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Early tolerance to the hypophagic effect of the cannabinoid receptor antagonist SR141716 does not impede blockade of an orexigenic stimulus
European Journal of Pharmacology 542 (2006) 116 – 120 www.elsevier.com/locate/ejphar
Early tolerance to the hypophagic effect of the cannabinoid receptor antagonist SR141716 does not impede blockade of an orexigenic stimulus Antonello E. Rigamonti, Claudio Giordani, Sara M. Bonomo, Silvano G. Cella, Eugenio E. Müller ⁎ Department of Medical Pharmacology, University of Milan, via Vanvitelli 32, 20129 Milan, Italy Received 3 March 2006; received in revised form 11 May 2006; accepted 19 May 2006 Available online 26 May 2006
Abstract The cannabinoid CB1 receptor antagonist SR141716 (Rimonabant®) is known to reduce food intake by central and peripheral mechanisms. Recently, SR141716 has been reported to block the orexigenic effect of ghrelin, a potent orexigenic peptide produced by the stomach. This study investigated whether in rats, made tolerant to the hypophagic effect of SR141716, the drug was still capable to block the orexigenic activity of another non-natural (hypothalamic) peptide, i.e., the growth hormone releasing peptide (GHRP) hexarelin, a ghrelin mimetic. In the acute experiments, each dose of SR141716 (1, 5 and 10 mg/kg i.p.) reduced food intake with respect to vehicle-treated rats, whereas hexarelin (160 μg/kg s.c.) markedly stimulated feeding. All doses of SR141716 were capable to reduce the orexigenic effect of the GHRP. A 15-day administration of SR141716 (10 mg/kg i.p.) reduced both food intake and body weight. Tolerance to the hypophagic effect of SR141716 developed within 5 days, but in contrast, body weight remained markedly below that of vehicle-treated group throughout the entire treatment period. Interestingly, despite development of tolerance to its hypophagic effect, SR141716 was capable to suppress the orexigenic effect of repeated hexarelin challenge tests performed throughout the chronic experiments. In conclusion, the results of the present study confirm and broaden the existence of a functional relationship between ghrelin and endocannabinoids in the control of food intake, and bespeak the ability of a CB1 receptor antagonist to suppress orexia caused by stimuli alien to direct stimulation of the cannabinoid system. Published by Elsevier B.V. Keywords: SR141716; Hexarelin; Food intake; Tolerance; Body weight
1. Introduction Ghrelin, a 28-amino-acid peptide, is synthesised in the periphery (mainly in the stomach) and in the brain (Kojima et al., 1999; Ghigo et al., 2005). It acts as an endogenous ligand of the growth hormone secretagogue (GHS) receptor (Arvat et al., 2002). Additionally, recent studies have shown that ghrelin reliably stimulates food intake in many animal models, including humans (Van der Lely et al., 2004). Ghrelin seems to exert its hyperphagic effect through activation of specific hypothalamic areas linked to the regulation of feeding and metabolism, particularly the paraventricular nucleus (Kojima and Kangawa, 2005). Direct injection of ghrelin into this site generates a robust feeding response (Wren et al., 2001b). Ghrelin induces feeding by stimulating the orexigenic neuropeptide Y, orexin and agouti⁎ Corresponding author. Tel.: +39 02 50317010 7012; fax: + 39 02 50317011. E-mail address: [email protected] (E.E. Müller). 0014-2999/$ - see front matter. Published by Elsevier B.V. doi:10.1016/j.ejphar.2006.05.028
related peptide pathways and, additionally, by inhibiting the anorexigenic cocaine and amphetamine-regulated transcript and peptides derived from pro-opiomelanocortin systems (Van der Lely et al., 2004). It may be reasonable, therefore, to anticipate functional interactions between ghrelin and other appetite- or energy-regulating systems. It is now well established that appetite is modulated by central endogenous cannabinoid systems (Cota et al., 2003). Administration of cannabis compounds such as Δ9-tetrahydrocannabinol (THC) stimulates appetite (Williams et al., 1998), while the cannabinoid CB1 receptor antagonist SR141716 (Rimonabant®) reduces food intake after systemic administration (Colombo et al., 1998). Furthermore, recent studies have shown that appetite is increased by the endogenous cannabinoid ligands, 2-arachidonoylglycerol and anandamide (Williams and Kirkham, 1999; Kirkham et al., 2002). As in the case of ghrelin, the paraventricular nucleus is also a sensitive site of action for the orexigenic actions of exogenous
A.E. Rigamonti et al. / European Journal of Pharmacology 542 (2006) 116–120
