JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2016, 67, 3,

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1 JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2016, 67, 3, M. WIERUCKA-RYBAK 1, M. WOLAK 1, M. JUSZCZAK 2, J. DROBNIK 3, E. BOJANOWSKA 1 THE INHIBITORY EFFECT OF COMBINATION TREATMENT WITH LEPTIN AND CANNABINOID CB1 RECEPTOR AGONIST ON FOOD INTAKE AND BODY WEIGHT GAIN IS MEDIATED BY SEROTONIN 1B AND 2C RECEPTORS 1 Department of Behavioral Pathophysiology, Institute of General and Experimental Pathology, Medical University of Lodz, Lodz, Poland; 2 Department of Pathophysiology and Experimental Neuroendocrinology, Institute of General and Experimental Pathology, Medical University of Lodz, Lodz, Poland; 3 Department of Neuropeptide Research, Institute of General and Experimental Pathology, Medical University of Lodz, Lodz, Poland. Previous studies reported that the co-injection of leptin and cannabinoid CB 1 receptor antagonists reduces food intake and body weight in rats, and this effect is more profound than that induced by these compounds individually. Additionally, serotonin mediates the effects of numerous anorectic drugs. To investigate whether serotonin interacts with leptin and endocannabinoids to affect food intake and body weight, we administered 5-hydroxytryptamine(HT) 1B and 5- hydroxytryptamine(ht) 2C serotonin receptor antagonists (3 mg/kg GR and 0.5 mg/kg SB , respectively) to male Wistar rats treated simultaneously with leptin (100 µg/kg) and the CB 1 receptor inverse agonist AM 251 (1 mg/kg) for 3 days. In accordance with previous findings, the co-injection of leptin and AM 251, but not the individual injection of each drug, resulted in a significant decrease in food intake and body weight gain. Blockade of the 5-HT 1B and 5-HT 2C receptors completely abolished the leptin- and AM 251-induced anorectic and body-weight-reducing effects. These results suggest that serotonin mediates the leptin- and AM 251-dependent regulation of feeding behavior in rats via the 5-HT 1B and 5-HT 2C receptors. Key words: leptin, cannabinoid CB 1 receptor agonists, endocannabinoids, hypophagia, 5-hydroxytryptamine 1B receptor, 5- hydroxytryptamine 2C receptor, body weight, food intake INTRODUCTION Appetite is controlled by a variety of neurotransmitters and hormones that enhance or attenuate each other s effects on brain feeding circuits. Therefore, it is postulated that antiobesity therapies should target orexigenic and anorexigenic systems. Leptin, the endocannabinoids, and serotonin have long been known to be involved in the control of energy balance in humans and other animals. Leptin, an established anorectic hormone, controls the energy balance by interacting with a variety of neurotransmitters regulating the hypothalamic as well as the hindbrain and mesolimbic neural circuits involved in food intake control, and by influencing peripheral metabolic and neuroendocrine mechanisms that control energy intake and expenditure (1, 2). Most commonly, obesity is related to leptin resistance, and several neurotransmitters and hormones act as leptin sensitizers, which might enhance the biological activity of leptin (2). Contrary to previous belief, recent publications suggest that cerebral metabolic and feeding circuits of animals with diet-induced obesity can respond to leptin normally (3, 4). This implies that anti-obesity therapies targeting leptin may be beneficial not only in subjects with leptin-deficiency, but also in those with hyperleptinemia associated with common obesity. The endocannabinoid system plays an important role in the maintenance of energy homeostasis through the regulation of appetite as well as lipid and glucose metabolism via the activation of the cannabinoid 1 (CB 1 ) receptor (5). Blockade of the CB 1 receptor decreases hunger, reduces body weight, and improves lipid and glucose metabolism in obese humans and animals (6). For example, AM 251, a CB 1 inverse agonist, suppresses food intake in rats provided with a high-fat high-carbohydrate diet and attenuates the hedonic aspects of food consumption (7). Among other systems, the endocannabinoid system emerges as an important co-modulator of leptin action. Several lines of evidence suggest that leptin may decrease the food intake through the inhibition of the endocannabinoid system in the hypothalamus of rodents (8-10). In agreement with these results, co-treatment with leptin and CB 1 receptor antagonists inhibits the food consumption and body weight gain in rats maintained on a high-calorie diet (11, 12). Interestingly, the defective function of both the cerebral cannabinoid system and leptin signaling may be involved in the pathogenesis of obesity (13). Serotonin is a key satiety factor causing hypophagia in humans and animals (14-16). Among all serotonin receptors, the 5-HT 1B and 5-HT 2C receptors play an essential role in the maintenance of energy homeostasis, and these receptors are potential targets for drugs influencing appetite (16-18). The 5-HT 1B receptor knockout

