1. Department of Pharmacology, University of California, Irvine, California 92697-4625, USA
2. Fundación Hospital Carlos Haya, Malaga, Spain
3. Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697-4625, USA
4. Institute of Medicinal Chemistry, University of Urbino, Italy

Correspondence to: Daniele Piomelli¹ Email: piomelli@uci.edu

Because OEA resembles natural compounds that activate PPARs, such as oleic acid^{3, 4, 5}, we considered that it may interact with a member of this nuclear receptor family^{6, 7, 8}. To test this idea, we engineered HeLa cells to express a luciferase reporter gene together with the ligand-binding domain (LBD) of human PPAR-, PPAR-/, PPAR- or retinoid-X receptor (RXR) fused to the yeast GAL4 DNA-binding domain⁹. In transactivation assays, OEA activated PPAR- with a half-maximal concentration (EC₅₀) of 120 1 nM and activated PPAR-/ with an EC₅₀ of 1.1 0.1 M (n = 16), whereas it had no effect on PPAR- or RXR (Fig. 1a).

Figure 1: OEA is a high-affinity PPAR- agonist.

a, b, Activation of human PPAR- by OEA. a, OEA activates human PPAR- (filled circles) and PPAR-/ (open triangles), but not PPAR- (filled squares) or RXR (open diamonds). b, Effects of OEA (filled circles), oleic acid (open squares), stearylethanolamide (filled triangles), myristylethanolamide (filled squares) and anandamide (open circles) on PPAR- activation. c, Saturation binding of [³H]OEA to purified mouse PPAR- LBD. Shown are total binding (filled diamonds), nonspecific binding (filled circles) and specific binding (filled triangles). The inset shows a Scatchard transformation (bound - [bound/free]) of the specific binding data. d, Competition binding of OEA to purified mouse PPAR- LBD. Results are the mean s.e.m. of 16 (a, b) or 10 (c, d) experiments.

High resolution image and legend (48K)

To study the structural selectivity of PPAR- activation, we tested several OEA analogues. As previously reported^{3, 4, 5}, oleic acid activated the receptor with micromolar potency (EC₅₀ = 10.3 0.21 M; n = 16), whereas stearylethanolamide, myristylethanolamide and anandamide¹⁰ had no effect (Fig. 1b). The synthetic agonists Wy-14643 (ref. 11) and GW7647 (ref. 12) engaged PPAR- with EC₅₀ values of 1.4 0.1 M and 150 20 nM, respectively (n = 5). Saturation binding studies showed that [³H]OEA associates with the purified LBD of mouse and human PPAR- (Fig. 1c), with dissociation constants (K_d) of 37.4 0.1 nM (mouse, n = 10) and 43.3 1.6 nM (human, n = 8). Binding competition experiments yielded a half-maximal inhibitory concentration (IC₅₀) for OEA of 120.0 10.7 nM (n = 10; Fig. 1d). The results indicate that OEA is a high-affinity agonist of PPAR-.

To test whether PPAR- activation contributes to the appetite-suppressing properties of OEA, we used mice deficient in PPAR- (PPAR-^-/- mice)^{13, 14}. OEA reduced feeding in wild-type mice, but not in PPAR-^-/- mice (Fig. 2a, b), although the two strains showed comparable tissue concentrations of OEA (Supplementary Table 1) and similar responses to other appetite suppressants, such as d-fenfluramine and cholecystokinin-8 (Fig. 2c). In addition to its short-term effects on feeding, OEA produces in rats a sustained inhibition of body weight gain¹. To determine the role of PPAR- in this response, we fed wild-type and PPAR-^-/- mice with a high-fat chow and, once they had become obese, treated them daily with OEA for 4 weeks. OEA reduced total food intake and suppressed body weight gain in wild-type mice, whereas it had no effect in PPAR-^-/- mice (Fig. 2d, e). Notably, whereas mutant mice receiving a standard chow maintain normal body weight^{13, 14}, those fed with a high-fat diet ate more and gained more weight than did wild-type mice (Fig. 2f). Thus, PPAR- expression may not be necessary only for the satiety-inducing and weight-reducing actions of OEA, but also for the maintenance of feeding homeostasis.

Figure 2: PPAR- mediates the appetite-suppressing and weight-reducing effects of OEA.

a–c, OEA reduces feeding in wild-type but not in PPAR-^-/- mice. a, b, Time course of the effects of OEA (filled squares; 10 mg per kg i.p.) and vehicle (open squares) on total food intake in wild-type (n = 6–11; a) and PPAR-^-/- (n = 5–8; b) mice. c, Effects of vehicle (V), d-fenfluramine (F; 4 mg per kg i.p.) and cholecystokinin-8 (C; 25 g per kg i.p.) on food intake in wild-type and PPAR-^-/- mice (n = 4–9). d, e, OEA reduces food intake and body weight gain in obese wild-type but not PPAR-^-/- mice. Shown are the effects of OEA (filled bars; 5 mg per kg i.p.) and vehicle (open bars) on food intake (d) and body weight gain (e). f, PPAR-^-/- mice fed with a high-fat diet develop hyperphagia. Food intake and body weight in wild-type and PPAR-^-/- mice before OEA treatment (n = 4–9). g, Effects of OEA (filled bars; 5 mg per kg i.p.) and vehicle (open bars) on serum cholesterol levels in obese wild-type and PPAR-^-/- mice. *P < 0.05 and **P < 0.001 (n = 6–8) by one-way ANOVA followed by Dunnett's test or Student's t-test (c–g).