and endogenous cannabinoids, and systemic cannabinoid receptor agonist and antagonist drugs induce c-fos expression in the paraventricular nucleus (Wenger et al., 2003). Recently, the CB1 antagonist SR141716 has been reported to reverse the orexigenic effect of a single injection of ghrelin into the paraventricular nucleus (Tucci et al., 2004). This finding provides the first evidence for a functional interaction between hypothalamic ghrelin and the endocannabinoids. Interestingly, even if SR141716 reduces dose-dependently both food intake and body weight in the early phase of a chronic treatment in the rat, tolerance to its hypophagic effect develops within 5 days, but despite this event, body weight remains markedly lower than that of vehicle-treated rats throughout the entire 14-day treatment period (Colombo et al., 1998). It is of note that tolerance is more rapid to occur in lean than in obese Zucker rats, suggesting that the obese state is more resistant to SR141716-induced tolerance (Vickers et al., 2003). The reasons of the maintenance of the body weight lowering effect of SR141716 are presently unknown, though some central and/or peripheral mechanisms have been invoked (Cota et al., 2003). The aim of the present work was to evaluate whether in rats, made tolerant to the hypophagic effect of SR141716, the drug was still capable to block the strong orexigenic activity of a stimulus alien to the cannabinoid system, i.e., the growth hormone releasing peptide (GHRP) hexarelin, a ghrelin mimetic (Papotti et al., 2000). If this was the case, the potential therapeutic applications of Rimonabant® might be accrued. 2. Materials and methods 2.1. Animals In either acute or chronic experiments (see below), Sprague– Dawley male rats (250–300 g; Charles-River, Calco, Italy) were used. They were singly housed in our facilities under controlled conditions (22 ± 2 °C, 65% humidity, artificial light from 0600 to 2000 h). A standard dry pellet diet (Mucedola S.p.A., Milan, Italy) and water were available ad libitum. Animals were previously accustomed to be handled and to receive intraperitoneal injections (about 15 days), reproducing the same next experimental set. All the experiments were performed in accordance with the Italian Guidelines for the Use of Animals in Medical Research.
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an i.p. injection of SR141716 (1, 5 and 10 mg/kg) or vehicle. After 2 h, rats received a s.c. injection of hexarelin (160 μg/kg) or saline, and immediately after treatment were placed in their cages with a pre-weighed amount of food. Food intake was measured each hour in the ensuing 4 h interval by an electronic balance, with values approximating the first decimal of g. Any effort was made to control food spillage into cage bedding. 2.3.2. Chronic experiments Thirty-six rats were randomly allocated to two experimental groups (vehicle or treated). They were given SR141716 (10 mg/ kg i.p.) or vehicle once daily for a 15-day period. All weighing and dosing took place at approximately 0900 h each day. Body weight and 24 h food intake were recorded daily for 15 days. Half of the animals of the vehicle and treated groups were exclusively used for hexarelin challenge test, which was performed on alternate days. Food intake post-hexarelin bolus was monitored with the same modalities and time intervals as referred previously for acute experiments. Baseline food intake during a 4-h period was evaluated in all animal groups before starting the chronic treatment. 2.4. Statistics All values are expressed as mean ± standard error. Data were analysed by two-way analysis of variance (ANOVA). Comparisons between individual groups were then made with Bonferroni's test. P < 0.05 was considered significant. To confirm the existence of a pharmacological antagonism between SR141716 and hexarelin, we compared the obtained data with these which would be arithmetically predicted from the opposing effects of the drugs when administered alone. The predicted outcome for the drug combination was calculated by subtracting the value of the decrease in (vehicle + saline) food intake produced by each dose of the CB1 antagonist from food intake stimulated by the GHS. Were the decreases in food intake produced by the antagonist the only contributing factor to the antagonist-induced dose–effect curve on food intake stimulated by hexarelin, these predicted values would have substantially overlapped the obtained values. 3. Results
2.2. Drugs
3.1. Acute experiments
N-piperidino-5(4 cholorophenyl)-1-(2,4-dicholophenyl)-4methylpyrazole carboxamide (SR141716; Sanofi-Aventis Research Center, Montpellier, France) was suspended in 10% Tween 80 vehicle solution (Sigma-Aldrich, Milan, Italy). Hexarelin (His–D-2-methyl-Trp–Ala–Trp–D-Lys–NH2, Europeptides, Argenteuil, France) was dissolved in isotonic saline.