2 458 mice are characterized by hyperphagia and excessive weight gain (19). Similarly, the 5-HT 2C receptor knockout mice are hyperphagic and obese (20), which suggests that the ablation of these receptors results in defective food intake regulation. On the other hand, 5-HT 1B and 5-HT 2C agonists reduce food intake in rodents and humans (21), thus they are involved in the maintenance of negative energy balance. Importantly, the 5-HT 2C receptor agonist, lorcaserin, has been approved for long-term obesity treatment in the United States (22). Serotonin interacts with other anorexigenic agents via the 5-HT 2C receptor. For example, this receptor mediates the anorectic action of urocortin (23), lipopolysaccharide (24), cholecystokinin, and glucagon-like peptide-1 (25). Furthermore, leptin utilizes the serotonergic pathways to regulate appetite (26). Leptin-serotonin interactions may be controlled by the leptin receptor in the raphe nuclei (27), where serotonergic pathways projecting to numerous hypothalamic feeding nuclei originate (28). Additionally, leptin and serotonin regulate food intake by modifying the activity of the hypothalamic melanocortin system; this interaction involves the serotonin 1B and 2C receptors (29, 30). Although not all studies have confirmed that there is a link between leptin and serotonin with regard to appetite regulation (31), the results of the numerous studies noted above have indicated that leptin-induced hypophagia may be mediated by serotonin. In addition, it has been shown that the endocannabinoid system can interact with the cerebral serotonergic system by inhibiting (32) or enhancing (33, 34) serotonin secretion following CB 1 receptor stimulation or inactivation, respectively. Not surprisingly, endocannabinoids and serotonin affect similar behaviors. For example, serotonin might be involved in the endocannabinoid system-dependent regulation of appetite (35) and, particularly, in the AM 251-dependent inhibition of appetite (34). Further studies revealed that the 5-HT 2C receptor regulates the functional interaction between the endocannabinoid and serotonergic systems in appetite control (36, 37). Although the significance of leptin, CB 1 receptor antagonists, and serotonin as signals for hypophagia is well recognized, little is known about the mechanisms by which the combined administration of leptin and AM 251 reduces food intake and body weight. Therefore, we investigated whether serotonin mediates the combined effects of leptin and AM 251 on food consumption and body weight. We injected antagonists of two serotonin receptors involved in food intake control, 5-HT 1B and 5-HT 2C, into rats together with the aforementioned pharmaceutical drugs, and measured the changes in food intake and body weight. Animals MATERIALS AND METHODS Adult male Wistar rats were collected from the breeding stock of Medical University of Lodz. We used rats weighing grams at the beginning of each experiment. Rats were housed individually in plastic cages in a temperature-controlled environment (20 22 C), and they were maintained under a 12/12 h light/dark cycle (lights off at 6.00 p.m.) with ad libitum access to food and water. On several days before the experiment, we handled the rats to accustom them to the experimental conditions and to attenuate the possible effects of stress on their feeding behavior. Rats were assigned randomly to either one of the 4 treatment groups in the first experiment or the 6 treatment groups in the second experiment (see Procedure). Food intake was monitored daily, and body weight was recorded at the beginning of the experiments and then twice, with 3-day intervals (for details, see the Procedure section). All procedures used in this study were reviewed and approved by the Ethics Committee of Medical University of Lodz. Drugs Drugs were dissolved 30 min before administration on each test day. AM 251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4- dichlorophenyl)-4-methyl-1h-pyrazole-3-carboxamide; Tocris Bioscience, Bristol, UK) was initially dissolved in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA), and then diluted in 0.9% saline using cremophore (TWEEN 80; Sigma, St. Louis, MO, USA) to a final DMSO:saline:TWEEN 80 ratio