NPY-induced feeding involves the action of a Y1-like receptor in rodents

Regulatory Peptides 75–76 (1998) 409–415

NPY-induced feeding involves the action of a Y1-like receptor in rodents a, a a a a Akio Kanatani *, Junko Ito , Akane Ishihara , Hisashi Iwaasa , Takahiro Fukuroda , a b b a Takehiro Fukami , Douglas J. MacNeil , Lex H.T. Van der Ploeg , Masaki Ihara a

Tsukuba Research Institute, Banyu Pharmaceutical Co. Ltd., Okubo 3, Tsukuba, 300 -2611 Japan b Merck Research Lab., Rahway, NJ 07065, USA

Received 7 November 1997; received in revised form 26 February 1998; accepted 2 March 1998

Abstract 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 (Ki 5 0.041 nM and 0.16 nM, respectively) and also a high affinity for the human Y4 receptor (Ki 5 0.33 nM), whereas it shows moderate affinities for the human Y2, Y5 and rat Y5 receptors (Ki values of 20–170 nM). Moreover, 1229U91 potently inhibits NPY-induced [Ca 21 ]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.  1998 Elsevier Science B.V. All rights reserved. Keywords: Neuropeptide Y; Pancreatic polypeptide; 1229U91; Y1 receptor; Y5 receptor; Feeding; Obesity

1. Introduction Neuropeptide Y (NPY) is a 36-amino acid polypeptide belonging to the pancreatic polypeptide family, which includes NPY, peptide YY (PYY) and pancreatic polypeptide (PP) [1,2]. NPY is highly concentrated in the hypothalamus [3,4], and is a potent ICV stimulant of feeding behavior [5–9]. Chronic administration of NPY into the brain results in hyperphagia and body weight gain, reduces energy expenditure, and increases lipogenic activity in the liver and adipose tissue [10,11]. It has been also reported that concentrations of NPY and its mRNA in the hypothalamus are markedly increased during food deprivation and in some genetic models of obesity in rodents [12–14,36]. Antibodies and antisense oligonucleotides to *Corresponding author. Tel: 1 81-298-77-2000; fax: 1 81-298-772027, e-mail: [email protected]

NPY significantly reduce food intake [38–41]. In support of a physiological role for NPY in food intake, it was also reported that NPY-deficient ob / ob mice are less obese than ob / ob mice. NPY-deficient ob / ob mice also exhibit reduced food intake when compared to ob / ob mice [37]. For this data it is inferred that NPY may be one of the major regulators of physiological feeding behavior. Recently, five distinct types of NPY receptors, Y1, Y2, Y4, Y5 and Y6 have been cloned [15–29]. Given the correlation between the in vitro functional and binding activity of different peptidic agonists at the Y5 receptor and their potent stimulation of food intake in rodent models, the Y5 receptor was proposed as a likely feeding receptor. In support of this model, mRNA encoding the Y5 receptor is predominantly expressed in the brain, including hypothalamic nuclei involved in feeding behavior [25]. 1229U91 has been reported as a potent NPY antagonist [31]. We recently reported that a peptidic Y1 antagonist,

0167-0115 / 98 / $ – see front matter  1998 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 98 )00096-2

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1229U91, significantly suppressed NPY-induced, fastinginduced and spontaneous feeding [32,33]. We show that 1229U91 is a potent antagonist for the Y1 receptor, while it acts as an agonist at the other NPY receptors including the Y5 receptor. Using the potent Y1 antagonist, 1229U91, we investigate the participation of Y1 and Y5 receptors in ingestive behavior.

2. Materials and methods

2.1. Materials 1229U91 [(IleGluProDprTyrArgLeuArgTyr-NH 2 ) 2 , cyclic (2,49),(29,4)-diamide)-amide] (Dpr: 2,3-diaminopropionic acid) was synthesized as described previously [32]. Human NPY (hNPY) and bovine PP (bPP) were purchased from Peptide Institute (Osaka, Japan). [ 125 I]PYY and [ 125 I]PP were from New England NuclearDuPont (Boston, MA). The culture reagents and bovine serum albumin (BSA) were obtained from GIBCO (Grand Island, NY). Bacitracin, phenylmethylsulfonyl fluoride (PMSF) and polyethylenimine were obtained from Sigma (St. Louis, MO). All other chemicals were of analytical grades.

