An anatomical and temporal portrait of physiological substrates for fatty acid amide hydrolase.

Fatty acid amide hydrolase (FAAH) regulates amidated lipid transmitters, including the endocannabinoid anandamide and its N-acyl ethanolamine (NAE) congeners and transient receptor potential channel agonists N-acyl taurines (NATs). Using both the FAAH inhibitor PF-3845 and FAAH(−/−) mice, we present a global analysis of changes in NAE and NAT metabolism caused by FAAH disruption in central and peripheral tissues. Elevations in anandamide (and other NAEs) were tissue dependent, with the most dramatic changes occurring in brain, testis, and liver of PF-3845-treated or FAAH(−/−) mice. Polyunsaturated NATs accumulated to very high amounts in the liver, kidney, and plasma of these animals. The NAT profile in brain tissue was markedly different and punctuated by significant increases in long-chain NATs found exclusively in FAAH(−/−), but not in PF-3845-treated animals. Suspecting that this difference might reflect a slow pathway for NAT biosynthesis, we treated mice chronically with PF-3845 for 6 days and observed robust elevations in brain NATs. These studies, taken together, define the anatomical and temporal features of FAAH-mediated NAE and NAT metabolism, which are complemented and probably influenced by kinetically distinguishable biosynthetic pathways that produce these lipids in vivo.

Gemini reverse-phase C18 column (5 µm, 4.6 mm × 50 mm; Phenomonex) together with a precolumn (C18, 3.5 µm, 2 mm × 20 mm). Mobile phase A was composed of 95:5 v/v H 2 O-MeOH, and mobile phase B was composed of 65:35:5 v/v/v i -PrOH-MeOH-H 2 O. Ammonium hydroxide (0.1%) was included to assist in ion formation in negative-ionization mode. The fl ow rate was 0.5 ml/ min, and the gradient consisted of 1.5 min 0% B, a linear increase to 100% B over 5 min, followed by an isocratic gradient of 100% B for 3.5 min before equilibrating for 2 min at 0% B (12 min total per sample). MS analysis was performed with an electrospray ionization (ESI) source. The capillary voltage was set to 3.0 kV and the fragmentor voltage was set to 70 V. The drying gas temperature was 350°C, the drying gas fl ow rate was 10 l/min, and the nebulizer pressure was 35 psi. d 8 -Arachidonic acid was quantifi ed by measuring the area under the peak in comparison to the PDA standard.

Measurement of tissue lipids
Tissues were weighed and subsequently Dounce homogenized in 2:1:1 v/v/v CHCl 3 -MeOH-Tris, pH 8.0 (8 ml) containing standards for NAEs and NATs (20 pmol d 4 -N -palmitoylethanolamine, Despite the aforementioned advances in our understanding of physiological substrates for FAAH, a comprehensive inventory of these lipids across multiple tissues from wild-type versus FAAH-disrupted animals has not yet been performed. Here, we describe a temporal and anatomical atlas of FAAH substrates following acute (i.e., pharmacological) versus chronic (i.e., genetic) blockade of this enzyme in mice. We fi nd that FAAH control over FA amide substrates is tissue specifi c and, in some cases, infl uenced by temporal factors that probably refl ect differences in the rate of substrate biosynthesis. These discoveries thus lend further support to the hypothesis that multiple pathways exist for the biosynthesis of both NAEs and NATs in vivo.

Pharmacological inhibition of FAAH
PF-3845 was prepared as a 1 mg/ml saline-emulphor emulsion by vortexing, sonicating, and gently heating neat compound directly into an 18:1:1 v/v/v solution of saline-ethanol-emulphor. FAAH( +/+ ) mice were administered PF-3845 at a volume of 10 l/g weight for a fi nal dose of 10 mg/kg. After the indicated amount of time, mice were anesthetized with isofl uorane and killed by decapitation, and tissues were fl ash frozen in liquid nitrogen. Animal experiments were conducted in accordance with the guidelines of the Public Health Service Policy on Human Care and Use of Laboratory Animals, and of the Institutional Animal Care and Use Committee of The Scripps Research Institute.