of 2:97:1 (v/v). Leptin (Bachem, Bubendorf, Switzerland), SB (6- chloro-2,3-dihydro-5-methyl-n-(6-((2-methyl-3-pyridinyl)oxy)- 3-pyridinyl)-1H-indole-1-carboxyamide dihydrochloride; Tocris Bioscience), the selective 5-HT 2C receptor antagonist (38) and GR (2 -methyl-4 -(5-methyl-(1,2,4)-oxadiazol-3- yl)biphenyl-4-carboxylic acid (4-methoxy-3-(4-methylpiperazin- 1-yl)phenyl)amide hydrochloride; Axon Medchem), the 5- HT 1B/1D antagonist (39), were dissolved in 0.9% sterile saline. All solutions, including drug combinations, were administered intraperitoneally with a single injection of 0.1 ml per 100 g body weight (b.w.). All drug doses indicated in the procedures (see below) were based on our previous results and on the findings of other laboratories (12, 40-43). Procedure Rats were fed standard rodent chow (LSM, Agropol, Motycz, Poland; energy value = 260 kcal/100 g). Rats received a preweighed amount (100 g) of food and tap water every day between 4.00 and 4.30 p.m. Food consumption was evaluated by measuring both the amount of food left in the container and that of the food spilled and collected from the bottom of the cage. Baseline food intake was recorded daily with 24-h intervals for 3 days before drug treatment. The rats were injected with the drugs once a day for the next 3 days, and food consumption was measured throughout this period 24 h after each injection. Body weight was measured at the beginning of the experiment, at the end of the 3-day-long pretreatment period, and, finally, at the end of the post-treatment period (the 3 rd measurement was performed 24 h after the last injection). The body weight measured at the last two time points was then compared with the initial body weight, and the differences were referred to as weight changes. To avoid errors arising from bias, the protocol included two blind experiments. In the first experiment, rats were divided randomly into experimental groups (n = 10 for each group), and we investigated the effects of leptin and AM 251 coadministration on food intake and body weight. Rats in various experimental groups were injected with 0.9% saline (control), 100 µg/kg b.w. leptin, 1 mg/kg b.w. AM 251, or 100 µg/kg leptin + 1 mg/kg AM 251. Immediately after the injection, the animals were placed into their cages with free access to food and water. In the second experiment, we investigated the effects of 5-HT 1B and 5-HT 2C serotonin receptor blockade on the leptin- and AM 251-induced changes in food consumption and body weight. To this end, rats were divided randomly into 6 groups (n = 7 in each group), receiving 0.9% saline, 100 µg/kg leptin + 1 mg/kg AM 251, 0.5 mg/kg b.w. SB , 3 mg/kg b.w. GR , SB AM leptin, or GR AM leptin. Statistical analysis Data were analyzed using the two-way repeated measures analysis of variance (ANOVA). The Bonferroni post-hoc multiple comparison test was used to determine differences between groups when significant primary effects (drug time) were found. Data were analyzed using the statistical software STATISTICA 10 (Statsoft, Cracow, Poland). For all analyses, groups were considered significantly different if P 0.05.

3 459 RESULTS Effects of leptin and AM 251 co-administration are shown in Fig. 1. ANOVA revealed a significant effect of time and drug time interaction (F 1,112 = 20.09, P < 0.001; F 3,112 = 4.05, P < 0.01; respectively) in the total daily calorie intake. Whereas there were no significant changes in the daily calorie intake when AM 251 or leptin were injected individually, leptin co-injected with AM 251 significantly decreased chow consumption compared with the basal value, leptin treatment alone, AM 251 treatment alone, or the control treatment (all P < 0.001; Fig. 1A). In addition, ANOVA revealed significant effects of time and drug time interaction (F 1,37 = 10.85, P < 0.01; F 3,37 = 4.24, P 0.05, respectively) in body weight changes. Leptin and AM 251 injected individually did not affect the body weight significantly. However, the decreased food intake after the co-administration of leptin and AM 251 was associated with a significant reduction in body weight compared with the pretreatment values in this group (P < 0.01), as well as compared with the body weight in the control and leptin-treated animals (both P < 0.01; Fig. 1B). Next, we investigated whether the blockade of serotonergic transmission affects the leptin and AM 251-dependent changes in food consumption and body weight (Fig. 2). ANOVA revealed a significant drug time interaction (F 5,101 = 3.59, P < 0.01) in food intake. The post-hoc test confirmed that leptin co-injected with AM 251 significantly reduced the food