IV. N-Arachidonoyl Glycine Biological Activity

NAGly was originally synthesized as part of a structure activity rela-tionship study of the endocannabinoid AEA (Burstein et al., 1997; Sheskinet al., 1997). NAGly differs from AEA by the oxidation state of the carbon bto the amido nitrogen (Fig. 8.1B); a modification that drastically reduces itsactivity at both cannabinoid receptors (Sheskin et al., 1997). NAGlyproduces antinociceptive and anti-inflammatory effects in a variety of painmodels (Burstein et al., 2000; Huang et al., 2001; Succar et al., 2007; Vuonget al., 2008). Studies by Kohno et al. (2006) found that low concentrations(EC50 � 20 nM) of NAGly activate GPR18, an orphan GPCR. Consistentwith the anti-inflammatory effects of NAGly, GPR18 is highly expressed inperipheral blood leukocytes and several hematopoietic cell lines. Bursteinet al. (2000) showed that at low concentrations NAGly induces proliferationof T cells, but suppresses production of IL-1b. NAGly also reduces prolif-eration of the Caco-2, human rectal carcinoma cell line (Gustafsson et al.,2009). In pancreatic b-cells, NAGly caused intracellular calcium mobiliza-tion and insulin release (Ikeda et al., 2005). This effect was blocked bythe L-type voltage-gated channel nitredipine (1 mM) or in the absence ofextracellular calcium. Additionally,NAGly inhibited the glycine transporter,GLYT2a through direct, noncompetitive interactions (Wiles et al., 2006).These data support the hypothesis that NAGly is an endogenous signalingmolecule with multiple biological activities.

V. N-Arachidonoyl Glycine Biosynthesis

Unlike the acyl glycerols and N-acyl ethanolamines, the biosynthesisof N-acyl glycines cannot logically be derived from phospholipid biochem-istry. Two primary pathways for the biosynthesis of the NAGly have beenproposed: (1) conjugation of arachidonyl CoA and glycine (Burstein et al.,


2002; Huang et al., 2001; McCue et al., 2008) and (2) oxygenation ofN-arachidonoyl ethanolamine via the sequential enzymatic reaction ofalcohol dehydrogenase (ADH) and aldehyde dehydrogenase (Bursteinet al., 2000).

Huang et al. (2001) proposed that NAGly is synthesized by the conden-sation of arachidonic acid (AA) with glycine based upon the formation ofdeuterated NAGly following incubations of brain membranes with deuter-ated AA and deuterated glycine. McCue et al. (2008) demonstrated thatNAGly is formed via cytochrome c acting on arachidonoyl CoA and glycinein support of this conjugation pathway. We have additional data to supportthis hypothesis; however, the AA that conjugates with glycine appears to bea result of the hydrolysis of AEA (Bradshaw et al., 2009). This evidenced byresults that show deuterium-labeled AEA (labeled on that arachidonoylchain) incubated in C6 glioma cells is converted to deuterium-labeledNAGly (Bradshaw et al., 2009). This pathway is blocked by the fatty acidamide hydrolase (FAAH) inhibitor, URB597 in vivo and in vitro (Bradshawet al., 2009). The paper by Mueller in this special edition suggests amitochondrial synthesome for the biosynthesis of oleamide, an importantlipid signaling acyl amine (Huitron-Resendiz et al., 2001; Martinez-Gonzalez et al., 2004). It is possible that NAGly biosynthesis is part of thismitochondrial enzyme complex and that the hydrolysis of AEA is a rate-limiting step.

An alternative pathway was proposed by Burstein et al. (2000) whospeculated that NAGly is produced by the oxidation of the ethanolaminein AEA, presumably through ADH. We have evidence to support thispathway as well in that deuterium-labeled AEA (deuterium on the ethanol-amine moiety; Fig. 8.1C) incubated in RAW264.7 cells is converted todeuterium-labeled NAGly (Fig. 8.1D; Bradshaw et al., 2009). This pathwayis illustrated by Mueller and Driscoll in this volume to demonstrate thepotential biosynthesis of N-oleoyl glycine from N-oleoyl ethanolamine.Therefore, there is evidence for both proposed pathways and they bothhave an N-acyl ethanolamine as the precursor molecule.