2.2. Cell culture CHO, LMtk- and COS-7 cells were obtained from the ATCC (Rockville, MD). CHO cells expressing recombinant human Y1, Y2 and Y4 receptors were grown in Iscove’s modified Dulbecco’s medium supplemented with 10% fetal bovine serum (FBS), penicillin-G (100 IU / ml), streptomycin (100 mg / ml) and G418 (1 mg / ml). LMtkcells expressing the recombinant human Y5 receptor were grown in Dulbecco’s modified Eagle’s medium (high glucose) with 10% FBS, penicillin-G (100 IU / ml), streptomycin (100 mg / ml) and G418 (0.8 mg / ml). COS-7 cells transiently expressing recombinant rat Y1 and Y5 receptors were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, penicillin-G (100 IU / ml) and streptomycin (100 mg / ml). These cells were grown in a 95% air, 5% CO 2 humidified atmosphere at 378C.

7.4) containing 10 mM MgCl 2 , 1 mM PMSF, 0.1% bacitracin and 0.5% BSA. The membranes (100–300 mg / ml) were incubated at 258C for 120 min with [ 125 I]PYY (25 pM) and [ 125 I]PP (25 pM), respectively. Bound and free peptides were separated by filtration using a GF / C glass filter (Whatman, UK)) presoaked with 0.3% polyethylenimine. The remaining radioactivity on the filter was TM quantitated using a Cobra (Packard, Japan). Specific binding of [ 125 I]PYY and [ 125 I]PP was defined as the difference between total binding and nonspecific binding in the presence of 1 mM PYY and PP, respectively. Binding of [ 35 S]GTPgS activated by NPY to membrane preparations was performed in 0.5 ml of the binding assay buffer containing 100 mM NaCl and 1 mM GDP in the presence of 100 nM NPY. The membranes (200–400 mg / ml) were incubated at 308C for 1 h with [ 35 S]GTPgS (150 pM). Separation of free ligand was performed applying the filtration methods mentioned above. The TM radioactivity of the filter was determined by TopCount TM (Packard, Japan) with Microscint -0 (Packard, Japan).

2.4. Measurement of intracellular calcium ion concentrations [Ca 21 ]i was measured fluorometrically using a Ca 21 sensitive fluorescent dye, fura-2. The cells expressing human NPY receptors were harvested using 0.25% trypsin and 0.02% EDTA. The cells (1.0 3 10 7 cells) were washed once with Krebs-Henseleit Hepes buffer containing 0.1% BSA (pH 7.4), suspended in 1 ml of the buffer and incubated with 2 mM fura-2 acetoxymethylester at 378C for 60 min. The fura-2-loaded cells were washed with the buffer and resuspended in 10 ml of the buffer. In a cuvette, 0.5 ml of the resultant suspension was stirred continuously at 378C during the measurement. Test compounds or vehicle were added 5 min before the addition of NPY and the related ligands, and fluorescent intensity at an emission wavelength of 500 nm and excitation wavelengths of 340 and 380 nm were monitored with a CAF-110 intracellular ion analyzer (JASCO, Tokyo, Japan), and [Ca 21 ]i values were calculated according to the previously reported method [34].

2.5. In vivo experimental protocols 2.3. Binding experiments Cells were washed with 50 mM HEPES buffer (pH 7.4) containing 20% sucrose, homogenized and centrifuged at 1000 3 g for 15 min. The supernatant was centrifuged at 100 000 3 g for 45 min. The pellets were resuspended in 5 mM HEPES buffer (pH 7.4) and centrifuged again. The membrane fraction was resuspended by a homogenizer in the same buffer and used for this study. Binding of [ 125 I]PYY and [ 125 I]PP to membrane preparations was performed in 0.2 ml of 25 mM Tris buffer (pH

Adult male Sprague-Dawley (SD) rats (7 weeks old, Charles River Japan, Japan, 280–350 g) were maintained in individual cages under controlled conditions of temperature (23628C) and light–dark cycle (7:00–19:00). Water and pellet food (CE-2, CLEA Japan Inc., Japan) were available ad libitum. Rats were anesthetized with sodium pentobarbital (50 mg / kg i.p., Dainabot, Japan). A permanent 21-gauge stainless steel cannula was stereotaxically implanted into the right lateral ventricle. The stereotaxic coordinates used were as follows: 0.9 mm posterior to the

