NPY receptors and opioidergic system are involved in NPY-induced feeding in goldfish

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1 Peptides 21 (2000) NPY receptors and opioidergic system are involved in NPY-induced feeding in goldfish Nuria de Pedro, Marcos A. López-Patiño, Ana I. Guijarro, M a Luisa Pinillos, M a Jesús Delgado*, Mercedes Alonso-Bedate Departamento de Biología Animal II, Facultad de Biología, Universidad Complutense, Madrid, Spain Received 17 May 2000; accepted 13 July 2000 Abstract The present study evaluated the effects of both intraperitoneal (i.p.) and intracerebroventricular administration of selective Y 1 [(Leu 31, Pro 34 )-NPY] and Y 2 [(Pro 13, Tyr 36 )-NPY (13 36)] receptor agonists on food intake in satiated goldfish. Food intake (FI) was significantly increased by central administration of the Y 1 agonist (1 g), but not by the Y 2 agonist, at 2 h postinjection. The feeding increase induced by (Leu 31, Pro 34 )-NPY was in a similar magnitude to that obtained after ICV injection of the neuropeptide Y, and both feeding stimulations were reversed by the NPY (27 36), a general NPY antagonist. The i.p. administration of the agonists either did not significantly modify (Y 2 agonist) or decreased (Y 1 agonist) food intake in goldfish. These data indicate that it is the Y 1 -like (similar to Y 1 and/or Y 5 ) receptor, and not Y 2, that is involved in the central modulation of the feeding behavior in goldfish. We also investigated the possible involvement of opioid peptides as mediators of the NPY stimulatory action on food intake in goldfish. The ICV administration of naloxone (10 g), a general opioid antagonist, blocked the NPY-induced feeding in goldfish, suggesting that the opioidergic system is involved in feeding regulation by NPY Published by Elsevier Science Inc. Keywords: NPY, neuropeptide Y; Fish; Goldfish; NPY receptors; Agonists; Antagonists; Food intake; Feeding; Opioid receptors; Naloxone 1. Introduction * Corresponding author. Tel.: ; fax: address: mjdelgad@bio.ucm.es (M.J. Delgado). Neuropeptide Y (NPY), a 36 amino acid peptide, is found in all vertebrates so far investigated and is highly conserved across a variety of species [19], which suggests that this neuropeptide plays key roles in ensuring basic physiological functions. Central administration of NPY induces a powerful increase in food intake in different groups of vertebrates: mammals, such as rat [13,41], mouse [27] rabbit [31], squirrel [4], pig [30] and sheep [25]; birds [34], and reptiles [28]. Recently, studies carried out in our laboratory have shown for the first time that NPY is also involved in the feeding central regulation in fish [23]. Particularly, we reported that ICV administration of NPY increases food intake in goldfish (Carassius auratus) at 2 h postinjection, without significant modifications of food consumption when this peptide was peripherally administered. Neuropeptide Y mediates their actions through G protein-coupled receptors called Y receptors. To date, multiple NPY receptor subtypes have been identified in mammals and they have been classified as Y 1,Y 2,Y 3,Y 4,Y 5 and Y 6 [1,3,20]. All of these receptors have been cloned, with the exception of the Y 3 subtype. The NPY receptor family contains three receptors that share a higher amino acid sequence identity to one another, and exhibit similar pharmacological properties, namely the Y 1 -Y 4 -Y 6 subfamily; and two receptors that are more divergent, Y 2 and Y 5.In fish, three NPY receptor genes have recently been cloned in the zebrafish (Danio rerio), called zy a,zy b and zy c [24, 35,43]. The zy a receptor displays a unique pharmacological profile, whereas zy b is quite similar to zy c (76%), and both are closely related to the cod (Gadus morrhua) Y b receptor [40]. These recently cloned NPY receptors in fish seem to be distinct from the mammalian receptor subtypes cloned to date [21]. However, it remains to be resolved if additional subtypes are still to be discovered in each lineage, or if fish and mammals have distinct repertoires of NPY receptor subtypes. The identity of NPY receptor subtypes involved in mediation of the feeding response by NPY-related compounds /00/$ see front matter 2000 Published by Elsevier Science Inc. PII: S (00)00303-X

