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Stearoylethanolamide exerts anorexic effects in mice via down-regulation of liver stearoyl-coenzyme A desaturase-1 mRNA expression

SALVATORE TERRAZZINO^*,1, FIORENZO BERTO^*, MAURIZIO DALLE CARBONARE^*, MICHELE FABRIS^*, ADRIANO GUIOTTO, DANIELE BERNARDINI and ALBERTA LEON^*

^* Research & Innovation (R&I) Company, Padova, Italy;
Department of Pharmaceutical Sciences, University of Padova, Padova, Italy; and
Department of Veterinary Clinical Sciences, School of Veterinary Medicine, University of Padova, Agripolis, Legnaro (PD), Italy

¹Correspondence: Research & Innovation (R&I) Company, via Svizzera 16, 35127 Padova, Italy. E-mail: info{at}researchinnovation.com

SPECIFIC AIMS

Given the recent demonstration that oleoylethanolamide (OEA, C18:1 ethanolamide), a cannabinoid receptor-inactive N-acylethanolamine, decreases food intake by activating the nuclear receptor PPAR (peroxisome proliferator-activated receptor ) in the periphery, we have evaluated the effects of saturated and unsaturated C18 N-acylethanolamines (C18:0; C18:1; C18:2) in mice feeding behavior. To gather information on the molecular mechanisms underlying these effects, we focused on the effects of stearoylethanolamide (SEA, C18:0 ethanolamide) on liver mRNA expression of stearoyl-CoA desaturase 1 (SCD-1) and PPARs.

PRINCIPAL FINDINGS

1. Anorexic effect of N-acylethanolamines in mice after i.p. administration
The effect of intraperitoneal (i.p.) administration of both saturated and unsaturated C:18 N-acylethanolamines was evaluated on feeding behavior in starved mice. A potent anorexic effect was observed after i.p. administration of stearoylethanolamide (SEA, C18:0 ethanolamide) at the dose of 25 mg/kg, which caused suppression of 1 h food consumption, while C18:1, C18:2, and C18:3 ethanolamides did not significantly modify food consumption. At the same dose, palmitoylethanolamide (PEA, C16:0 ethanolamide) was less potent than SEA but still effective in reducing food intake.

2. SEA displays a markedly acute dose-dependent anorexic effect in mice
Feeding behavior response was evaluated in starved mice after i.p. administration of SEA at doses of 3, 10, and 25 mg/kg (Fig. 1 ). While i.p. administration of 3 mg/kg of SEA does not modify food consumption, at the dose of 10 and 25 mg/kg SEA significantly reduces 1 h food intake compared with vehicle-treated mice. When assessed 2 h after i.p. SEA administration, serum leptin levels were not different from vehicle-treated mice.

View larger version (15K): [in this window] [in a new window]   Figure 1. Effect of i.p. administration of different doses of SEA on eating behavior response in starved mice. Compounds or vehicle were i.p. administered at t = 0 h; food was presented at t = 1 h and food consumption measured at t = 2 h. Each histogram is the mean ± SE for 6 mice. ***P < 0.001 compared with vehicle-treated mice (ANOVA, followed by Student-Newman-Keuls’ test).

3. Oral administration of SEA is effective in reducing mice feeding behavior
Feeding behavior response to SEA was evaluated in starved mice after per os administration at doses of 5, 10, and 25 mg/kg. In addition, one animal group received an equimolar suspension of stearic acid and ethanolamine equivalent to the highest dose of SEA. Whereas suspension of stearic acid and ethanolamine equivalent of the highest dose of SEA does not modify food consumption, SEA administered at 25 mg/kg significantly reduces 1 h food intake compared with vehicle-treated mice. The anorexic response was not associated to sedation or illness, as evaluated by investigators who were not aware of the experimental setting. It is noteworthy that no significant changes in body temperature were observed after per os administration of SEA even at a dose of 100 mg/kg. To evaluate whether anorexic activity of SEA was associated to changes on serum factors acting on energy homeostasis, biochemical analyses were conducted on serum samples obtained 2 h after per os administrations. Among the biochemical parameters considered (glucose, cholesterol, triglycerides, urea nitrogen, creatinine, creatine kinase, aspartate aminotranferase, alanine aminotranferase, and lactate dehydrogenase), no significant changes were observed in mice treated with SEA at the various doses compared with vehicle-treated mice. Serum leptin levels were also unaffected by the treatment.

