ARTICLESAnandamide, the naturally occurring amide of arachidonic acid with ethanolamine, meets all key criteria of an endogenous cannabinoid substance 1 : it is released on demand by stimulated neurons 2,3 ; it activates cannabinoid receptors with high affinity 1 ; and it is rapidly eliminated through a two-step process consisting of carrier-mediated transport followed by intracellular hydrolysis 2,4 . Anandamide hydrolysis is catalyzed by the enzyme fatty acid amide hydrolase (FAAH), a membrane-bound serine hydrolase 5,6 that also cleaves other bioactive fatty acid ethanolamides such as oleoylethanolamide 7 and palmitoylethanolamide 8 . Mutant mice lacking the gene encoding FAAH (Faah) cannot metabolize anandamide 9 and, although fertile and generally normal, show signs of enhanced anandamide activity at cannabinoid receptors such as reduced pain sensation 9 . This is suggestive that drugs targeting FAAH may heighten the tonic actions of anandamide, while possibly avoiding the multiple and often unwanted effects produced by ∆ 9 -tetrahydrocannabinol (∆ 9 -THC) and other direct-acting cannabinoid agonists 10,11 . To test this hypothesis, potent, selective and systemically active inhibitors of intracellular FAAH activity are needed. However, most current inhibitors of this enzyme lack the target selectivity and biological availability required for in vivo studies [12][13][14] , whereas newer compounds, though promising, have not yet been characterized 15,16 . Thus, the therapeutic potential of FAAH inhibition remains essentially unexplored. Lead identification and optimizationDespite its unusual catalytic mechanism 6 , FAAH is blocked by a variety of serine hydrolase inhibitors, including compounds with activated carbonyls 16 . Therefore we examined whether esters of carbamic acid such as the anti-cholinesterase agent carbaryl (compound 1; Table 1) might inhibit FAAH activity in rat brain membranes. Although compound 1 was ineffective, its positional isomer 2 produced a weak inhibition of FAAH (half-maximal inhibitory concentration (IC 50 ) = 18.6 ± 0.7 µM; mean ± s.e.m., n = 3), which was enhanced by replacing the N-methyl substituent with a cyclohexyl group (compound 3; IC 50 = 324 ± 31 nM). The aryl ester 4, the benzyloxyphenyl group of which can be regarded as an elongated bioisosteric variant of the naphthyl moiety of compound 2, inhibited the activity of FAAH with a potency (IC 50 = 396 ± 63 nM) equivalent to that of compound 3. A conformational analysis of compound 4 revealed families of accessible conformers differing mainly in the torsion angle around the O-CH 2 bond, with substituents in anti or gauche conformations (data not shown). As the latter conformations more closely resembled the shape of the naphthyl derivative 3, we hypothesized that they might be responsible for the interac-

Oleylethanolamide (OEA) is a naturally occurring lipid that regulates satiety and body weight. Although structurally related to the endogenous cannabinoid anandamide, OEA does not bind to cannabinoid receptors and its molecular targets have not been defined. Here we show that OEA binds with high affinity to the peroxisome-proliferator-activated receptor-alpha (PPAR-alpha), a nuclear receptor that regulates several aspects of lipid metabolism. Administration of OEA produces satiety and reduces body weight gain in wild-type mice, but not in mice deficient in PPAR-alpha. Two distinct PPAR-alpha agonists have similar effects that are also contingent on PPAR-alpha expression, whereas potent and selective agonists for PPAR-gamma and PPAR-beta/delta are ineffective. In the small intestine of wild-type but not PPAR-alpha-null mice, OEA regulates the expression of several PPAR-alpha target genes: it initiates the transcription of proteins involved in lipid metabolism and represses inducible nitric oxide synthase, an enzyme that may contribute to feeding stimulation. Our results, which show that OEA induces satiety by activating PPAR-alpha, identify an unexpected role for this nuclear receptor in regulating behaviour, and raise possibilities for the treatment of eating disorders.