2 1496 N. de Pedro et al. / Peptides 21 (2000) is unclear. Initially, it was suggested that NPY-induced feeding in mammals was mediated by Y 1 receptors [22]. Recent studies have pointed to the involvement of either the Y 5 or other, currently unidentified receptor subtypes, in the control of feeding by NPY [11,37]. The neuroendocrine control of food intake and energy balance is a complex process controlled by many overlapping integrated pathways. There is some information on the possible interaction between NPY and opioid peptides in feeding regulation. It has been documented that opioid agonists increase food intake when centrally administered in different vertebrates groups [29], including fish [6,9]. Several studies have shown that peripheral and central administration of opioid antagonists blocks the NPY-induced feeding increase in rats [16,18,33,36,38]. These studies suggest that NPY can depend upon an opioid pathway to increase feeding. Whether similar neurochemical processes also operate in fish remain to be elucidated. To date, the mechanisms governing the stimulatory action of NPY on feeding in fish have not been deeply investigated. Recently, we have reported that feeding stimulation by NPY is mediated through specific receptors in goldfish [23], like in mammals [1,2]. The present experiments focused on investigating the effects of different selective NPY agonists on food intake in goldfish and on identifying the particular receptor subtype involved in NPY-induced feeding. Moreover, taking in mind the stimulatory effect of opioids on feeding in teleosts [6,9], we also investigate the involvement of this neuropeptidergic system as a possible mediator of the NPY action on food intake in C. auratus. 2. Method 2.1. Animals Experiments were performed in goldfish, Carassius auratus, ( g body weight, bw) provided by a commercial supplier in Madrid. Fish were acclimated to a 12L:12D photoperiod and a 21 2 C water temperature in 50 liters aquaria, with a constant flow of filtered water. Feeding consisted in Sera Biogran pellets at a daily ration of 1% bw at 10:00 11:00 h. Animals were acclimated to these conditions for at least 15 days before the experiments were carried out Drugs and treatment Porcine NPY; (Leu 31, Pro 34 )-NPY; (Pro 13, Tyr 36 )-NPY (13 36); naloxone (SIGMA) and (D-Tyr 27,36, D-Thr 32 )- NPY (27 36) (RBI) were dissolved in teleost saline (20 mg Na 2 CO 3 per 100 ml of 0.6% NaCl). The intracerebroventricular (ICV) injections were accomplished with a 0.3 mm Microlance needle connected to a 5 l Hamilton microsyringe with a 18 Venocath cannula. The i.p. (IP) injections were carried out with a1mlsyringe and a 0.3 mm Microlance needle, close to the ventral midline posterior to pelvic fins. The ICV and IP procedure and the accuracy of injection placement into the ventricular regions of the fish brain were previously established [5] Experimental design To ensure that the fish were satiated at the time of the feeding test, the animals were fed 1 h before the injections. The goldfish were anesthetized in water containing tricaine methanesulphonate (MS-222, 1:10,000) and injected between 10:30 11:30 h. Fish recovered equilibrium and normal swimming activity in anesthetic-free water within 1 2 min after the injections. A detailed description of the food intake quantification