Preparations of mouse tissue proteomes
Tissues were Dounce-homogenized in PBS, pH 7.5, followed by a low-speed spin (1,400 g , 5 min) to remove debris. The supernatant was then subjected to centrifugation (64,000 g , 45 min) to provide the cytosolic fraction in the supernatant and the membrane fraction as a pellet. The pellet was washed and resuspended in PBS buffer by sonication. Total protein concentration in each fraction was determined using a protein assay kit (Bio-Rad). Samples were stored at Ϫ 80°C until use.

Fluorophosphonate-rhodamine labeling of tissue proteomes
Tissue proteomes were diluted to 1 mg/ml in PBS, and fl uorophosphonate-rhodamine (FP-Rh) was added at a fi nal concentration of 1 µM in a 50 µl total reaction volume. After 30 min at 25°C, the reactions were quenched with 4× SDS-PAGE loading buffer, boiled for 5 min at 90°C, subjected to SDS-PAGE, and visualized in-gel using a fl atbed fl uorescence scanner (Hitachi).
Anandamide hydrolysis assays d 8 -Anandamide (100 µM) was incubated with tissue membranes (50 µg) in PBS (100 µl) at 37°C for 30 min. The reactions were quenched by the addition of 300 µl 2:1 v/v CHCl 3 -MeOH, doped with 5 nmol PDA, vortexed, then centrifuged (1,400 g , 3 min) to separate the phases. Thirty microliters of the resultant organic phase was injected onto an Agilent 1100 series liquid chromatography-MSD SL instrument. LC separation was achieved with a Pharmacological and genetic disruption of FA amide hydrolase (FAAH) in mice. A: Anandamide hydrolysis activity from tissue membranes prepared from control mice, mice treated with PF-3845 (10 mg/kg, i.p., 3 h), or FAAH( Ϫ / Ϫ ) mice. B, C: Serine hydrolase activity profi les using the active site-directed probe fl uorophosphonate-rhodamine (FP-Rh) from brain (B) or testis (C) membranes prepared from mice as described in EXPERIMENTAL PROCEDURES. FAAH migrates as an ‫ف‬ 60 kDa singlet, indicated by the arrowhead; protein samples were treated with PNGaseF to remove N -linked glycans prior to analysis. For A, ** P < 0.01; *** P < 0.001 for control versus PF-3845 or control versus FAAH( Ϫ / Ϫ ). Data are presented as the means ± standard deviation, n = 3-4/group. The capillary was set to 4 kV, the fragmentor was set to 100 V, the delta EMV was set to +300, and the collision energy was set to 11 V for NAE metabolites. The drying gas temperature was 350°C, the drying gas fl ow rate was 11/min, and the nebulizer pressure was 35 psi. NAEs were quantifi ed by measuring the area under the peak in comparison to the average area of the deuterated standards.
For measurements of NATs, LC separation was the same as for NAEs except that 0.1% ammonium hydroxide was included to assist in ion formation in negative-ionization mode. The following MS parameters were used to measure the indicated NATs (precursor ion, product ion): C16:0 (362.6, 80), C18:0 (390.6, 2 pmol d 4 -anandamide, and 100 pmol C15:0-NAT). The mixture was vortexed and then centrifuged (1,400 g , 10 min). The organic layer was removed, dried under a stream of N 2 , resolubilized in 2:1 v/v CHCl 3 -MeOH (120 µl), and 10 µl of this resolubilized lipid was injected onto an Agilent G6410B QQQ instrument. For measurements of NAEs, LC separation, mobile phase A, and mobile phase B were exactly the same as for the enzyme activity assays, except that 0.1% formic acid was included to assist in ion formation in positive ionization mode. The fl ow rate for each run started at 0.1 ml/min with 0% B. At 5 min, the solvent was immediately changed to 60% B with a fl ow rate of 0.4 ml/min and increased linearly to 100% B over 10 min. This was followed by an isocratic gradient of 100% B for 5 min at 0.5 ml/min before equilibrating for 3 min at 0% B at 0.5 ml/min (23 min total per sample). The following MS parameters were used to measure the indicated NAEs (precursor ion, product an ‫ف‬ 60 kDa FP-Rh-labeled protein in brain and testis of control mice, but not PF-3845-treated or FAAH( Ϫ / Ϫ ) mice ( Fig. 1B, C ). In other tissues, additional serine hydrolases with overlapping masses precluded an assessment of FAAH inactivation using gel-based profi ling with FP-Rh (see supplementary Fig. I). These tissue profi les, however, could still be used to confi rm the remarkable selectivity that PF-3845 displays for FAAH, because no additional FP-Rh-labeled serine hydrolases other than FAAH were affected by this compound.