In the acute experiments, the SR141716 (vehicle, 1, 5 and 10 mg/kg) × hexarelin (saline and 160 μg/kg) ANOVA yielded a significant main effect on food intake, for SR141716 [df = 3; F = 23.31; P < 0.001], for hexarelin [df = 1; F = 9.74; P < 0.002] and SR141716 × hexarelin [df = 3; F = 10.13; P < 0.001], indicating that food intake changed significantly under different treatments and that SR141716 and hexarelin significantly interacted. Precisely, SR141716 reduced food intake in respect to vehicle-treated rats (P < 0.001 for each dose vs. vehicle), whereas hexarelin markedly stimulated feeding (P < 0.002 vs. saline). All doses of SR141716 were capable to reduce the
2.3. Experimental procedure 2.3.1. Acute experiments Animals were randomly allocated to 8 treatment groups of 9 animals each. On the test day, at 0900 h, each animal received
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orexigenic effect of the GHRP (P < 0.001 for each dose vs. saline), which was completely abolished by the highest dose (Fig. 1). A further confirmation of the existence of a pharmacological antagonism between SR141716 and hexarelin derived from the evaluation of the arithmetically predicted values of the antagonist-induced dose–effect curve on food intake stimulated by hexarelin. They were divorced from the obtained values (data not shown). 3.2. Chronic experiments The effect of SR141716 on food intake over the course of the chronic experiments is shown in Fig. 2A, whereas Fig. 2B depicts daily body weight. The SR141716 (vehicle and 10 mg/ kg) × time ANOVA yielded a significant main effect on either food intake or body weight, for SR141716 [food intake: df = 1; F = 22.36; P < 0.001; body weight: df = 1; F = 3.91; P < 0.05], for time [food intake: df = 14; F = 3.57; P < 0.001; body weight: df = 14; F = 149.22; P < 0.001] and SR141716 × time [df = 14; F = 7.74; P < 0.001; body weight: df = 14; F = 26.25; P < 0.001], indicating that food intake and body weight changed significantly across the time and that vehicle- and SR141716-treated rats displayed quantitative differences in food intake and body weight over the time. Precisely, on the first treatment day, SR141716 markedly inhibited food intake in comparison to vehicle-treated rats (Fig. 2A). However, on continuing treatment, tolerance to the feeding suppressant effect of SR141716 developed within 5 days (Fig. 2A). Body weight paralleled the initial reduction of food intake in SR141716-treated rats; however, in contrast to the rapid return of food intake to baseline values, SR141716-treated rats failed to reach the body weight of vehicle-treated animals throughout the entire treatment period (Fig. 2B). The SR141716 (vehicle and 10 mg/kg) × time ANOVA yielded a significant main effect on food intake stimulated by hexarelin, for SR141716 [df = 1; F = 39.64; P < 0.001], time [df = 6; F = 8.52; P < 0.001] and SR141716 × time [df = 6; F = 6.55; P < 0.001], indicating that food intake stimulated by hexarelin changed significantly across the time and that vehicle-
Fig. 1. Four-hour cumulative food intake in rats (n = 9 per group) treated acutely with SR141716 (vehicle, 1, 5 and 10 mg/kg i.p.) or with hexarelin (saline and 160 μg/kg s.c.). ⁎P < 0.001 vs. vehicle–hexarelin; °P < 0.001 vs. vehicle–saline.
Fig. 2. One-day cumulative food intake (panel A) and body weight (panel B) in rats (n = 9 per group) treated chronically with SR141716. ⁎Significance of at least P < 0.05 vs. respective vehicle value.
and SR141716-treated rats displayed quantitative differences in food intake over the time. In fact, SR141716-treated rats, when challenged with hexarelin, exhibited a strong inhibition of the
Fig. 3. Four-hour cumulative food intake stimulated by hexarelin in rats given chronic administration of either vehicle or SR141716. Baseline values of food intake (before vehicle or SR141716 chronic treatment and with saline) are reported. Time 0 refers to timing of hexarelin challenge test in rats which then will be treated with vehicle or SR141617. Each point following time 0 represents 4-h cumulative food intake after a hexarelin challenge test, performed on alternate days throughout the chronic treatment. ⁎P < 0.001 vs. vehicle-treated rats.
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orexigenic effect of the GHRP in all tests performed throughout the experiment, whereas animals of vehicle-injected group did not (Fig. 3). All statistical comparisons of chronic experiments are reported in the corresponding figures.
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In conclusion, the results of the present study confirm and broaden the existence of a functional relationship between ghrelin and endocannabinoids in the control of food intake (Tucci et al., 2004) and bespeak the ability of a CB1 receptor antagonist to suppress orexia caused by stimuli alien to direct stimulation of the cannabinoid system.