in goldfish has been previously reported [5]. Briefly, individual goldfish received an excess of preweighed food (5% body weight) and food intake (FI) was evaluated at 2 (in all experiments), and 8 (experiments 1.1, 1.2 and 2) h postinjection as FI W i -(W f F), where W i is the initial dry food weight, W f is the remaining dry food weight and F is a correction factor calculated to determine the effect of water dissolution on food pellets during the feeding time Experiment 1. Involvement of Y 1 receptors in the feeding regulation by NPY Three experiments were carried out. Experiment 1.1: animals were ICV injected with either 1 l teleost saline alone (control group, n 10) or containing the Y 1 agonist (Leu 31, Pro 34 )-NPY (experimental groups) at doses of 0.33 (n 9),1(n 9) and 3.3 (n 10) g. Experiment 1.2: fish were divided into three groups (n 8/group): a) control group, IP injected with 10 l saline/g bw, and b) (Leu 31, Pro 34 )-NPY groups, IP injected with 0.1 and 0.33 g (Leu 31, Pro 34 )-NPY/g bw. Experiment 1.3: four groups (n 9/group) of fish received two sequential ICV injections: a) control group: two injections of 1 l saline; b) Y 1 agonist group: saline followed by (Leu 31, Pro 34 )-NPY (1 g) injection; c) NPY general antagonist group: 1 l NPY (27 36) (5 g) injection followed by 1 l saline injection; d) NPY (27 36) (Leu 31, Pro 34 )-NPY injection: antagonist (5 g) injection followed by Y 1 agonist (1 g) injection Experiment 2. Involvement of Y 2 receptors in the feeding regulation Experiment 2.1: fish were ICV injected with either 1 l teleost saline by itself (control group) or containing the Y 2 agonist (Pro 13, Tyr 36 )-NPY (13 36) (experimental groups) at doses of 0.33 (n 7),1(n 8) and 3.3 (n 9) g. Experiment 2.2: fish were divided into three groups (n 8/group): a) control group, IP injected with 10 l saline/g bw, and b) Y 2 groups, IP injected with two different doses of (Pro 13, Tyr 36 )-NPY (13 36), 0.1 and 0.33 g/g bw.

3 N. de Pedro et al. / Peptides 21 (2000) Fig. 1. Food intake (mg) following intracerebroventricular (ICV) injection of 1 l saline alone (n 10) or containing the Y 1 agonist (Leu 31, Pro 34 )-NPY, at the doses 0.33 (n 9),1(n 9) and 3.3 (n 10) g, and at 0 2 (top), 2 8 (middle) and 0 8 (bottom) h postinjection in goldfish (Carassius auratus). The x-axis represents absolute doses of the agonist. Data are expressed as mean SEM. ** P 0.01 compared to control group (SNK test). Fig. 2. Effect of i.p. (IP) injection of the Y 1 agonist (Leu 31, Pro 34 )-NPY (0.1 and 0.33 g/g bw) on food intake (mg) in the teleost C. auratus at 0 2 (top), 2 8 (middle) and 0 8 (bottom) h postinjection (n 8/group). Data are expressed as mean SEM. * P 0.05; ** P 0.01 compared to control group (SNK test) Experiment 3. Involvement of opioid receptors as mediators of NPY-induced feeding stimulation Four groups of fish received two sequential ICV injections: a) control group: two injections of 1 l saline (n 16); b) NPY group: saline followed by NPY (1 g) injection (n 17); c) naloxone group: 1 l naloxone (10 g) injection followed by 1 l saline injection (n 8); d) naloxone NPY injection: naloxone (10 g) injection followed by NPY (1 g) injection (n 8) Statistical analysis Data were analyzed by an ANOVA followed by Student- Newman-Keuls (SNK) multiple range test for multigroup

