[1] 17w NPY-induced feeding involves the action of a Y1-like receptor in rodents a , a a a a

[1] 211w We have reported that the potent peptidic Y1 antagonist, 1229U91, significantly suppressed NPY-induced and spontaneous feeding [32,33]. However, information on the precise selectivity of 1229U91 for NPY receptors is lacking. The Y5 receptor has been considered a key receptor for feeding regulation. In the present study we showed that 1229U91 has high affinities for the human and rat Y1 receptors (K 5 0.041 nM and 0.16 nM, respectively) and also a high affinity for the human Y4 receptor (K 5 0.33 nM), whereas it shows moderate i i affinities for the human Y2, Y5 and rat Y5 receptors (K values of 20-170 nM). Moreover, 1229U91 potently inhibits NPY-induced i 21[Ca ]i increases in cells expressing human Y1 receptors. In contrast, 1229U91 is an agonist at other NPY receptors like the Y2, Y4 and Y5 receptors. Intracerebroventricular (ICV)-injected 1229U91 (30 mg / head) significantly suppressed human NPY-induced feeding in SD rats, while 1229U91 only moderately inhibited bovine pancreatic polypeptide (bPP; an in vivo Y5 agonist)-induced feeding. These results indicate that the food intake evoked by NPY might be mediated by the Y1 receptor, rather than the Y5 receptor. Thus, the Y1 receptor or possibly a novel Y1-like receptor sensitive to 1229U91 may play a key role in the regulation of NPY-induced feeding.

[1] 259w NPY significantly reduce food intake [38][39][40][41]. In support of a physiological role for NPY in food intake, it was also Neuropeptide Y (NPY) is a 36-amino acid polypeptide reported that NPY-deficient ob / ob mice are less obese than belonging to the pancreatic polypeptide family, which ob / ob mice. NPY-deficient ob / ob mice also exhibit reincludes NPY, peptide YY (PYY) and pancreatic poly-duced food intake when compared to ob / ob mice [37]. For peptide (PP) [1,2]. NPY is highly concentrated in the this data it is inferred that NPY may be one of the major hypothalamus [3,4], and is a potent ICV stimulant of regulators of physiological feeding behavior. feeding behavior [5][6][7][8][9]. Chronic administration of NPY Recently, five distinct types of NPY receptors, Y1, Y2, into the brain results in hyperphagia and body weight gain, Y4, Y5 and Y6 have been cloned [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29]. Given the reduces energy expenditure, and increases lipogenic activi-correlation between the in vitro functional and binding ty in the liver and adipose tissue [10,11]. It has been also activity of different peptidic agonists at the Y5 receptor reported that concentrations of NPY and its mRNA in the and their potent stimulation of food intake in rodent hypothalamus are markedly increased during food depriva-models, the Y5 receptor was proposed as a likely feeding tion and in some genetic models of obesity in rodents receptor. In support of this model, mRNA encoding the Y5 [12][13][14]36]. Antibodies and antisense oligonucleotides to receptor is predominantly expressed in the brain, including hypothalamic nuclei involved in feeding behavior [25].

[2] 115w 1229U91, significantly suppressed NPY-induced, fasting-7.4) containing 10 mM MgCl , 1 mM PMSF, 0.1% 2 induced and spontaneous feeding [32,33]. We show that bacitracin and 0.5% BSA. The membranes (100-300 mg / 125 1229U91 is a potent antagonist for the Y1 receptor, while ml) were incubated at 258C for 120 min with [ I]PYY 125 it acts as an agonist at the other NPY receptors including (25 pM) and [ I]PP (25 pM), respectively. Bound and the Y5 receptor. Using the potent Y1 antagonist, 1229U91, free peptides were separated by filtration using a GF / C we investigate the participation of Y1 and Y5 receptors in glass filter (Whatman, UK)) presoaked with 0.3% polyingestive behavior.