4. Discussion Acknowledgment In the present study, single administration of SR141716 to ad libitum fed rats induced a consistent reduction of food intake during the 4-h post-injection period, in accordance with previous data of the literature (Colombo et al., 1998). Importantly, our results confirmed the existence of a functional interaction between the endocannabinoid system and GHRP/ ghrelin in the control of appetite (Tucci et al., 2004). Specifically, the orexigenic effect of an s.c. injection of hexarelin, an agonist of GHS receptor (Papotti et al., 2000), was prevented by acute pretreatment with the cannabinoid CB1 receptor antagonist SR141716. Collectively, these findings would imply that an endocannabinoid tone is instrumental to the expression of ghrelin hyperphagia and that the endogenous cannabinoid system is another downstream or upstream target for the direct or indirect orexigenic actions of ghrelin within the hypothalamus (Di et al., 2003; Cani et al., 2004; Tucci et al., 2004). In the chronic experiments, SR141716 was capable of reducing food intake only during the initial 5 days, as from day 6 on the amount of food eaten tended to return to pretreatment values. It is noteworthy that (1) the lowering effect of SR141716 on body weight persisted almost unchanged throughout the chronic experiment, pointing to the existence of additional actions of the compound which are not mediated by a direct inhibition of food intake (e.g., increase of energy expenditure) (Cota et al., 2003); (2) SR141716, despite development of tolerance to its hypophagic effect, proved capable to cancel the orexigenic effect of repeated hexarelin challenge tests performed throughout the chronic experiments. This apparent paradox may be explained by the existence of a functional interaction between the endocannabinoid and the GHRP/ghrelin. Alternatively, the endocannabinoid tone may contribute to maintain the hedonic value of food intake (Saper et al., 2002; Harrold and Williams, 2003; Fride et al., 2005) triggered by orexigenic peptides, such as ghrelin (Carlini et al., 2004). This effect, which depends from a direct action of ghrelin at the reward system (i.e., at nucleus accumbens) (Naleid et al., 2005), would not be susceptible to tolerance. Irrespective of the possible interpretation of these findings, exogenous administration of hexarelin may be viewed as mimicking the pre-prandial elevation of endogenous plasma ghrelin levels, one of the most potent hunger signals in humans (Wren et al., 2001a; DelParigi et al., 2002). In other words, SR141716 should be highly effective in the control of eating disorders or endocrine-metabolic diseases, featuring hyperphagic episodes of various origins (bulimia nervosa, binge eating, Prader-Willi syndrome) (Bizzarri et al., 2004; Monteleone et al., 2005).
We thank warmly Sanofi-Aventis Research Center, Milan, Italy, for providing SR141716. Personal thanks are given to Dr. T. Croci for his unabated collaboration. Supported in part by FIRB Project RBNE01JKLF. References Arvat, E., Broglio, F., Amaretti, G., Benso, A., Giordano, R., Deghenghi, R., Ghigo, E., 2002. Ghrelin and synthetic GH secretagogues. Best Pract. Res. Clin. Endocrinol. Metab. 16, 505–517. Bizzarri, C., Rigamonti, A.E., Giannone, G., Berardinelli, R., Cella, S.G., Cappa, M., Muller, E.E., 2004. Maintenance of a normal meal-induced decrease in plasma ghrelin levels in children with Prader-Willi syndrome. Horm. Metab. Res. 36, 164–169. Cani, P.D., Montoya, M.L., Neyrinck, A.M., Delzenne, N.M., Lambert, D.M., 2004. Potential modulation of plasma ghrelin and glucagon-like peptide-1 by anorexigenic cannabinoid compounds, SR141716A (rimonabant) and oleoylethanolamide. Br. J. Nutr. 92, 757–761. Carlini, V.P., Varas, M.M., Cragnolini, A.B., Schioth, H.B., Scimonelli, T.N., de Barioglio, S.R., 2004. Differential role of the hippocampus, amygdala, and dorsal raphe nucleus in regulating feeding, memory, and anxiety-like behavioral responses to ghrelin. Biochem. Biophys. Res. Commun. 313, 635–641. Colombo, G., Agabio, R., Diaz, G., Lobina, C., Reali, R., Gessa, G.L., 1998. Appetite suppression and weight loss after the cannabinoid antagonist SR 141716. Life Sci. 63, 113–117. Cota, D., Marsicano, G., Lutz, B., Vicennati, V., Stalla, G.K., Pasquali, R., Pagotto, U., 2003. Endogenous cannabinoid system as a modulator of food intake. Int. J. Obes. Relat. Metab. Disord. 27, 289–301. DelParigi, A., Tschop, M., Heiman, M.L., Salbe, A.D., Vozarova, B., Sell, S.M., Bunt, J.C., Tataranni, P.A., 2002. High circulating ghrelin: a potential cause for hyperphagia and obesity in Prader-Willi syndrome. J. Clin. Endocrinol. Metab. 87, 5461–5464. Di, S., Malcher-Lopes, R., Halmos, K.C., Tasker, J.G., 2003. Nongenomic glucocorticoid inhibition via endocannabinoid release in the hypothalamus: a fast feedback mechanism. J. Neurosci. 23, 4850–4857. Fride, E., Bregman, T., Kirkham, T.C., 2005. Endocannabinoids and food intake: newborn suckling and appetite regulation in adulthood. Exp. Biol. Med. 230, 225–234. Ghigo, E., Broglio, F., Arvat, E., Maccario, M., Papotti, M., Muccioli, G., 2005. Ghrelin: more than a natural GH secretagogue and/or an orexigenic factor. Clin. Endocrinol. 62, 1–17. Harrold, J.A., Williams, G., 2003. The cannabinoid system: a role in both the homeostatic and hedonic control of eating? Br. J. Nutr. 90, 729–734. Kirkham, T.C., Williams, C.M., Fezza, F., Di Marzo, V., 2002. Endocannabinoid levels in rat limbic forebrain and hypothalamus in relation to fasting, feeding and satiation: stimulation of eating by 2-arachidonoyl glycerol. Br. J. Pharmacol. 136, 550–557. Kojima, M., Kangawa, K., 2005. Ghrelin: structure and function. Physiol. Rev. 85, 495–522. Kojima, M., Hosoda, H., Date, Y., Nakazato, M., Matsuo, H., Kangawa, K., 1999. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402, 656–660. Monteleone, P., Martiadis, V., Rigamonti, A.E., Fabrazzo, M., Giordani, C., Muller, E.E., Maj, M., 2005. Investigation of peptide YY and ghrelin responses to a test meal in bulimia nervosa. Biol. Psychiatry 57, 926–931.