4 1498 N. de Pedro et al. / Peptides 21 (2000) Fig. 3. Food intake (mg) after ICV injection of saline (control); the Y 1 agonist (Leu 31, Pro 34 )-NPY, 1 g; the NPY antagonist NPY (27 36), 5 g; and both, NPY (27 36) (5 g) (Leu 31, Pro 34 )-NPY (1 g) at 2 h postinjection in goldfish (C. auratus) (n 9/group). Data are expressed as mean SEM. * P 0.05; ** P 0.01 (SNK test). comparisons. A probability level of P 0.05 was considered statistically significant. 3. Results Fig. 1 summarizes the effects of ICV administration of different doses of the Y 1 agonist on food intake in goldfish. FI was significantly increased by 1 g ofy 1 agonist with respect to the control group at 2 h postinjection, F(3,32) 12.1, P During the discrete interval 2 8 h, none of the doses tested significantly modified food intake, F(3,34) 0.007, P An increasing trend, although not statistically significant, was observed in cumulative food intake at 8h(1 g), F(3,31) 1.09, P As can be observed in Fig. 2, the IP administration of the Y 1 agonist (Leu 31 -Pro 34 )-NPY induced a decrease of food intake at any of the time intervals studied: 0 2 h (both doses, 0.1 and 0.33 g/g bw), F(2,20) 12.12, P 0.01; 2 8 h (0.33 g/g bw dose), F(2,20) 2.91, P 0.05; and 0 8 h (0.33 g/g bw dose), F(2,20) 4.08, P Fig. 3 shows the effects of the ICV administration of the NPY general antagonist (D-Tyr 27 36, D-Thr 32 -NPY (27 36) either by itself or in combination with the Y 1 agonist. The NPY antagonist by itself did not significantly modify food intake in goldfish. However, it counteracted the stimulatory action of the Y 1 agonist on food consumption in goldfish, F(3,36) 5.95, P In contrast with the effect of the Y 1 agonist, the Y 2 agonist (Pro 13, Tyr 36 )-NPY (13 36) did not significantly alter food intake in goldfish either after the ICV (Fig. 4) or the IP (Fig. 5) injections at any of the tested doses and the time intervals studied. Results obtained from experiment 3 about the possible role of opioid peptides as mediators of eating induced by NPY in goldfish are presented in Fig. 6. As can be observed, naloxone (10 g) totally blocked the feeding increase induced by NPY, reaching food intake values similar to those observed in the control group, F(3,44) 9.798, P Discussion The present study confirms the orexigenic actions of NPY previously described in goldfish [23]. We have also demonstrated that the food intake stimulation with (Leu 31, Pro 34 )-NPY (Y 1 agonist), but not with NPY (13 36) (Y 2 agonist), occurs in a similar magnitude to that obtained with the endogenous peptide. The ICV administration of the Y 1 agonist (1 g) increased food intake at 2 h postinjection, but

5 N. de Pedro et al. / Peptides 21 (2000) Fig. 4. Food intake (mg) following intracerebroventricular (ICV) injection of 1 l saline (n 10) or containing the Y 2 agonist (Pro 13, Tyr 36 )-NPY (13 36), at the doses 0.33 (n 7),1(n 8) and 3.3 (n 9) g, and at 0 2 (top), 2 8 (middle) and 0 8 (bottom) h postinjection in goldfish (Carassius auratus). The x-axis represents absolute doses of the agonist. Data are expressed as mean SEM. Fig. 5. Effect of i.p. (IP) injection of the Y 2 agonist (Pro 13, Tyr 36 )-NPY (13 36) (0.1 and 0.33 g/g bw) on food intake (mg) in the teleost C. auratus at 0 2 (top), 2 8 (middle) and 0 8 (bottom) h postinjection (n 8/group). Data are expressed as mean SEM. not during the next discrete time interval (2 8 h), as occurs after NPY central injection. Thus, we suggest that both ligands act at a short time interval in goldfish. Moreover, the NPY receptor antagonist, NPY (27 36), reversed the feeding increase induced by both NPY and (Leu 31, Pro 34 )-NPY. These findings lead us to discard any involvement of Y 2 receptors in the control of food intake and strongly suggest that Y 1 -like receptors could mediate the central action of NPY on feeding in goldfish. Several reports have indicated the important role of hypothalamic Y 1 receptors in mediating NPY-induced feeding in mammals. Selective Y 1 receptor agonists injected directly into the hypothalamus or in the third ventricle increased food intake in rat [14], whereas selective Y 2 receptor agonists were ineffective in producing an increase in feeding [42]. Moreover, Y 1 receptor antagonists inhibited the eating response evoked by NPY in rat [15].