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lated [Ca 21 ]i increases in cells expressing the hY5 receptor (Fig. 2, Table 1). Moreover, 1229U91 did not inhibit 1 mM bPP-stimulated [Ca 21 ]i increases in the cells (Table 1). Subsequently, 1229U91 is a potent and selective Y1 antagonist while it acts as an agonist at the Y5 receptor.

bregma, 1.2 mm bilateral to the midsagittal sinus and 1.5 mm ventral to the brain surface. After one week of recovery, fully satiated rats were used in experiments. Groups of ten animals received ICV injections of hNPY, bPP, 1229U91, a mixture of these compounds, or vehicle (10 mM phosphate-buffered saline containing 10 ml of 0.05% BSA), and their food intake was monitored. The experiments were performed between 9:00 and 11:30 a.m. Results are given as mean6S.E. Statistical significance of the differences between groups was calculated using ANOVA followed by Benfferoni’s test.

3.3. Effects of 1229 U91 on hNPY- and bPP-induced feeding in SD rats ICV-injected hNPY and bPP significantly induced food 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 bPP-induced feeding. In addition, 1229U91 had no effect on feeding behavior in the satiated SD rats, when tested alone (data not shown). The effects of ICV 1229U91 administration appear selective for the control of food intake since 1229U91 administration did not cause any remarkable changes in other behavior, including sedation and barrel-rolling. In satiated SD rats, ICV-injected bovine PP (Y4 and Y5 agonist) stimulated food intake (Fig. 4B). It has been reported that rat PP (Y4-selective agonist) did not elicit a feeding response [25], implying that the bovine PP-induced food intake is most likely mediated through its action on the Y5 receptor. We therefore concluded that the hypophagic effects of 1229U91 on feeding behavior might be predominantly mediated through its action of the Y1 or Y1-like receptors, rather than the Y5 receptor.

3. Results

3.1. Binding affinity and selectivity of 1229 U91 1229U91 had high binding affinities for the hY1 and rY1 receptors (Ki 5 0.041 and 0.16 nM, respectively) and also a high affinity for the hY4 receptor (Ki 5 0.33 nM) (Table 1). Furthermore, 1229U91 showed moderate affinities for the hY2, hY5 and rY5 receptors with Ki values of 20 nM, 48 nM and 170 nM, respectively (Table 1).

3.2. Effects of 1229 U91 on functional assays 1229U91 potently inhibited 10 nM NPY-induced calcium increase in CHO cells expressing hY1 receptor (IC 50 value of 3.2 nM) without any evidence of partial agonism (Fig. 1, Table 1) Furthermore, 1229U91 also inhibited 10 mM bPP-induced response in the cells (IC 50 value of 3.6 nM) (Table 1). In addition, 1229U91 suppressed 100 nM NPY-activated [ 35 S]GTPgS G-protein binding to the membranes of COS-7 cells transiently expressing the rY1 receptor (IC 50 value of 6.3 nM)(Table 1). 1229U91 behaved as an agonist on the hY2, hY4, hY5 and rY5 receptors (Figs. 2 and 3, Table 1). We failed to observe any significant inhibition of 1229U91 on 100 nM NPY-stimu-

4. Discussion We earlier reported that 1229U91 is a potent Y1 antagonist which can suppress NPY- and fasting-induced feeding in SD rats in a dose-dependent manner [32]. 1229U91 also potently attenuated spontaneous feeding in Zucker fatty rats, when compared to lean littermates [33].

Table 1 In vitro profiles of 1229U91 for cloned NPY receptors hY1

rY1

hY2

hY4 a

hY5

rY5

0.09 0.30 . 1000 20

180 660 0.09 0.33

0.70 23 1.9 48

0.72 9.0 1.6 170

125

hNPY pNPY(13–36) bPP 1229U91

[ I]PYY binding (Ki :nM) 0.45 1.05 38 270 42 23 0.041 0.16

1229U91

NPY-induced [Ca 21 ]i increase (IC 50 :nM) 3.2 6.3 b . 10 000

NT

. 10 000

. 10 000 b

(Agonism:EC 50 nM)

bPP-induced [Ca 21 ]i increase (IC 50 :nM) 3.6 NT NT (4300)