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Naleid, A.M., Grace, M.K., Cummings, D.E., Levine, A.S., 2005. Ghrelin induces feeding in the mesolimbic reward pathway between the ventral tegmental area and the nucleus accumbens. Peptides 26, 2274–2279. Papotti, M., Ghe, C., Cassoni, P., Catapano, F., Deghenghi, R., Ghigo, E., Muccioli, G., 2000. Growth hormone secretagogue binding sites in peripheral human tissues. J. Clin. Endocrinol. Metab. 85, 3803–3807. Saper, C.B., Chou, T.C., Elmquist, J.K., 2002. The need to feed: homeostatic and hedonic control of eating. Neuron 36, 199–211. Tucci, S.A., Rogers, E.K., Korbonits, M., Kirkham, T.C., 2004. The cannabinoid CB1 receptor antagonist SR141716 blocks the orexigenic effects of intrahypothalamic ghrelin. Br. J. Pharmacol. 143, 520–523. Van der Lely, A.J., Tschop, M., Heiman, M.L., Ghigo, E., 2004. Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin. Endocr. Rev. 25, 426–457. Vickers, S.P., Webster, L.J., Wyatt, A., Dourish, C.T., Kennett, G.A., 2003. Preferential effects of the cannabinoid CB1 receptor antagonist, SR 141716, on food intake and body weight gain of obese (fa/fa) compared to lean Zucker rats. Psychopharmacology 167, 103–111.
Wenger, T., Ledent, C., Tramu, G., 2003. The endogenous cannabinoid, anandamide, activates the hypothalamo-pituitary-adrenal axis in CB1 cannabinoid receptor knockout mice. Neuroendocrinology 78, 294–300. Williams, C.M., Kirkham, T.C., 1999. Anandamide induces overeating: mediation by central cannabinoid (CB1) receptors. Psychopharmacology 143, 315–317. Williams, C.M., Rogers, P.J., Kirkham, T.C., 1998. Hyperphagia in pre-fed rats following oral delta9-THC. Physiol. Behav. 65, 343–346. Wren, A.M., Seal, L.J., Cohen, M.A., Brynes, A.E., Frost, G.S., Murphy, K. G., Dhillo, W.S., Ghatei, M.A., Bloom, S.R., 2001a. Ghrelin enhances appetite and increases food intake in humans. J. Clin. Endocrinol. Metab. 86, 5992–5995. Wren, A.M., Small, C.J., Abbott, C.R., Dhillo, W.S., Seal, L.J., Cohen, M.A., Batterham, R.L., Taheri, S., Stanley, S.A., Ghatei, M.A., Bloom, S.R., 2001b. Ghrelin causes hyperphagia and obesity in rats. Diabetes 50, 2540–2547.