[3] 16w ethylenimine. The remaining radioactivity on the filter was TM quantitated using a Cobra (Packard, Japan). Specific

[4] 210w 125 125 binding of [ I]PYY and [ I]PP was defined as the 2. Materials and methods difference between total binding and nonspecific binding in the presence of 1 mM PYY and PP, respectively. 35 2.1. Materials Binding of [ S]GTPgS activated by NPY to membrane preparations was performed in 0.5 ml of the binding assay 1229U91 [(IleGluProDprTyrArgLeuArgTyr-NH ) , buffer containing 100 mM NaCl and 1 mM GDP in the 2 2 cyclic (2,49),(29,4)-diamide)-amide] (Dpr: 2,3-diaminoppresence of 100 nM NPY. The membranes (200-400 35 ropionic acid) was synthesized as described previously mg / ml) were incubated at 308C for 1 h with [ S]GTPgS [32]. Human NPY (hNPY) and bovine PP (bPP) were (150 pM). Separation of free ligand was performed purchased from Peptide Institute (Osaka, Japan). applying the filtration methods mentioned above. The TM 125 125 [ I]PYY and [ I]PP were from New England Nuclearradioactivity of the filter was determined by TopCount TM DuPont (Boston, MA). The culture reagents and bovine (Packard, Japan) with Microscint -0 (Packard, Japan). serum albumin (BSA) were obtained from GIBCO (Grand Island, NY). Bacitracin, phenylmethylsulfonyl fluoride 2.4. Measurement of intracellular calcium ion (PMSF) and polyethylenimine were obtained from Sigma concentrations (St. Louis, MO). All other chemicals were of analytical grades. 21 21

[5] 292w [Ca ]i was measured fluorometrically using a Casensitive fluorescent dye, fura-2. The cells expressing 2.2. Cell culture human NPY receptors were harvested using 0.25% trypsin 7 and 0.02% EDTA. The cells (1.0 3 10 cells) were washed CHO, LMtk-and COS-7 cells were obtained from the once with Krebs-Henseleit Hepes buffer containing 0.1% ATCC (Rockville, MD). CHO cells expressing recombi-BSA (pH 7.4), suspended in 1 ml of the buffer and nant human Y1, Y2 and Y4 receptors were grown in incubated with 2 mM fura-2 acetoxymethylester at 378C Iscove's modified Dulbecco's medium supplemented with for 60 min. The fura-2-loaded cells were washed with the 10% fetal bovine serum (FBS), penicillin-G (100 IU / ml), buffer and resuspended in 10 ml of the buffer. In a cuvette, streptomycin (100 mg / ml) and G418 (1 mg / ml). LMtk-0.5 ml of the resultant suspension was stirred continuously cells expressing the recombinant human Y5 receptor were at 378C during the measurement. Test compounds or grown in Dulbecco's modified Eagle's medium (high vehicle were added 5 min before the addition of NPY and glucose) with 10% FBS, penicillin-G (100 IU / ml), strepthe related ligands, and fluorescent intensity at an emission tomycin (100 mg / ml) and G418 (0.8 mg / ml). COS-7 cells wavelength of 500 nm and excitation wavelengths of 340 transiently expressing recombinant rat Y1 and Y5 reand 380 nm were monitored with a CAF-110 intracellular ceptors were grown in Dulbecco's modified Eagle's 21 ion analyzer (JASCO, Tokyo, Japan), and [Ca ]i values medium supplemented with 10% FBS, penicillin-G (100 were calculated according to the previously reported IU / ml) and streptomycin (100 mg / ml). These cells were method [34]. grown in a 95% air, 5% CO humidified atmosphere at 2 378C.

[1] 79w We earlier reported that 1229U91 is a potent Y1 receptor (IC value of 6.3 nM)(Table 1). 1229U91 antagonist which can suppress NPY-and fasting-induced 50 behaved as an agonist on the hY2, hY4, hY5 and rY5 feeding in SD rats in a dose-dependent manner [32]. receptors (Figs. 2 and 3, Table 1). We failed to observe any 1229U91 also potently attenuated spontaneous feeding in significant inhibition of 1229U91 on 100 nM NPY-stimu-Zucker fatty rats, when compared to lean littermates [33].

[2] 209w Table 1 In vitro profiles of 1229U91 for cloned NPY receptors a hY1 rY1 hY2 hY4 hY5 rY5 125 [ I]PYY binding (K :nM) i hNPY 0.45 1.05 0.09 180 0.70 0.72 pNPY(13-36) 38 270 0.30 660 23 9.0 bPP 42 23 . 1000 0.09 1.9 1.6 1229U91 0.041 0.16 20 0.33 48 170 21 NPY-induced [Ca ]i increase (IC :nM) 50 b b 1229U91 3.2 6.3 . 10 000 NT . 10 000 . 10 000 21 bPP-induced [Ca ]i increase (IC :nM) 50 3.6 NT NT ND . 10 000 NT b (Agonism:EC nM) (4300) (2.2) (6600) (10 000) 50 a 125 [ I]PP binding. b 35 [ S]GTPgS binding. ND 5 not detected, NT 5 not tested. 21 Fig. 2. Effects of 1229U91 on mobilization of [Ca ]i in LMtk-cells expressing human Y5 receptors. (A) Agonistic effects of NPY, bPP and 1229U91. (B) 21 1229U91 antagonism against 100 nM NPY-induced [Ca ] increase in the cells. Different concentrations of 1229U91 were added 5 min prior to the i addition of 100 nM NPY at 378C. The results are expressed as a percentage of the maximal 1 mM (A) or 100 nM (B) effects, respectively. Each point shows the mean6SE from three independent experiments performed in duplicate.