Accumulation and distribution of NAEs in FAAH-disrupted animals
We next examined the effects of genetic or pharmacological blockade of FAAH on NAE accumulation in mouse tissues. Anandamide (C20:4 NAE) was highly elevated (>8-fold) in brain, liver, and testis of FAAH( Ϫ / Ϫ ) or PF-3845-treated mice, and was modestly elevated in some but not all of the other tissues analyzed ( Tables 1, 2 , Fig. 2 ). Curiously, the accumulation of anandamide following FAAH disruption was not correlated with the basal concentrations of this lipid or the FAAH enzyme itself. For instance, FAAH disruption caused dramatic elevations in anandamide in testis, but not kidney ( Table 1 ), despite both tissues possessing high basal anandamide concentrations ( Table 1 ) and FAAH activity ( Fig. 1 ).
Some organs with substantial FAAH activity, such as WAT, did not exhibit changes in NAEs following FAAH blockade ( Table 1 ). Conversely, other tissues that lack FAAH activity, such as heart, showed signifi cant (albeit modest) elevations in several NAEs in FAAH( Ϫ / Ϫ ) or PF-3845-treated mice.

FAAH activity in mouse tissues
We compared anandamide hydrolysis activity across a panel of tissues from FAAH(+/+) and ( Ϫ / Ϫ ) mice, as well as FAAH(+/+) mice treated with vehicle or the selective FAAH inhibitor PF-3845 (10 mg/kg, i.p., 3 h) ( 19 ). Anandamide hydrolysis rates did not differ between naïve and vehicle-treated FAAH(+/+) mice, so, for the sake of clarity, we merged these groups to give one consolidated control group.
Anandamide hydrolysis was highest in brain, testis, liver, kidney, and white adipose tissue (WAT) of FAAH(+/+) mice, with lower concentrations detected in spleen and lung, and negligible activity in brown adipose tissue and heart ( Fig. 1A ). Virtually all of the observed anandamide hydrolysis activity was ablated in tissues from FAAH( Ϫ / Ϫ ) mice or mice treated with PF-3845, indicating that FAAH is the principal enzyme responsible for anandamide hydrolysis in mammalian tissues, at least under the assay conditions employed. We also analyzed gel-resolvable, active serine hydrolases in these tissues using the serine hydrolasedirected, activity-based probe FP-Rh ( 20,21 ). Consistent with previous studies ( 22,23 ), active FAAH was detected as Fig. 3. Tissue-specifi c differences in NAE accumulation. A-C: Average fold-change in various NAEs between PF-3845-treated (10 mg/kg, i.p., 3 h) or FAAH( Ϫ / Ϫ ) mice versus control mice in brain (A), testis (B), and liver (C). The values used for this graph were obtained from Table 1 and Table 2. n.d., not detected .  Table 1 and Table 2 .  Such changes could be explained by transport of NAEs from distal sites of production to organs where FAAH is not expressed. Consistent with this model, most NAEs were signifi cantly elevated in plasma from FAAH( Ϫ / Ϫ ) or PF-3845treated mice ( Table 1 ). More generally, these fi ndings indicate that the amounts of FAAH activity displayed by tissues are not predictive of the changes in NAEs observed in these organs following FAAH disruption.