Oleylethanolamide (OEA) is a natural analogue of the endogenous cannabinoid anandamide. Like anandamide, OEA is produced in cells in a stimulus-dependent manner and is rapidly eliminated by enzymatic hydrolysis, suggesting a function in cellular signalling 1. However, OEA does not activate cannabinoid receptors and its biological functions are still unknown 2. Here we show that, in rats, food deprivation markedly reduces OEA biosynthesis in the small intestine. Administration of OEA causes a potent and persistent decrease in food intake and gain in body mass. This anorexic effect is behaviourally selective and is associated with the discrete activation of brain regions (the paraventricular hypothalamic nucleus and the nucleus of the solitary tract) involved in the control of satiety. OEA does not affect food intake when injected into the brain ventricles, and its anorexic actions are prevented when peripheral sensory fibres are removed by treatment with capsaicin. These results indicate that OEA is a lipid mediator involved in the peripheral regulation of feeding

Summary The association between fat consumption and obesity underscores the need to identify physiological signals that control fat intake. Previous studies have shown that feeding stimulates small-intestinal mucosal cells to produce the lipid messenger oleoylethanolamide (OEA) which, when administered as a drug, decreases meal frequency by engaging peroxisome proliferator-activated receptors-α (PPAR-α). Here we report that duodenal infusion of fat stimulates OEA mobilization in the proximal small intestine, whereas infusion of protein or carbohydrate does not. OEA production utilizes dietary oleic acid as a substrate and is disrupted in mutant mice lacking the membrane fatty-acid transporter CD36. Targeted disruption of CD36 or PPAR-α abrogates the satiety response induced by fat. The results suggest that activation of small-intestinal OEA mobilization, enabled by CD36-mediated uptake of dietary oleic acid, serves as a molecular sensor linking fat ingestion to satiety.

Fatty acid amide hydrolase (FAAH) is an intracellular serine enzyme that catalyzes the hydrolysis of bioactive fatty acid ethanolamides such as anandamide and oleoylethanolamide (OEA). Genetic deletion of the faah gene in mice elevates brain anandamide levels and amplifies the effects of this endogenous cannabinoid agonist. Here, we show that systemic administration of the selective FAAH inhibitor URB597 (cyclohexyl carbamic acid 3Ј-carbamoyl-biphenyl-3-yl ester; 0.3 mg/kg i.p.) increases anandamide levels in the brain of rats and wild-type mice but has no such effect in FAAH-null mutants. Moreover, URB597 enhances the hypothermic actions of anandamide (5 mg/kg i.p.) in wild-type mice but not in FAAH-null mice. In contrast, the FAAH inhibitor does not affect anandamide or OEA levels in the rat duodenum at doses that completely inhibit FAAH activity. In addition, URB597 does not alter the hypophagic response elicited by OEA (5 and 10 mg/kg i.p.), which is mediated by activation of peroxisome proliferator-activated receptor type-␣. Finally, exogenously administered OEA (5 mg/kg i.p.) was eliminated at comparable rates in wild-type and FAAH Ϫ/Ϫ mice. Our results indicate that URB597 increases brain anandamide levels and magnifies anandamide responses by inhibiting intracellular FAAH activity. The results also suggest that an enzyme distinct from FAAH catalyzes OEA hydrolysis in the duodenum, where this lipid substance acts as a local satiety factor.The endogenous cannabinoid anandamide (Devane et al., 1992;Di Marzo et al., 1994), the satiety factor oleoylethanolamide (OEA) (Rodríguez de Fonseca et al., 2001;Fu et al., 2003), and the analgesic and anti-inflammatory factor palmitoylethanolamide (PEA) (Kuehl et al., 1957;Calignano et al., 1998;Jaggar et al., 1998) are all members of the fatty acid ethanolamide (FAE) family of lipid mediators. FAEs are found in most mammalian tissues, where they are thought to be stored as the phospholipid precursor N-acylphosphatidylethanolamine (Schmid et al., 1996;Piomelli, 2003) and to be produced in a stimulus-dependent manner by activation of an N-acylphosphatidylethanolamine-specific phospholipase D (Okamoto et al., 2004).After release from cells, polyunsaturated FAEs such as anandamide may be eliminated via a two-step process consisting of high-affinity transport into cells (Di Marzo et al., 1994;Beltramo et al., 1997;Hillard et al., 1997;Fegley et al., 2004;Ortega-Gutierrez et al., 2004) followed by intracellular degradation, catalyzed by fatty acid amide hydrolase (FAAH) (Cravatt and Lichtman, 2002). On the other hand, monounsaturated and saturated FAEs such as OEA and PEA are poor substrates for anandamide transport, and their inactivation may be primarily mediated by intracellular hydrolysis catalyzed by FAAH and/or by a distinct but as-yet-uncharacterized FAE amidase that operates at acid pH values (Ueda et al., 1999(Ueda et al., , 2001 ABBREVIATIONS:OEA, oleoylethanolamide; PEA, palmitoylethanolamide; FAE, fatty acid ethanolamide; FAAH, fatty acid amide hydrolase...