[3] 25w An accurate interpretation of this data requires knowledge models [28-30]. Finally, the pharmacological action of of the exact selectivity of 1229U91 for the NPY receptors.

[4] 41w NPY, PYY and PYY3-36 at the Y3 receptor, which has not We showed that 1229U91 has high affinities for Y1 and Y4 yet been cloned, indicates that the Y3 receptor dose not receptors and moderate affinities for Y2 and Y5 receptors.

[5] 52w play a crucial role in food intake. The action of 1229U91 Furthermore, 1229U91 is an agonist for all of these NPY at the Y1 and Y5 receptors was therefore evaluated in receptors except for the Y1 receptor, on which it acts as an more detail. antagonist. Delineating which NPY receptors are involved

[6] 176w The Y5 receptor has been proposed to be involved in the in NPY-induced feeding, we considered the following: the control of NPY-induced feeding since the rank order Y2 and Y4 agonists fail to modulate feeding after ICV-potency of NPY and the related ligands to stimulate food injection in rats [6,25,35] and therefore will not be further intake correlated reasonably well with their pharmacoconsidered. The recently cloned mouse Y6 receptor is logical binding profiles at the Y5 receptor [25]. We showed absent from the rat genome and this receptor therefore that an in vivo Y5 agonist, bPP, significantly induced food does not require further consideration in rat food intake intake following its ICV-injection. 1229U91 is a weak Fig. 4. Effects of 1229U91 on the response to 5 mg hNPY or bPP. ** P , 0.01 compared with rats injected with NPY alone. The graphs show the cumulative food intake in Sprague-Dawley rats for 2 h after ICV injection of drugs. Data are represented as the mean6SE. n 5 9-11 rats / group (ANOVArep followed by Benfferoni test).

[7] 264w agonist for the Y5 receptor with a moderate binding Y1 receptors, suggesting that NPY-induced feeding might affinity. 1229U91 failed to significantly suppress bPP-be predominantly caused by the action of Y1 receptors. induced feeding mediated by the Y5 receptor. Moreover, In addition to a proposed role for the Y1 receptor in we did not detect any antagonistic activity of 1229U91 on NPY-induced feeding, it is conceivable that an additional NPY-induced and bPP-induced signal transductions as yet undefined NPY receptor plays a major role in the through the Y5 receptor even at high doses tested in control of NPY-induced food intake. This consideration is 21 35 [Ca ]i response and [ S]GTPgS binding experiments. fueled by the observation that PYY(3-36) which does not These results indicate that the anorexigenic effect of recognize the Y1 receptor is the most potent feeding 1229U91 might not be mediated through its action at the stimulant of the NPY-related ligands [35]. The use of Y5 receptor. From the data, we infer that the Y5 receptor 1229U91 does not therefore allow us to discriminate its does not seem to play a crucial role in the regulation of action at the Y1 receptor from its action at a novel Y1-like NPY-induced feeding in rats. Conclusive evidence for this receptor and additional experimentation is needed to model will require the use of selective Y5 antagonists in resolve this issue. rodent models of food intake. These results suggest that We conclude that 1229U91 is a potent Y1 antagonist 1229U91 inhibits food intake through inhibition of typical with a moderate agonistic activity for the Y5 receptor.

[8] 7w pharmacology of a recombinantly expressed neuropeptide receptor.

[9] 12w Deduced from the effects of 1229U91 on feeding behavior Mol Pharmacol 1992;41:817-21.

[10] 9w in rodents, the Y1 receptor or novel Y1-like receptor,

[11] 29w [18] Eva C, Oberrto A, Sprengel R, Genazzani E. The murine NPY-1 sensitive to 1229U91, seems to play a key role in NPYreceptor gene-structure and delineation of tissue-specific expression.