High resolution image and legend (67K)

Does OEA modulate feeding through direct activation of PPAR-? To address this question, we used three synthetic PPAR- agonists of different potency^{11, 12}: Wy-14643 and GW7674, which engage PPAR- in vitro with potencies comparable to that of OEA, inhibited food intake in C57BL/6J mice (Fig. 3a), whereas the weaker agonist clofibrate did not (25–100 mg per kg body weight; data not shown). Meal pattern analyses showed that the effects of Wy-14643 and GW7647 were due to prolonged eating latency, rather than to changes in meal size or post-meal interval (Fig. 3b). This response is identical to that elicited by OEA (Fig. 3b) and is indicative of a satiety-inducing action².

Figure 3: PPAR- agonists mimic the appetite-suppressing effects of OEA.

a, Effects of vehicle (open squares), Wy-14643 (filled triangles; 40 mg per kg i.p.) and GW7647 (open circles; 20 mg per kg i.p.) on food intake in C57BL/6J mice (vehicle, n = 40; drugs, n = 4–7). b, Effects of vehicle (V), Wy-14643 (W; 40 mg per kg i.p.), GW7647 (G₁; 20 mg per kg i.p.) and OEA (O; 10 mg per kg i.p.) on feeding latency, first meal size (MS) and first post-meal interval (PMI) in C57BL/6J mice (vehicle, n = 40; drugs, n = 4–7). c, Effects of vehicle (V), OEA (O; 10 mg per kg i.p.) and d-fenfluramine (F; 3 mg per kg subcutaneous) on food intake in control (sham) and vagotomized (vag) rats. d, e, Time course of the effects of vehicle (open symbols) and Wy-14643 (filled symbols; 40 mg per kg i.p.) on food intake in control (d) and vagotomized (e) rats. f, Lack of effect of the PPAR-/ agonist GW501516 (G₂; 5 mg per kg i.p.) and PPAR- agonist ciglitazone (C; 15 mg per kg i.p.) on food intake in C57BL/6J mice (n = 4–6). g, h, Time course of the effects of vehicle (open symbols) and Wy-14643 (filled symbols; 40 mg per kg i.p.) on food intake in wild-type (g) and PPAR-^-/- (h) mice (n = 7–11). *P < 0.05, **P < 0.001 and ***P < 0.0001 by one-way ANOVA followed by Dunnett's test or Student's t-test with Bonferroni's correction.

High resolution image and legend (34K)

OEA is thought to produce satiety by activating peripheral sensory fibres¹. Accordingly, in rats in which these fibres had been removed either by cutting the vagus nerve below the diaphragm or by treatment with the neurotoxin capsaicin¹, OEA had no effect on food intake (Fig. 3c and data not shown). These procedures also prevented the effect of Wy-14643 (Fig. 3d, e , and Supplementary Table 2), whereas they did not affect that of d-fenfluramine, which reduces appetite through a central mechanism (Fig. 3c).

The close correspondence between the effects of OEA and those of synthetic PPAR- agonists is reinforced by two findings. First, potent agonists of PPAR-/ (GW501516)¹⁵ and PPAR- (ciglitazone)¹⁶ did not affect feeding in C57BL/6J mice (Fig. 3f); and second, PPAR-^-/- mice did not respond to Wy-14643 (Fig. 3g, h). We cannot exclude the possibility that PPAR-/, which weakly recognizes OEA in vitro (Fig. 1a), may contribute to some OEA responses; however, the fact that the PPAR-/ agonist GW501516 does not affect food intake implies that the role of PPAR-/ in OEA signalling, if any, is distinct from that of PPAR-.

To test further the ability of OEA to activate PPAR-, we examined its effects on the expression of PPAR- target genes. We focused first on the small intestine, which is a likely site of action of OEA¹ and contains large amounts of PPAR-^{17, 18}. In the jejunum of C57BL/6J mice, OEA increased the expression of three genes regulated by PPAR- (Fig. 4a–c): those encoding PPAR-, fatty acid translocase (FAT/CD36) and fatty acid transport protein 1 (FATP1)^{17, 19}. A similar effect was observed in the liver (Fig. 4e–g) and the duodenum, but not in the ileum (Supplementary Figs 1 and 2). By contrast, OEA did not affect the expression of three additional genes that are not controlled by PPAR- (Fig. 4d).