Accumulation and distribution of NATs in FAAH-disrupted animals
As has been observed in previous studies ( 17,18 ), longchain saturated NATs, such as C22:0, were highly elevated in brains of FAAH( Ϫ / Ϫ ) mice, whereas liver and kidney tissues from these animals showed large increases (5-100-fold) in polyunsaturated (C20:4, C22:6) NATs ( Tables 3, 4 , and Fig. 4 ). Other saturated and monounsaturated NATs were also elevated in liver, but to a more modest degree. In contrast, polyunsaturated NATs were unaltered, or, in the case of C20:4, paradoxically lower in brains from FAAH( Ϫ / Ϫ ) mice ( Table 4 ). Acute inhibitor treatment produced greater elevations in polyunsaturated NATs in liver than were observed in FAAH( Ϫ / Ϫ ) animals, suggesting that chronic FAAH blockade may feed back to inhibit NAT biosynthesis in this organ.
Whereas the predominant NATs in liver and brain of FAAH( Ϫ / Ϫ ) mice were polyunsaturated ( ‫ف‬ 5-10 nmol/g) and long-chain, saturated ( ‫ف‬ 700 pmol/g), respectively, shorter chain NATs were the major species that accumulated in lung, with C18:0 and C18:1 NATs showing the   Table 1 . chronic versus acute PF-3845-treated mice ( Table 2 ). These data contrast with the kinetic profi les for shorter chain and mono/poly-unsaturated NAEs, which accumulated quickly in brain and peripheral tissues following FAAH disruption and then plateaued at new steady-state concentrations that are apparently sustained throughout life [based on comparisons with FAAH( Ϫ / Ϫ ) mice]. The markedly different kinetic profi les observed for long-chain saturated versus shorter chain/unsaturated NAEs/NATs indicate that these subsets of FA amides are probably produced by distinct biosynthetic pathways.

DISCUSSION
Several previous studies have reported changes in NAE and NAT lipids in rodents with pharmacological or genetic disruptions of FAAH ( 6,12,17,18,24,25 ). Most of these reports focus on a single or select number of tissues, and few, if any, have compared acute versus chronic disruption of FAAH in a quantitative manner. Here, we set out to establish an anatomical inventory of NAE and NAT metabolism across a broad range of central and peripheral tissues from FAAH inhibitor-treated and FAAH( Ϫ / Ϫ ) mice. This analysis revealed that despite the presence of FAAH in most tissues, there were marked tissue-specifi c differences in the accumulation of both NAEs and NATs in FAAH-disrupted animals. Most tissues that express FAAH showed some elevations in a subset of the NAEs or NATs, but each had a distinct lipid profi le.
highest absolute concentrations ( ‫ف‬ 500 pmol/g) ( Table  3 ). In the remaining peripheral organs, the NATs as a group were largely unchanged except for one or two individual species in each organ. For example, testis and spleen each showed ‫ف‬ 4-fold and ‫ف‬ 5-fold elevations in C22:0 NAT and C22:6 NAT, respectively, but not the other NATs. NATs, like NAEs, were unchanged in WAT.
It is noteworthy that liver and plasma NAT profi les were quite similar ( Fig. 5A, B ). This fi nding could point to the existence of a NAT transport system that transfers NATs from liver to the circulation, where these lipids could then be delivered to other tissues. We speculate that this might occur in spleen, for instance, which displayed a NAT profi le similar that seen in liver, with principal elevations in polyunsaturated NATs.