6 1500 N. de Pedro et al. / Peptides 21 (2000) Fig. 6. Food intake (mg) in C. auratus at 2 h after the ICV injection of the opioid peptides general antagonist, naloxone (10 g), alone or in combination with NPY (1 g). Data are expressed as mean SEM. ** P 0.01 (SNK test). The Y 1 receptor subtype exhibits high affinity for full length NPY molecules or full length analogues such as (Leu 31, Pro 34 )-NPY, and low affinity for the carboxyl-terminal fragments, such as NPY (13 36). In contrast, Y 2 subtype binds C-terminal fragments with high affinity [2,3]. The fact that the Y 1 agonist (Leu 31, Pro 34 )-NPY, but not the Y 2 agonist NPY (13 36), increases food intake in fish, support the general agreement in mammals that the entire molecule is required for the stimulatory action of the neuropeptide [2]. Nevertheless, recent studies have shown that deletion of the amino terminal tyrosine residue results in NPY (2 36), a peptide even more potent than NPY in stimulating feeding in rats [39]. These results led to the hypothesis that an atypical Y 1 receptor would mediate the feeding response, probably Y 5, which binds NPY (2 36) with high affinity. It is likely that both Y 1 and Y 5 receptors together contribute to the NPY-mediated regulation of food intake, or even that a novel receptor functionally distinct from the recognized receptor subtypes in mammals (but with characteristics of both Y 1 and Y 5 receptors) may be involved in feeding [11,12,37]. Keeping in mind all this data and the higher affinity of (Leu 31, Pro 34 )-NPY for Y 1 and Y 5 receptors rather than for Y 2, and that the cloned receptors in fish are distinct from receptor subtypes described in mammals [21,40], we suggest that Y 1 -like (similar to Y 1 and/or Y 5 ) receptors would mediate the effects of NPY on feeding in goldfish. Nevertheless, the lack of selective antagonists commercially available for fish receptors to date does not allow us to demonstrate such a conclusion. Particular mechanisms for explaining the food intake reduction observed after i.p. administration of the Y 1 agonist (Leu 31, Pro 34 )-NPY are unknown at present. (Leu 31 - Pro 34 )-NPY which has traditionally been used to define Y 1 receptors also binds with high affinity to Y 4 receptors [8]. Y 4 receptors are predominantly expressed in peripheral tissues and exhibit high affinity for the pancreatic polypeptide (PP) [1,3]. Thus, it can be suggested that feeding decrease induced by IP administration of (Leu 31 - Pro 34 )-NPY in goldfish could be mediated by the Y 4 receptor. Further studies would be necessary to elucidate this subject. Finally, although (Leu 31 - Pro 34 )-NPY can bind to Y 1,Y 5, and Y 4, we discarded the idea that the feeding stimulation after ICV injection of this ligand could be via Y 4 receptors, on the basis that such receptor subtypes are practically undetectable in the brain [1,3]. It is known that NPY interacts with the opioid peptides in regulating feeding behavior in mammals [26]. This idea is

7 N. de Pedro et al. / Peptides 21 (2000) supported by a neural anatomic interaction between hypothalamic NPY and the opioid system. Synaptic connections between NPY nerve terminals and opioid-containing neurons has been observed in the rat hypothalamus [11]; NPY stimulates -endorphin release from the hypothalamus [32]; and opioid agonists and antagonists increase and decrease, respectively, hypothalamic expression of NPY [17], indicating a possible feedback monitoring NPY-opioid interaction. -endorphin stimulates food intake in C. auratus [6,9], and so our hypothesis states that opioids can act as mediators in the orexigenic effect of NPY. To test this possibility we used naloxone, a nonselective opioid antagonist that blocks the -endorphin-induced feeding increase in goldfish [6]. Our results from the present study show that NPYinduced feeding is antagonized by naloxone. This is in agreement with previous reports in rats, where central and peripheral administrations of naloxone reduces the NPYinduced food intake increase [16,18,33,36,38]. Thus, we suggest that in fish NPY can also regulate food intake via an opioid related pathway. The use of selective opioid receptor antagonists has suggested that mu and kappa (but not delta) opioid receptors would mediate the feeding stimulatory action of NPY in rats [16,18]. To date, it is known that the -endorphin effects on feeding in fish are mediated via mu receptors [7], which could represent the opioid receptor subtype involved in the feeding action of NPY in goldfish. A possible model of the pathways of NPY-opioids circuitry controlling feeding in fish would be the following: NPY binding through the Y 1 -like receptor would induce the synthesis and/or release of endogenous -endorphin, which would increase food intake via receptors. 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