Figure 4: OEA regulates gene expression through PPAR- activation.

a–d, Effects of vehicle (V), Wy-14643 (W; 30 mg per kg i.p.) and OEA (O; 10 mg per kg i.p.) on messenger RNA levels of PPAR- (a), FAT/CD36 (b), FATP1 (c) and PPAR-/, PPAR- and I-FABP (d) in the jejunum of wild-type and PPAR-^-/- mice (n = 5). e–g, Effects of vehicle (V), Wy-14643 (W; 30 mg per kg i.p.) and OEA (O; 10 mg per kg i.p.) on mRNA levels of PPAR- (e), FAT/CD36 (f) and L-FABP (g) in the liver of wild-type and PPAR-^-/- mice (n = 5). h, Transrepression of iNOS expression by OEA (O; 10 mg per kg i.p.) and Wy-14643 (W; 30 mg per kg i.p.) in the jejunum of C57BL/6J mice (n = 5). mRNA levels are expressed in arbitrary units. *P < 0.05 and **P < 0.001 by one-way ANOVA followed by Dunnett's test.

High resolution image and legend (64K)

Underscoring the role of PPAR- in these responses, we found that Wy-14643 mimicked the effects of OEA (Fig. 4a–g) and that neither OEA nor Wy-14643 changed gene expression in PPAR-^-/- mice (Fig. 4a–c, e–g). In addition to stimulating transcription, PPAR- also represses genes such as that encoding inducible nitric oxide synthase (iNOS)²⁰. Accordingly, in the jejunum of C57BL/6J mice, OEA and Wy-14643 decreased iNOS expression (Fig. 4h). These results indicate that OEA closely mimics the genomic actions of PPAR- agonists.

If OEA enhances expression of PPAR- target genes, it should also reproduce the corrective effects of PPAR- agonists on serum lipid levels^{21, 22, 23}. Consistent with this prediction, in genetically obese Zucker (fa/fa) rats, a 2-week regimen of OEA administration decreased serum cholesterol and triglyceride along with food intake and body weight gain (Supplementary Fig. 3). In addition, 4 weeks of OEA treatment lowered cholesterol in obese wild-type mice²⁴, but not in PPAR-^-/- mice (Fig. 2g). We conclude that long-term OEA administration causes metabolic changes similar to those produced by PPAR- agonists, which are abrogated by PPAR- deletion.

The functional similarity between OEA and PPAR- agonists suggests that OEA is a natural ligand for PPAR-. This possibility is supported by the concerted regulation of OEA synthesis and expression of PPAR- and iNOS. In the small intestine of C57Bl/6J mice, OEA concentrations were significantly lower at night (01:30), when the mice feed, than during the day (16:30), when they are satiated and resting (Fig. 5a, b). The expression of PPAR- paralleled OEA concentrations, whereas that of its transrepression target iNOS showed the opposite pattern (Fig. 5c, d). Notably, daytime intestinal concentrations of OEA were in the range needed to activate PPAR- fully in vitro ( 300 nM), suggesting that they may be adequate to engage the receptor in vivo.

Figure 5: Circadian regulation of intestinal OEA synthesis and PPAR- expression.

Food intake (a), OEA content (b), and PPAR- (c) and iNOS (d) mRNA levels at 01:30 (filled bars) and 16:30 (open bars) in free-feeding C57Bl/6J mice maintained on a 12-h/12-h dark/light cycle.

High resolution image and legend (20K)

In conclusion, our findings indicate that OEA shows all of the defining characteristics of an endogenous PPAR- agonist: first, it binds with high affinity to PPAR-; second, it mimics the actions of synthetic agonists in a PPAR--dependent manner; and last, under appropriate conditions, it reaches tissue concentrations that are sufficient to activate PPAR-. The results also suggest that PPAR- activation not only mediates OEA-induced weight stabilization, which may be expected from the metabolic roles of this receptor^{6, 7, 8}, but is also responsible for OEA-induced satiety, a behavioural role that was not previously attributed to PPAR-. The molecular mechanism underlying this response is still undefined, but one possibility is that it may involve the transcriptional regulation of intestinal NO production. Intestinal epithelial cells contain the enzyme iNOS and generate significant amounts of its product, NO, which may act as a peripheral appetite-stimulating signal^{25, 26}. The ability of OEA to reduce iNOS expression through PPAR- suggests that downregulation of NO signalling may contribute to the persistent satiety-inducing actions of this lipid mediator.

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Supplementary Information

Supplementary information accompanies this paper.

Acknowledgements

We thank L. Stein for comments; L. Giron, N. Heyrani, N. Izadi and K. Nguyen for help with experiments; M. Guzmán for critically reading the manuscript; and F. Valiño for synthesizing fatty acid ethanolamides. This research was supported by grants (to D.P.) from the National Institute on Drug Abuse. Further support came from the Fondo de Investigación Sanitaria.

Competing interests statement

The authors declare  competing financial interests.