Early tolerance to the hypophagic effect of the cannabinoid receptor antagonist SR141716 does not impede blockade of an orexigenic stimulus Antonello E. Rigamonti, Claudio Giordani, Sara M. Bonomo, Silvano G. Cella, Eugenio E. Müller ⁎ Department of Medical Pharmacology, University of Milan, via Vanvitelli 32, 20129 Milan, Italy Received 3 March 2006; received in revised form 11 May 2006; accepted 19 May 2006 Available online 26 May 2006
Abstract The cannabinoid CB1 receptor antagonist SR141716 (Rimonabant®) is known to reduce food intake by central and peripheral mechanisms. Recently, SR141716 has been reported to block the orexigenic effect of ghrelin, a potent orexigenic peptide produced by the stomach. This study investigated whether in rats, made tolerant to the hypophagic effect of SR141716, the drug was still capable to block the orexigenic activity of another non-natural (hypothalamic) peptide, i.e., the growth hormone releasing peptide (GHRP) hexarelin, a ghrelin mimetic. In the acute experiments, each dose of SR141716 (1, 5 and 10 mg/kg i.p.) reduced food intake with respect to vehicle-treated rats, whereas hexarelin (160 μg/kg s.c.) markedly stimulated feeding. All doses of SR141716 were capable to reduce the orexigenic effect of the GHRP. A 15-day administration of SR141716 (10 mg/kg i.p.) reduced both food intake and body weight. Tolerance to the hypophagic effect of SR141716 developed within 5 days, but in contrast, body weight remained markedly below that of vehicle-treated group throughout the entire treatment period. Interestingly, despite development of tolerance to its hypophagic effect, SR141716 was capable to suppress the orexigenic effect of repeated hexarelin challenge tests performed throughout the chronic experiments. In conclusion, the results of the present study confirm and broaden the existence of a functional relationship between ghrelin and endocannabinoids in the control of food intake, and bespeak the ability of a CB1 receptor antagonist to suppress orexia caused by stimuli alien to direct stimulation of the cannabinoid system. Published by Elsevier B.V. Keywords: SR141716; Hexarelin; Food intake; Tolerance; Body weight
1. Introduction Ghrelin, a 28-amino-acid peptide, is synthesised in the periphery (mainly in the stomach) and in the brain (Kojima et al., 1999; Ghigo et al., 2005). It acts as an endogenous ligand of the growth hormone secretagogue (GHS) receptor (Arvat et al., 2002). Additionally, recent studies have shown that ghrelin reliably stimulates food intake in many animal models, including humans (Van der Lely et al., 2004). Ghrelin seems to exert its hyperphagic effect through activation of specific hypothalamic areas linked to the regulation of feeding and metabolism, particularly the paraventricular nucleus (Kojima and Kangawa, 2005). Direct injection of ghrelin into this site generates a robust feeding response (Wren et al., 2001b). Ghrelin induces feeding by stimulating the orexigenic neuropeptide Y, orexin and agouti⁎ Corresponding author. Tel.: +39 02 50317010 7012; fax: + 39 02 50317011. E-mail address: [email protected] (E.E. Müller). 0014-2999/$ - see front matter. Published by Elsevier B.V. doi:10.1016/j.ejphar.2006.05.028
related peptide pathways and, additionally, by inhibiting the anorexigenic cocaine and amphetamine-regulated transcript and peptides derived from pro-opiomelanocortin systems (Van der Lely et al., 2004). It may be reasonable, therefore, to anticipate functional interactions between ghrelin and other appetite- or energy-regulating systems. It is now well established that appetite is modulated by central endogenous cannabinoid systems (Cota et al., 2003). Administration of cannabis compounds such as Δ9-tetrahydrocannabinol (THC) stimulates appetite (Williams et al., 1998), while the cannabinoid CB1 receptor antagonist SR141716 (Rimonabant®) reduces food intake after systemic administration (Colombo et al., 1998). Furthermore, recent studies have shown that appetite is increased by the endogenous cannabinoid ligands, 2-arachidonoylglycerol and anandamide (Williams and Kirkham, 1999; Kirkham et al., 2002). As in the case of ghrelin, the paraventricular nucleus is also a sensitive site of action for the orexigenic actions of exogenous
A.E. Rigamonti et al. / European Journal of Pharmacology 542 (2006) 116–120
and endogenous cannabinoids, and systemic cannabinoid receptor agonist and antagonist drugs induce c-fos expression in the paraventricular nucleus (Wenger et al., 2003). Recently, the CB1 antagonist SR141716 has been reported to reverse the orexigenic effect of a single injection of ghrelin into the paraventricular nucleus (Tucci et al., 2004). This finding provides the first evidence for a functional interaction between hypothalamic ghrelin and the endocannabinoids. Interestingly, even if SR141716 reduces dose-dependently both food intake and body weight in the early phase of a chronic treatment in the rat, tolerance to its hypophagic effect develops within 5 days, but despite this event, body weight remains markedly lower than that of vehicle-treated rats throughout the entire 14-day treatment period (Colombo et al., 1998). It is of note that tolerance is more rapid to occur in lean than in obese Zucker rats, suggesting that the obese state is more resistant to SR141716-induced tolerance (Vickers et al., 2003). The reasons of the maintenance of the body weight lowering effect of SR141716 are presently unknown, though some central and/or peripheral mechanisms have been invoked (Cota et al., 2003). The aim of the present work was to evaluate whether in rats, made tolerant to the hypophagic effect of SR141716, the drug was still capable to block the strong orexigenic activity of a stimulus alien to the cannabinoid system, i.e., the growth hormone releasing peptide (GHRP) hexarelin, a ghrelin mimetic (Papotti et al., 2000). If this was the case, the potential therapeutic applications of Rimonabant® might be accrued. 2. Materials and methods 2.1. Animals In either acute or chronic experiments (see below), Sprague– Dawley male rats (250–300 g; Charles-River, Calco, Italy) were used. They were singly housed in our facilities under controlled conditions (22 ± 2 °C, 65% humidity, artificial light from 0600 to 2000 h). A standard dry pellet diet (Mucedola S.p.A., Milan, Italy) and water were available ad libitum. Animals were previously accustomed to be handled and to receive intraperitoneal injections (about 15 days), reproducing the same next experimental set. All the experiments were performed in accordance with the Italian Guidelines for the Use of Animals in Medical Research.