[12] 37w FEBS Lett 1992;314:285-8. [19] Rose PM, Fernandes P, Lynch JS, Frazier ST, Fisher SM, Kodukula K, Kienzle B, Seethala R. Cloning and functional expression of a cDNA encoding a human type 2 neuropeptide Y receptor. J Biol

[1] 18w Adult male Sprague-Dawley (SD) rats (7 weeks old, Cells were washed with 50 mM HEPES buffer (pH 7.4)

[2] 113w Charles River Japan, Japan, 280-350 g) were maintained containing 20% sucrose, homogenized and centrifuged at in individual cages under controlled conditions of tempera-1000 3 g for 15 min. The supernatant was centrifuged at ture (23628C) and light-dark cycle (7:00-19:00). Water 100 000 3 g for 45 min. The pellets were resuspended in 5 and pellet food (CE-2, CLEA Japan Inc., Japan) were mM HEPES buffer (pH 7.4) and centrifuged again. The available ad libitum. Rats were anesthetized with sodium membrane fraction was resuspended by a homogenizer in pentobarbital (50 mg / kg i.p., Dainabot, Japan). A permathe same buffer and used for this study. nent 21-gauge stainless steel cannula was stereotaxically 125 125

[3] 71w Binding of [ I]PYY and [ I]PP to membrane prepa-implanted into the right lateral ventricle. The stereotaxic rations was performed in 0.2 ml of 25 mM Tris buffer (pH coordinates used were as follows: 0.9 mm posterior to the 21 bregma, 1.2 mm bilateral to the midsagittal sinus and 1.5 lated [Ca ]i increases in cells expressing the hY5 receptor mm ventral to the brain surface. After one week of (Fig.

[4] 97w 2, Table 1). Moreover, 1229U91 did not inhibit 1 21 recovery, fully satiated rats were used in experiments. mM bPP-stimulated [Ca ]i increases in the cells (Table Groups of ten animals received ICV injections of hNPY, 1). Subsequently, 1229U91 is a potent and selective Y1 bPP, 1229U91, a mixture of these compounds, or vehicle antagonist while it acts as an agonist at the Y5 receptor. (10 mM phosphate-buffered saline containing 10 ml of 0.05% BSA), and their food intake was monitored. The 3.3. Effects of 1229U91 on hNPY-and bPP-induced experiments were performed between 9:00 and 11:30 a.m.

[5] 31w feeding in SD rats Results are given as mean6S.E. Statistical significance of the differences between groups was calculated using ICV-injected hNPY and bPP significantly induced food ANOVA followed by Benfferoni's test.

[6] 296w intake in satiated SD rats (Fig. 4). ICV 1229U91 (30 mg / head) suppressed hNPY-induced feeding in the SD rats by 89%. In contrast, 1229U91 did not significantly affect 3. Results bPP-induced feeding. In addition, 1229U91 had no effect on feeding behavior in the satiated SD rats, when tested 3.1. Binding affinity and selectivity of 1229U91 alone (data not shown). The effects of ICV 1229U91 administration appear selective for the control of food 1229U91 had high binding affinities for the hY1 and rY1 intake since 1229U91 administration did not cause any receptors (K 5 0.041 and 0.16 nM, respectively) and also remarkable changes in other behavior, including sedation i a high affinity for the hY4 receptor (K 5 0.33 nM) (Table and barrel-rolling. i 1). Furthermore, 1229U91 showed moderate affinities for In satiated SD rats, ICV-injected bovine PP (Y4 and Y5 the hY2, hY5 and rY5 receptors with K values of 20 nM, agonist) stimulated food intake (Fig. 4B). It has been i 48 nM and 170 nM, respectively (Table 1). reported that rat PP (Y4-selective agonist) did not elicit a feeding response [25], implying that the bovine PP-in-3.2. Effects of 1229U91 on functional assays duced food intake is most likely mediated through its action on the Y5 receptor. We therefore concluded that the 1229U91 potently inhibited 10 nM NPY-induced cal-hypophagic effects of 1229U91 on feeding behavior might cium increase in CHO cells expressing hY1 receptor (IC be predominantly mediated through its action of the Y1 or 50 value of 3.2 nM) without any evidence of partial agonism Y1-like receptors, rather than the Y5 receptor. (Fig. 1, Table 1) Furthermore, 1229U91 also inhibited 10 mM bPP-induced response in the cells (IC value of 3.6 50 nM) (Table 1). In addition, 1229U91 suppressed 100 nM 4. Discussion