intake at later time points of the experiment compared with the basal consumption at the beginning of the experiment (P < 0.01), and compared with the food intake after control treatment (P < 0.001), the coinjection of SB AM leptin (P < 0.001), and the co-administration of GR AM leptin (P 0.05). Neither SB nor GR , administered alone or with leptin and AM 251, had a significant effect on food intake (Fig. 2A). Furthermore, ANOVA revealed a significant drug time interaction (F 5,34 = 4.53; P < 0.01) and the effect of time (F 5,34 = 8.18; P < 0.001) on body weight changes. The 5-HT 2C receptor antagonist SB and 5-HT 1B antagonist GR had no effect on body weight when injected individually. On the other hand, in rats with a suppressed calorie intake, co-treatment with leptin and AM 251 was accompanied by a significant weight loss A Daily food intake (kcal) CONTROL AM LEP AM+LEP B Body weight changes (g) CONTROL AM LEP AM+LEP DAYS #, +,! Fig. 1. Effects of single or combined treatment with 100 µg/kg leptin (LEP) and 1 mg/kg AM 251 (AM) on the average daily calorie intake (panel A) over the 3-day-long pretreatment (white bars) and post-treatment (colored bars) periods. Body weight was measured at the beginning of the experiment and at the end of the preand post-treatment period to calculate body weight change (panel B). The arrow indicates the day of the first injection. Data represent mean ± standard error of mean. P < 0.001; significant differences in body weight were found compared with the initial value in the same group (#), and compared with the body weight of saline-injected controls (+) and leptininjected rats (!) (all P < 0.01).

4 460 Daily food intake (kcal) CONTROL SBS GR AM+LEP SB+AM+LEP GR+AM+LEP CONTROL GR AM+LEP SB+AM+LEP SB GR+AM+LEP Body weight changes (g) Days Fig. 2. Effects of 5-HT 1B receptor and 5-HT 2C receptor blockade by 3 mg/kg GR (GR) and 0.5 mg/kg SB (SB), respectively, on the average daily calorie intake (panel A) over the 3-day-long pretreatment (white bars) and post-treatment (colored bars) periods in rats co-treated with 100 µg/kg leptin (LEP) and 1 mg/kg AM 251 (AM). Body weight was measured at the beginning of the experiment and at the end of the pre- and post-treatment period to calculate body weight change (panel B). The arrow indicates the day of the first injection. Data represent mean ± standard error of mean. P < 0.05, P < 0.01, P < 0.001; significant differences in body weight change were found compared with that in saline-injected controls ( # P < 0.001), as well as in LEP + AM + GR-injected ( + P < 0.05) and LEP + AM + SB-injected (! P < 0.001) rats. compared with the control (P < 0.001), SB AM leptin (P < 0.001), and GR AM leptin treatments (P 0.05; Fig. 2B). DISCUSSION We have demonstrated that co-administration of leptin and AM 251 suppressed food intake and reduced body weight in rats fed a standard diet, even if these compounds were administered at doses that are subanorectic individually. However, we have shown for the first time that this suppressant effect was totally abolished by the simultaneous injection of 5-HT 2C and 5-HT 1B receptor blockers. These results indicate that rats with blocked 5-HT 1B and 5-HT 2C receptors are not sensitive to the hypophagic and body weight-reducing effects of leptin and AM 251 co-treatment. Earlier studies demonstrated that AM 251 injected individually at a dose employed in our study did not suppress the daily food intake in rats fed single (no-choice) standard (44) or high-fat diets (45). In addition, this treatment was not able to reduce body weight regardless of the diet used (12, 43-45). Therefore, our results are consistent with these findings, and

5 461 they prove that we used AM 251 at a subanorectic dose. Similarly, leptin administered alone at a relatively low dose did not produce any significant hypophagic or body weight reducing effects in the present study, and this is consistent with previous results (46, 47). In contrast, the concomitant administration of leptin and AM 251 at subanorectic doses resulted in the reduction of both body weight and food intake. Similarly, the additive effects of these drugs administered at subanorectic doses have been reported previously in rats fed highcarbohydrate or high-fat diets (12). Therefore, leptin and AM 251 reciprocally enhance each other s effects on food consumption and body mass, and their co-administration causes a negative energy balance in rats maintained on normal or highcalorie diets. Due to this additive enhancement, a combined therapy might allow the reduction of body weight and food consumption at lower drug doses; therefore, it