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an i.p. injection of SR141716 (1, 5 and 10 mg/kg) or vehicle. After 2 h, rats received a s.c. injection of hexarelin (160 μg/kg) or saline, and immediately after treatment were placed in their cages with a pre-weighed amount of food. Food intake was measured each hour in the ensuing 4 h interval by an electronic balance, with values approximating the first decimal of g. Any effort was made to control food spillage into cage bedding. 2.3.2. Chronic experiments Thirty-six rats were randomly allocated to two experimental groups (vehicle or treated). They were given SR141716 (10 mg/ kg i.p.) or vehicle once daily for a 15-day period. All weighing and dosing took place at approximately 0900 h each day. Body weight and 24 h food intake were recorded daily for 15 days. Half of the animals of the vehicle and treated groups were exclusively used for hexarelin challenge test, which was performed on alternate days. Food intake post-hexarelin bolus was monitored with the same modalities and time intervals as referred previously for acute experiments. Baseline food intake during a 4-h period was evaluated in all animal groups before starting the chronic treatment. 2.4. Statistics All values are expressed as mean ± standard error. Data were analysed by two-way analysis of variance (ANOVA). Comparisons between individual groups were then made with Bonferroni's test. P < 0.05 was considered significant. To confirm the existence of a pharmacological antagonism between SR141716 and hexarelin, we compared the obtained data with these which would be arithmetically predicted from the opposing effects of the drugs when administered alone. The predicted outcome for the drug combination was calculated by subtracting the value of the decrease in (vehicle + saline) food intake produced by each dose of the CB1 antagonist from food intake stimulated by the GHS. Were the decreases in food intake produced by the antagonist the only contributing factor to the antagonist-induced dose–effect curve on food intake stimulated by hexarelin, these predicted values would have substantially overlapped the obtained values. 3. Results
2.2. Drugs
3.1. Acute experiments
N-piperidino-5(4 cholorophenyl)-1-(2,4-dicholophenyl)-4methylpyrazole carboxamide (SR141716; Sanofi-Aventis Research Center, Montpellier, France) was suspended in 10% Tween 80 vehicle solution (Sigma-Aldrich, Milan, Italy). Hexarelin (His–D-2-methyl-Trp–Ala–Trp–D-Lys–NH2, Europeptides, Argenteuil, France) was dissolved in isotonic saline.
In the acute experiments, the SR141716 (vehicle, 1, 5 and 10 mg/kg) × hexarelin (saline and 160 μg/kg) ANOVA yielded a significant main effect on food intake, for SR141716 [df = 3; F = 23.31; P < 0.001], for hexarelin [df = 1; F = 9.74; P < 0.002] and SR141716 × hexarelin [df = 3; F = 10.13; P < 0.001], indicating that food intake changed significantly under different treatments and that SR141716 and hexarelin significantly interacted. Precisely, SR141716 reduced food intake in respect to vehicle-treated rats (P < 0.001 for each dose vs. vehicle), whereas hexarelin markedly stimulated feeding (P < 0.002 vs. saline). All doses of SR141716 were capable to reduce the
2.3. Experimental procedure 2.3.1. Acute experiments Animals were randomly allocated to 8 treatment groups of 9 animals each. On the test day, at 0900 h, each animal received
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orexigenic effect of the GHRP (P < 0.001 for each dose vs. saline), which was completely abolished by the highest dose (Fig. 1). A further confirmation of the existence of a pharmacological antagonism between SR141716 and hexarelin derived from the evaluation of the arithmetically predicted values of the antagonist-induced dose–effect curve on food intake stimulated by hexarelin. They were divorced from the obtained values (data not shown). 3.2. Chronic experiments The effect of SR141716 on food intake over the course of the chronic experiments is shown in Fig. 2A, whereas Fig. 2B depicts daily body weight. The SR141716 (vehicle and 10 mg/ kg) × time ANOVA yielded a significant main effect on either food intake or body weight, for SR141716 [food intake: df = 1; F = 22.36; P < 0.001; body weight: df = 1; F = 3.91; P < 0.05], for time [food intake: df = 14; F = 3.57; P < 0.001; body weight: df = 14; F = 149.22; P < 0.001] and SR141716 × time [df = 14; F = 7.74; P < 0.001; body weight: df = 14; F = 26.25; P < 0.001], indicating that food intake and body weight changed significantly across the time and that vehicle- and SR141716-treated rats displayed quantitative differences in food intake and body weight over the time. Precisely, on the first treatment day, SR141716 markedly inhibited food intake in comparison to vehicle-treated rats (Fig. 2A). However, on continuing treatment, tolerance to the feeding suppressant effect of SR141716 developed within 5 days (Fig. 2A). Body weight paralleled the initial reduction of food intake in SR141716-treated rats; however, in contrast to the rapid return of food intake to baseline values, SR141716-treated rats failed to reach the body weight of vehicle-treated animals throughout the entire treatment period (Fig. 2B). The SR141716 (vehicle and 10 mg/kg) × time ANOVA yielded a significant main effect on food intake stimulated by hexarelin, for SR141716 [df = 1; F = 39.64; P < 0.001], time [df = 6; F = 8.52; P < 0.001] and SR141716 × time [df = 6; F = 6.55; P < 0.001], indicating that food intake stimulated by hexarelin changed significantly across the time and that vehicle-
Fig. 1. Four-hour cumulative food intake in rats (n = 9 per group) treated acutely with SR141716 (vehicle, 1, 5 and 10 mg/kg i.p.) or with hexarelin (saline and 160 μg/kg s.c.). ⁎P < 0.001 vs. vehicle–hexarelin; °P < 0.001 vs. vehicle–saline.