5. Orally administered SEA decreases liver stearoyl-CoA desaturase-1 mRNA expression
Following the same schedule used for feeding experiments, starved mice were treated with per os administration of SEA (25 mg/kg) or vehicle. Mice were sacrificed 2 h later and the liver of each animal was immediately recovered for real-time RT-PCR quantification of stearoyl-CoA desaturase-1 (SCD-1) and PPARs (PPAR, PPARß, and PPAR). Orally administered SEA at the dose of 25 mg/kg significantly decreased liver SCD-1 mRNA expression, whereas PPAR, PPARß, and PPAR mRNA expression was not significantly affected compared with vehicle-treated mice (Fig. 2 ).

View larger version (23K): [in this window] [in a new window]   Figure 2. Effect of per os administration of vehicle and SEA (25 mg/kg) on liver PPAR , PPARß, PPAR, and SCD-1 mRNA expression in starved mice. SEA or vehicle were per os administered at t = 0 h and food was presented to overnight-fasted mice at t = 1 h. Animals were sacrificed at t = 2 h; the liver was immediately taken and stored at –80°C until real-time RT-PCR analysis. To correct for both RNA quantity and reverse transcription efficiency, the number of copies of the target gene in each sample was normalized by dividing with the respective number of copies of housekeeping gene PBGD. Data are presented as mean ± SE of 5 or 6 mice for each group. **P < 0.01 compared with vehicle-treated mice (unpaired t test).

CONCLUSIONS AND SIGNIFICANCE

In the present paper we provide evidence that, among endocannabinoid-like compounds, stearoylethanolamide (SEA, C18:0 ethanolamide) displays a potent anorexic activity in mice. In contrast, at the same dose, C18:1, C18:2, and C18:3 ethanolamides did not significantly modify 1 h food consumption. On the other hand, palmitoylethanolamide (PEA, C16:0 ethanolamide) was less potent than SEA but still effective in reducing food intake, indicating that saturation and chain length are determinant features underlying the pharmacological efficacy of the N-acylethanolamines in acutely reducing food intake. Oral administration of SEA was also effective in reducing feeding behavior, an effect ascribed less to the integrity of the molecule. This, together with the observation that oral administration of expected breakdown products of SEA (i.e., ethanolamine or stearic acid) are inactive in reducing food intake when administered at equimolar doses, raises the possibility that orally administered SEA is actively absorbed as such from the intestinal tract. The latter is of particular pharmacological relevance, as such a route of administration is not accompanied by unwanted side effects. In fact, although SEA has been reported to exert behavioral cannabinoid-like effects after its intraperitoneal administration, we observed that orally administered SEA had no effect neither on body temperature even when administered at very high doses, nor any effect on animal motility. It is noteworthy that the anorexic effect of the orally administered SEA was not accompanied by changes in either serum glucose or serum leptin levels. Molecular mechanisms underlying the anorexic activity of N-acylethanolamines are still largely unknown, but it is possible that SEA may induce some changes in the liver of genes involved in the regulation of lipid metabolism. Given the recent evidence that the delayed anorexic activity of OEA in rats is mediated by PPAR activation, which is a key nuclear receptor involved in the regulation of several aspects of lipid metabolism in the liver, one possibility is that SEA, given its structural similarity with OEA, activates PPAR. However, in line with previous in vitro findings reporting that SEA is not effective in activating PPAR, we show here that the in vivo anorexic effect of SEA is not accompanied by changes (at least in the liver) of PPAR mRNA expression. Analogously, no changes were found in liver mRNA expression of other PPARs (PPARß and PPAR) shown to be involved in lipid and glucose metabolism. On the other hand, endocannabinoids have been linked to leptin, which, in turn, has been shown to affect expression of SCD-1, the central rate-limiting lipogenic enzyme catalyzing synthesis of monounsaturated fatty acids. The observation provided here that the anorexic response to orally administered SEA is accompanied with down-regulation of liver SCD-1 mRNA expression is particularly relevant. SCD-1-deficient mice, in fact, show increased energy expenditure, reduced body adiposity, increased insulin sensitivity, and are resistant to diet-induced obesity. Because of this, SCD-1 has recently been proposed as a molecular target for treating obesity. In sum, our results show that, among non-eicanosoid-like N-acylethanolamides, SEA exerts 1) anorexic effects in mice after both i.p. and oral administration. In particular, the anorexic effect of oral administration is 2) ascribable to the molecule itself and not to its metabolites, 3) not associated with changes of serum leptin levels or hematochemical parameters, and, finally, 4) accompanied by down-regulation of liver SCD-1 mRNA expression in the absence of changes in liver PPAR expression. Although further studies are warranted, especially with regard to the molecular mechanisms involved, the novel observation provided here that SEA reduces food intake in mice in a structurally selective manner—in turn, correlated with down-regulation of liver SCD-1 mRNA expression—has the potential to provide new insights into a class of lipid mediators with properties suitable for the pharmacological treatment of overeating dysfunctions.

View larger version (38K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1080fje;

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