might decrease the risk of side effects. Next, we investigated the possible role of serotonergic transmission in regulating food intake and body weight changes caused by leptin and AM 251 co-treatment. We demonstrated that the blockade of the 5-HT 1B and 5-HT 2C receptors completely abolished the anorectic and body-mass reducing effects of leptin co-injected with AM 251. Therefore, the normal activity of these receptors is necessary for the aforementioned metabolic effects of leptin and the CB 1 receptor antagonist AM 251. This finding supports the hypothesis that the hypophagic effects of leptin and CB 1 receptor antagonists require the activation of the serotonergic pathways. Previous studies suggested that CB 1 receptor blockade enhances the anorectic action of leptin (11, 12), and serotonin mediates the action of leptin and endocannabinoids on energy homeostasis (34, 48). In particular, pharmacological inactivation of the 5-HT 2C receptor by SB was found to abolish the anorectic effects of centrally injected leptin or AM 251 in rats (37, 49). Other studies (31), however, contradicted the thesis that serotonin is involved in a leptin-dependent effect on appetite. Hence, our work supports the view that serotonin mediates the action of leptin and endocannabinoids on neural circuits controlling food intake and demonstrates for the first time that serotonin is involved in the interaction of leptin and AM 251 via the 5-HT 1B and 5-HT 2C receptors. Single administration of serotonin antagonists did not affect feeding behavior, which is consistent with previous findings showing that single administration of GR and SB , used at the same or similar doses as in the present study, inhibited the hypophagic effects of serotonin 5-HT 1B and 5-HT 2C receptor agonists or fenfluramine in rats without affecting food intake (21, 38, 41, 42, 50). This suggests that the doses of both serotonin receptor antagonists employed in our study effectively influenced the activity of their specific receptors, even though they did not affect food intake and body weight. Centrally injected leptin and AM 251 increase serotonin secretion in the hypothalamus (34, 51) and thus serotonin might mediate the anorectic action of these compounds via the 1B and 2C receptors, which have been detected in the hypothalamic feeding nuclei (29, 30). Stimulation of the former receptor attenuates orexigenic drive, whilst the stimulation of the latter activates the anorexigenic neurons promoting hypophagia (28). Consequently, the pharmacological blockade of these receptors might prevent the serotonin-mediated action of leptin and AM 251 on feeding circuits, which we indeed found in our study. Furthermore, synergy between leptin and CB 1 antagonists in modulating serotonin secretion might explain why these compounds have no effects on food intake and body weight when they are used separately. Apparently, leptin and AM 251 injected individually at relatively low doses cannot activate serotonin secretion at sufficient levels and cause anorexia. However, if these drugs are applied together, they can additively increase serotonin levels in the hypothalamus, which results in hypophagia due to the adequate stimulation of the aforementioned serotonin receptors. Another CB 1 receptor inverse agonist, rimonabant, also enhances the anorectic effect of leptin (11) and increases serotonin release in the brain (52), which supports our hypothesis. In summary, we have shown that the simultaneous administration of leptin and a CB 1 receptor antagonist induces hypophagia and body weight-reduction by modulating the 5- HT 2C and 5-HT 1B receptor-mediated serotonin signaling in rats. 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7 463 interleukin-1 beta-, and leptin-induced anorexia. Pharmacol Biochem Behav 2003; 74: Clifton PG, Lee MD, Dourish CT. Similarities in the action of Ro , a 5-HT2C receptor agonist and d- fenfluramine on feeding patterns in the rat. Psychopharmacology 2000; 152: Telles MM, Guimaraes RB, Ribeiro EB. Effect of leptin on the acute feeding-induced hypothalamic serotonergic stimulation in normal rats. Regul Pept 2003; 115: Tzavara ET, Davis RJ, Perry KW, et al. The CB1 receptor antagonist SR141716A selectively increases monoaminergic neurotransmission in the medial prefrontal cortex: implications for therapeutic actions. Br J Pharmacol 2003; 138: Received: January 21, 2016 Accepted: April 15, 2016 Author s address: Prof. Ewa Bojanowska, Department of Behavioral Pathophysiology, Institute of General and Experimental Pathology, Medical University of Lodz, 60 Narutowicza Street; Lodz, Poland. ewa.bojanowska@umed.lodz.pl