Fig. 2. One-day cumulative food intake (panel A) and body weight (panel B) in rats (n = 9 per group) treated chronically with SR141716. ⁎Significance of at least P < 0.05 vs. respective vehicle value.
and SR141716-treated rats displayed quantitative differences in food intake over the time. In fact, SR141716-treated rats, when challenged with hexarelin, exhibited a strong inhibition of the
Fig. 3. Four-hour cumulative food intake stimulated by hexarelin in rats given chronic administration of either vehicle or SR141716. Baseline values of food intake (before vehicle or SR141716 chronic treatment and with saline) are reported. Time 0 refers to timing of hexarelin challenge test in rats which then will be treated with vehicle or SR141617. Each point following time 0 represents 4-h cumulative food intake after a hexarelin challenge test, performed on alternate days throughout the chronic treatment. ⁎P < 0.001 vs. vehicle-treated rats.
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orexigenic effect of the GHRP in all tests performed throughout the experiment, whereas animals of vehicle-injected group did not (Fig. 3). All statistical comparisons of chronic experiments are reported in the corresponding figures.
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In conclusion, the results of the present study confirm and broaden the existence of a functional relationship between ghrelin and endocannabinoids in the control of food intake (Tucci et al., 2004) and bespeak the ability of a CB1 receptor antagonist to suppress orexia caused by stimuli alien to direct stimulation of the cannabinoid system.
4. Discussion Acknowledgment In the present study, single administration of SR141716 to ad libitum fed rats induced a consistent reduction of food intake during the 4-h post-injection period, in accordance with previous data of the literature (Colombo et al., 1998). Importantly, our results confirmed the existence of a functional interaction between the endocannabinoid system and GHRP/ ghrelin in the control of appetite (Tucci et al., 2004). Specifically, the orexigenic effect of an s.c. injection of hexarelin, an agonist of GHS receptor (Papotti et al., 2000), was prevented by acute pretreatment with the cannabinoid CB1 receptor antagonist SR141716. Collectively, these findings would imply that an endocannabinoid tone is instrumental to the expression of ghrelin hyperphagia and that the endogenous cannabinoid system is another downstream or upstream target for the direct or indirect orexigenic actions of ghrelin within the hypothalamus (Di et al., 2003; Cani et al., 2004; Tucci et al., 2004). In the chronic experiments, SR141716 was capable of reducing food intake only during the initial 5 days, as from day 6 on the amount of food eaten tended to return to pretreatment values. It is noteworthy that (1) the lowering effect of SR141716 on body weight persisted almost unchanged throughout the chronic experiment, pointing to the existence of additional actions of the compound which are not mediated by a direct inhibition of food intake (e.g., increase of energy expenditure) (Cota et al., 2003); (2) SR141716, despite development of tolerance to its hypophagic effect, proved capable to cancel the orexigenic effect of repeated hexarelin challenge tests performed throughout the chronic experiments. This apparent paradox may be explained by the existence of a functional interaction between the endocannabinoid and the GHRP/ghrelin. Alternatively, the endocannabinoid tone may contribute to maintain the hedonic value of food intake (Saper et al., 2002; Harrold and Williams, 2003; Fride et al., 2005) triggered by orexigenic peptides, such as ghrelin (Carlini et al., 2004). This effect, which depends from a direct action of ghrelin at the reward system (i.e., at nucleus accumbens) (Naleid et al., 2005), would not be susceptible to tolerance. Irrespective of the possible interpretation of these findings, exogenous administration of hexarelin may be viewed as mimicking the pre-prandial elevation of endogenous plasma ghrelin levels, one of the most potent hunger signals in humans (Wren et al., 2001a; DelParigi et al., 2002). In other words, SR141716 should be highly effective in the control of eating disorders or endocrine-metabolic diseases, featuring hyperphagic episodes of various origins (bulimia nervosa, binge eating, Prader-Willi syndrome) (Bizzarri et al., 2004; Monteleone et al., 2005).
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