VI. N-Palmitoyl Glycine Biological Activity

An additional member of the N-acyl glycine family was identified inthe Walker group. Rimmerman et al. (2008) showed that N-palmitoylglycine (PalGly; Fig. 8.2A) is produced throughout the body and plays arole in sensory neuronal signaling. The authors showed that PalGly isproduced following cellular stimulation and occurs in high levels in ratskin and spinal cord. PalGly was upregulated in FAAH knockout (KO)

N-palmitoyl glycine(PalGly)

A

OH

O

NH

O

NH

O

O

OH

N-linoleoyl glycine(LinGly)

D

N-stearoyl glycine(StrGly)

OHNH

O

O

C

OHNH

O

O

N-oleoyl glycine(OlGly)

B

NH

O

O

OHE

N-docosahexaenoyl glycine(DocGly)

Figure 8.2 Molecular structures of five N-acyl glycines: (A) N-palmitoyl glycine(PalGly), which was recently identified (Rimmerman); four other N-acyl glycinesidentified here: (B) N-oleoyl glycine (OlGly), (C) N-stearoyl glycine (StrGly),(D) N-linoleoyl glycine (LinGly), and (E) N-docosahexaenoyl glycine (DocGly).


mice suggesting a pathway for enzymatic regulation. PalGly potently inhib-ited heat-evoked firing of nociceptive neurons in rat dorsal horn. In addi-tion, PalGly induced transient calcium influx in native adult DRG cells andin a DRG-like cell line (F-11). The effect of PalGly on the latter was


characterized by strict structural requirements, PTX sensitivity, and depen-dence on the presence of extracellular calcium. PalGly-induced calciuminflux was blocked by the nonselective calcium channel blockers rutheniumred, SKF96365, and La3þ. Furthermore, PalGly contributed to the produc-tion of nitric oxide (NO) through calcium-sensitive nitric oxide synthase(NOS) enzymes present in F-11 cells, and was inhibited by the NOSinhibitor 7-NI.

VII. N-Palmitoyl Glycine Biosynthesis

PalGly is comprised of an 18-carbon saturated fatty acid that is amide-linked to glycine (Fig. 8.2A) and, therefore, has structural similarities to thephospholipid-derived N-acyl ethanolamines. Several biosynthetic pathwaysfor the production of PalGly are possible. Historically glycine conjugationwas investigated in the context of glycineN-acylase (Schachter and Taggart,1954). The enzyme was purified from the mitochondria of bovine liver, wasglycine specific, and active with aliphatic short and medium carbon chains(2–10 carbons), and aromatic acyl thioesters, yielding only short andmedium chain N-acyl glycines (Schachter and Taggart, 1954). Anotherglycine-conjugating enzyme, bile acid CoA:amino acid N-acyl transferase(BACAT), was found in microsomes and peroxisomes and shown to con-jugate bile acids mainly to glycine and taurine amino acids (O’Byrne et al.,2003). Furthermore, it was shown that human BACAT can conjugatesaturated 16–20 carbon fatty acids to glycine in vitro. The enzyme wasfound in liver and gallbladder mucosa with lower expression in skin andlung. However, the activity of BACATwith fatty acid CoAs was only 20% ofthe activity reported for bile acids. Recently, Mueller and Driscoll (2007)demonstrated the production of several acyl glycines (including PalGly) via theenzyme cytochrome c acting in the presence of hydrogen peroxide, glycine,and acyl CoAs in vitro.

Based on its structural similarity to NAGly, the alcohol dehydrogenasebiosynthetic scheme may also be a viable biosynthetic route. Further inves-tigations are needed to determine the production of PalGly via this pathway.

VIII. PalGly Metabolism

We recently observed that both the FAAH inhibitor URB597treated rats and FAAH KO mice have increased brain levels of PalGly(Rimmerman et al., 2008). These findings suggest that FAAH may be a


major metabolic pathway for the hydrolysis of saturated fatty acid glycinemolecules. In vitro metabolism of PalGly was demonstrated using a bacterialcytochrome P450 (CYPBM-3). PalGly bound the enzyme with higheraffinity than any other tested compound. The products of the enzymaticoxidation were o-1-, o-2-, and o-3-monohydroxylated metabolites ofPalGly (Haines et al., 2001).