Differences in FA amide amounts following acute versus chronic FAAH disruption
A curious disconnect was observed for NAT profi les in brain, where long-chain saturated NATs were highly elevated in FAAH( Ϫ / Ϫ ) mice but not in acutely FAAH-inhibited animals ( Fig. 4A ). This phenomenon has been reported previously and attributed to a potentially slow biosynthetic pathway for NATs in the nervous system ( 18 ). We set out to test this hypothesis by treating mice with PF-3845 chronically for 6 days (10 mg/kg, i.p., once-per-day dosing) and then measuring brain lipids. This treatment regime produced substantial increases in saturated NATs (e.g., C18:0, C22:0) that approximated the amounts observed in FAAH( Ϫ / Ϫ ) mice ( Table 4 ). Interestingly, a similar, albeit less-pervasive trend was observed for NAEs, where C22:0 NAE also accumulated to signifi cantly higher amounts in NAEs and NATs is strongly infl uenced by the host tissue. Such differences in FAAH substrate accumulation provide evidence for the existence of multiple biosynthetic pathways for NAEs and NATs in vivo, some of which are highly active (polyunsaturated NAE and NAT production in brain/testis and liver, respectively), whereas others are much slower (long-chain saturated NAE and NAT production in brain). The molecular characterization of these pathways promises to deliver new enzymatic targets whose perturbation should enrich our understanding of the physiological functions of individual branches of the FA amide family.
These differences may refl ect the presence of distinct sets of biosynthetic enzymes for NAEs and NATs, which may shape the rate of accumulation and acyl chain distribution of FAAH substrates. The production of long-chain saturated and monounsaturated NAEs in the brain has already been characterized to occur by the action of N -acylphosphatidylethanolamine phospholipase D (NAPE-PLD), an enzyme that liberates NAEs from an unusual class of N -acylated phospholipids ( 26,27 ). However, the pathways that produce other subsets of NAEs and NATs remain poorly characterized. Our data point to anatomical locations where these different biosynthetic pathways are probably operational. For instance, both brain and testis appear to possess an enzymatic route to rapidly generate polyunsaturated NAEs, including anandamide. That testis can furthermore accumulate anandamide without substantial elevations in shorter chain NAEs suggests that at least two additional NAPE-PLD-independent NAE biosynthetic pathways may exist, one for polyunsaturated NAEs and the other for shorter chain saturated and mono-unsaturated NAEs.
Similar complexity was found for NATs, where liver appears to contain a highly active biosynthetic pathway for polyunsaturated NATs, whereas brain has a much slower process for producing long-chain saturated NATs. In vitro evidence suggests that NATs may originate from the enzymatic conjugation of taurine with fatty acyl-CoAs, analogous to the formation of bile salts ( 18,28 ). If this is the endogenous pathway for NAT production in liver, it would be interesting to examine whether the acyl-CoA fraction of the liver metabolome shows selective decreases in arachidonoyl-and docosahexaenoyl-CoA in FAAH-disrupted animals. These lipids have been typically reported in the range of less than 10 nmol/g in liver ( 29,30 ), and in our data, the C20:4/C22:6 NATs increase by a comparable stoichiometry. We hypothesize that different enzymes catalyze the acyltransferase reaction in liver and brain to account for their markedly distinct NAT profi les following FAAH inactivation. Finally, the accumulation of NATs in brain tissue of FAAH-disrupted animals over the course of days rather than hours is a cogent reminder that slow metabolic pathways exist in mammals and may only reveal themselves following prolonged perturbations of up-or downstream enzymes.
We were particularly intrigued by the similarity of NAT profi les in liver and plasma from FAAH-disrupted animals, which showed nearly identical acyl chain distributions (differing only in the absolute concentration of each NAT). Such an observation is reminiscent of endocrines secreted by the liver, such as insulin-like growth factor-1 (IGF-1) ( 31 ) or thrombopoietin ( 32,33 ) and suggests that NATs may also function as endocrine-like signaling molecules. How NATs are released from liver and taken up into other tissues remains unknown, but they could be substrates for organic anion transporters, which are responsible for bile salt uptake ( 34 ).
In summary, our near-comprehensive anatomical portrait of physiological substrates for FAAH underscores key features of FA amide metabolism in mammals, emphasizing, in particular, that FAAH control over specifi c