Oleoylethanolamide (OEA) is a lipid mediator that inhibits food intake by activating the nuclear receptor peroxisome proliferator-activated receptor-␣. In the rodent small intestine OEA levels decrease during food deprivation and increase upon refeeding, suggesting that endogenous OEA may participate in the regulation of satiety. Here we show that feeding stimulates OEA mobilization in the mucosal layer of rat duodenum and jejunum but not in the serosal layer from the same intestinal segments in other sections of the gastrointestinal tract (stomach, ileum, colon) or in a broad series of internal organs and tissues (e.g. liver, brain, heart, plasma). Feeding also increases the levels of other unsaturated fatty acid ethanolamides (FAEs) (e.g. linoleoylethanolamide) without affecting those of saturated FAEs (e.g. palmitoylethanolamide). Feeding-induced OEA mobilization is accompanied by enhanced accumulation of OEA-generating N-acylphosphatidylethanolamines (NAPEs) increased activity and expression of the OEA-synthesizing enzyme NAPE-phospholipase D, and decreased activity and expression of the OEAdegrading enzyme fatty acid amide hydrolase. Immunostaining studies revealed that NAPE-phospholipase D and fatty acid amide hydrolase are expressed in intestinal enterocytes and lamina propria cells. Collectively, these results indicate that nutrient availability controls OEA mobilization in the mucosa of the proximal intestine through a concerted regulation of OEA biosynthesis and degradation.The gastrointestinal tract controls key aspects of feeding behavior through both neuronal and humoral mechanisms. Among the regulatory signals released by the gut, peptides such as cholecystokinin (CCK), 3 glucagon-like peptide-1, and ghrelin have attracted the most attention (1), but recent evidence indicates that lipid-derived messengers such as oleoylethanolamide (OEA) may also be involved (2). OEA is synthesized in the small intestine of various vertebrate species, where its levels decrease during food deprivation and increase upon refeeding (3-5). The possibility that these fluctuations represent a satiety signal is suggested by experiments in rodents which show that pharmacological administration of OEA delays meal initiation and prolongs the interval between successive meals, resulting in a persistent inhibition of food intake (3, 6 -8). These anorexiant effects are strikingly different from those elicited by traditional satiety factors such as CCK, which reduce meal size without affecting the interval between meals (9). Moreover, the hypophagic actions of OEA differ from those exerted by glucagon-like peptide-1 (10) and corticotropin-releasing factor (11) in that they are not accompanied by behavioral signs of malaise and anxiety or by changes in circulating corticosterone levels (3, 12). The molecular mechanism through which OEA inhibits feeding has been partially elucidated. In vitro studies have shown that OEA is a high affinity agonist of peroxisome proliferator-activated receptor-␣ (PPAR-␣) (13), a nuclear receptor pres...

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Oleoylethanolamide (OEA) is a structural analog of the endogenous cannabinoid anandamide, which does not activate cannabinoid receptors. The biosynthesis of OEA in rat small intestine is increased by feeding and reduced by fasting. Moreover, OEA decreases food intake in food-deprived rats via a mechanism that requires intact sensory fibers (Rodríguez de Fonseca, 2001). These results suggest that OEA may contribute to the peripheral regulation of feeding. In the present study, we have investigated the effects of systemic OEA administration (1-20 mg/kg, intraperitoneal) on meal pattern in free-feeding and food-deprived rats. In free-feeding animals, OEA delayed feeding onset in a dose-dependent manner, but had no effect on meal size or postmeal interval. In food-deprived animals, OEA both delayed feeding onset and reduced meal size. The selective effects of OEA in free-feeding rats are strikingly different from those of the serotonergic anorexiant d-fenfluramine (which delayed feeding and reduced meal size) and the intestinal peptide cholecystokinin (which reduced meal size). These results suggest that OEA may participate in the regulation of satiety and may provide a chemical scaffold for the design of novel appetite-suppressing medications.