IX. Identification and Characterization of

Additional Members of the N-Acyl Glycines

The recognition of the growing family of N-acyl amides (Farrell andMerkler, 2008; Tan et al., 2006) has been made possible by the developingfield of lipidomics. Lipidomics is the lipid corollary to proteomics forproteins with the exception that there is no genomic template fromwhich to predict lipids in the way that novel proteins are extrapolatedfrom the genome. The lipidomics of the N-acyl amides is based on theknowledge that there are a certain number of predominate fatty acids (acylchains) and amines (e.g., amino acids) that could possibly form conjuga-tions. Here, using lipidomics approaches similar to those used to identifyPalGly (Rimmerman et al., 2008), we continue with the identification ofthe endogenous N-acyl glycines with the isolation and characterization ofN-oleoyl glycine (OlGly; Fig. 8.2B), N-stearoyl glycine (StrGly; Fig. 8.2C),N-linoleoyl glycine (LinGly; Fig. 8.2D), and N-docosahexaenoyl glycine(DocGly; Fig. 8.2E).

Comparisons of a partially purified brain matrix and the product ion scanof a synthesized OlGly standard demonstrate that there is an identicalmolecular species of lipid compound in the mammalian brain (Fig. 8.3).Equivalent analyses were performed for each of the additional N-acylglycines listed and the molecular ion and fragment ions in parts per million(ppm) for each is shown in Table 8.1.

To determine the distribution of each of these N-acyl glycines through-out the body, extraction, purification, and HPLC/MS/MS methodswere optimized for each compound using the synthetic standards with amethodology previously described (Rimmerman et al., 2008). Figure 8.4shows an example of a chromatogram of an OlGly standard and of thisHPLC/MS/MSmethod used on purified spinal cord extracts. Twelve tissuetypes (skin, lung, spinal cord, ovary, kidney, liver, spleen, brain, smallintestine, uterus, testes, and heart) were subjected to lipid extraction andpartial purification as previously outlined for PalGly and the amounts of

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N-oleoyl glycine standard

Mass/z

Inte

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(C

PS

) 247.2419

265.2524

265.2536

247.2431

76.0393

76.0393

340.2846

340.2842

Figure 8.3 Mass spectrums of brain lipid extract match those of synthetic N-oleoylglycine (OlGly). Product ion scan mass spectrum of the OlGly synthetic standard(gray peaks) positively charged molecular ion in which the arrows are directed to thepeaks of the proposed molecular and fragment ions and labeled with the calculatedexact mass. The black peaks indicated the product ion scan mass spectrum of the brainlipid extract tuned to the positively charged OlGly molecular ion in which the arrowsare directed to the peaks of the proposed molecular and fragment ions and labeled withthe calculated exact mass.


each novel lipid quantified (Fig. 8.5A–D). StrGly had the highest levels ofthe four novelN-acyl glycines reported here, with levels that were similar toPalGly reported earlier (Rimmerman et al., 2008). Additionally, like PalGly,but unlike NAGly, the levels in the skin, lung, and spinal cord were thehighest in StrGly, OlGly, and DocGly. LinGly levels differed in that therelative levels in the spinal cord were much less than the other N-acylglycines measured here. The fairly ubiquitous nature of these moleculessuggests that they have potential roles either as signaling molecules or asprecursors to other bioactive lipids or both. The identification of OlGly asan endogenous molecule that is measured throughout the body lendsevidence to the hypothesis that it is produced endogenously and, therefore,available to be the precursor for oleamide as summarized in this volume byMueller and Driscoll.

Finally, the metabolism of these novel N-acyl glycines was examinedafter the systemic injection of the FAAH inhibitor, URB597 (Piomelli et al.,2006). Levels of each N-acyl glycine were measured from rat striatum aftersystemic vehicle (DMSO) or URB597 i.p. injection (3 mg/kg). We foundthat both StrGly and OlGly were significantly increased in striatum after theFAAH blocker was present for 30 min (Fig. 8.6). LinGly levels were not

Table 8.1 Molecular and fragmentation ions of N-acyl glycines

Molecular and fragment ions

(ppm difference from

theoretical exact mass) Proposed formulae Proposed fragmentation

N-oleoyl glycine

340.2846 (�0.061) C20H38NO3 MHþ

294.2801 (�9.9) C19H36NO MHþ–CH2O2

265.2536 (�10.2) C18H33O MHþ–C2H5NO2

247.2431 (�11.0) C18H31 MHþ–C2H7NO3

76.0393 (�0.065) C2H6NO2 MHþ–C18H32O

N-stearoyl glycine

342.3001 (2.1020) C20H40NO3 MHþ

324.2970 (20.7994) C20H38NO2 MHþ–H2O

296.2993 (13.3642) C19H38NO MHþ–CH2O2

267.2636 (19.4229) C18H35O MHþ–C2H5NO2

76.0391 (9.9097) C2H6NO2 MHþ–C18H34O

N-linoleoyl glycine

338.2703 (3.9294) C20H36NO3 MHþ

245.2203 (�24.7841) C18H29 MHþ–C2H7NO3

76.0401 (10.4556) C2H6NO2 Mþ–C18H30O

N-docosahexaenoyl glycine

336.2681 (�2.2543) C24H36NO3 MHþ

76.0396 (3.38802) C2H6NO2 MHþ–C22H30O

Mass measurements of the molecular and fragment ions as measured using nano-HPLC coupled to a C18capillary column with a QSTAR pulsar Q-TOF detector. Proposed formulae of these ionswere extrapolated by the fragmentation patterns and determinations for four putative endogenousN-acyl glycines were found in lipid extracts of murine brain and liver. The parts per million (ppm)differences from the theoretical exact masses of each molecular ion and fragment ion are represented inparenthesis.


detected in striatum and DocGly levels were just at detection limits and nosignificant differences were measured. This demonstrates that these endog-enous N-acyl glycines are metabolically regulated by FAAH. The biosyn-thesis of these molecules is still under investigation.

X. Biological Activity of Novel N-Acyl Glycines

There is developing evidence for biological activity of these novelN-acyl glycines. Chaturvedi et al. (2006) suggest that OlGly possess biologicactivity that is independent of its conversion to oleamide. Recent data by

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per

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Figure 8.4 Chromatograms of N-oleoyl glycine (OlGly) and spinal cord matrix.A 100 fmol standard of OlGly was analyzed using the API 3000 LC/MS/MS MRMmethod that optimizes on the molecular ion 338 and the fragment ion 74 in negative ionmode and a chromatographic gradient optimized for C18 column retention and releaseof the standard (gray line) and an overlay of a chromatogram from the injection of amethanolic extract partially purified on C18 solid phase extraction columns using thesame MRM method (black line).


Burstein et al. (2007) demonstrate that NAGly, DocGly, and LinGly sup-press proliferation of the murine macrophage cell line, RAW264.7, whereasOlGly and PalGly had no effect. Additionally, his work shows that NAGly,DocGly, and LinGly increase PGJ2 immunoreactivity, whereas again OlGlyand PalGly had no effect.

XI. Conclusions

Lipidomics is a field that is broadening our view of the molecular worldto include growing numbers of lipid signaling molecules. Many of theselipids will undoubtedly provide new insights into old questions while otherswill provide broad platforms for new questions. The family of N-acylglycines presented here is merely a sampling of what is to come in thediscovery of novel lipids. Basic combinatorial math of as few as 7 fatty acidchains and 20 amino acids yields 140 novel N-acyl amino acids. MichaelWalker’s last 8 years were dedicated to this search and he lived long enoughto see 54 novel lipids elucidated in biological tissue in his laboratory. Hisdream lives on in all those he trained and in the larger community of scientistswho continue to isolate and characterize novel endogenous lipids.

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Figure 8.5 N-acyl glycines are found throughout the CNS and body. Constitutive production of N-oleoyl glycine (OlGly), N-stearoyl glycine(StrGly), N-linoleoyl glycine (LinGly), and N-docosahexaenoyl glycine (DocGly) was measured in partially purified lipid extractsusing LC/MS/MSoptimized to each of the synthetic standards. As a matter of weight standardization, dry tissue weights were estimated from the ratio of lyophilized towet weight of each type of tissue that underwent methanolic lipid extraction and partial purification on C18 solid phase extraction columns directlyafter dissection. The 12 types of tissue are listed on the x-axis in relative order of production per gram dry weight.

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Figure 8.6 FAAH metabolism of acyl glycines. Comparison of production levels ofN-stearoyl glycine (StrGly), N-oleoyl glycine (OlGly), N-linoleoyl glycine (LinGly),and N-docosahexaenoyl glycine (DocGly) in rat striatum: Vehicle (gray bars) and3 mg/kg URB597 i.p. (black bars). Brains were removed and striatum dissected30 min after injection. Tissue underwent methanolic extraction and partial purificationon C18 solid phase extraction columns before eluants were measured with LC/MS/MSas outlined in Figs. 8.3–8.5.


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