ND (2.2)

. 10 000 (6600)

NT (10 000)b

a

[ 125 I]PP binding. [ 35 S]GTPgS binding. ND 5 not detected, NT 5 not tested.

b

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Fig. 1. Effects of 1229U91 on mobilization of [Ca 21 ]i in CHO cells expressing human Y1 receptors. (A) Agonistic effects of NPY, bPP and 1229U91. (B) 1229U91 antagonism against 10 nM NPY-induced [Ca 21 ] i increase in the cells. Different concentrations of 1229U91 were added 5 min prior to the addition of 10 nM NPY at 378C. The results are expressed as a percentage of the maximal 100 nM (A) or 10 nM (B) effects, respectively. Each point shows the mean6SE from three independent experiments performed in duplicate.

Fig. 2. Effects of 1229U91 on mobilization of [Ca 21 ]i in LMtk- cells expressing human Y5 receptors. (A) Agonistic effects of NPY, bPP and 1229U91. (B) 1229U91 antagonism against 100 nM NPY-induced [Ca 21 ] i increase in the cells. Different concentrations of 1229U91 were added 5 min prior to the 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.

An accurate interpretation of this data requires knowledge of the exact selectivity of 1229U91 for the NPY receptors. We showed that 1229U91 has high affinities for Y1 and Y4 receptors and moderate affinities for Y2 and Y5 receptors. Furthermore, 1229U91 is an agonist for all of these NPY receptors except for the Y1 receptor, on which it acts as an antagonist. Delineating which NPY receptors are involved in NPY-induced feeding, we considered the following: the Y2 and Y4 agonists fail to modulate feeding after ICVinjection in rats [6,25,35] and therefore will not be further considered. The recently cloned mouse Y6 receptor is absent from the rat genome and this receptor therefore does not require further consideration in rat food intake

models [28–30]. Finally, the pharmacological action of NPY, PYY and PYY3-36 at the Y3 receptor, which has not yet been cloned, indicates that the Y3 receptor dose not play a crucial role in food intake. The action of 1229U91 at the Y1 and Y5 receptors was therefore evaluated in more detail. The Y5 receptor has been proposed to be involved in the control of NPY-induced feeding since the rank order potency of NPY and the related ligands to stimulate food intake correlated reasonably well with their pharmacological binding profiles at the Y5 receptor [25]. We showed that an in vivo Y5 agonist, bPP, significantly induced food intake following its ICV-injection. 1229U91 is a weak

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Fig. 3. Agonistic activity of 1229U91 on mobilization of [Ca 21 ]i in the cells expressing human Y2 and Y4 receptors. (A) Agonistic effects of NPY, pNPY13-36 and 1229U91 on Y2 receptors. (B) Agonistic effects of NPY, bPP and 1229U91 on Y4 receptors. The results are expressed as a percentage of the maximal increase in [Ca 21 ]i by 100 nM NPY (A) or 100 nM bPP (B). Each point shows the mean6SE from three independent experiments performed in duplicate.

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).

agonist for the Y5 receptor with a moderate binding affinity. 1229U91 failed to significantly suppress bPPinduced feeding mediated by the Y5 receptor. Moreover, we did not detect any antagonistic activity of 1229U91 on NPY-induced and bPP-induced signal transductions through the Y5 receptor even at high doses tested in [Ca 21 ]i response and [ 35 S]GTPgS binding experiments. These results indicate that the anorexigenic effect of 1229U91 might not be mediated through its action at the Y5 receptor. From the data, we infer that the Y5 receptor does not seem to play a crucial role in the regulation of NPY-induced feeding in rats. Conclusive evidence for this model will require the use of selective Y5 antagonists in rodent models of food intake. These results suggest that 1229U91 inhibits food intake through inhibition of typical

Y1 receptors, suggesting that NPY-induced feeding might be predominantly caused by the action of Y1 receptors. In addition to a proposed role for the Y1 receptor in NPY-induced feeding, it is conceivable that an additional as yet undefined NPY receptor plays a major role in the control of NPY-induced food intake. This consideration is fueled by the observation that PYY(3–36) which does not recognize the Y1 receptor is the most potent feeding stimulant of the NPY-related ligands [35]. The use of 1229U91 does not therefore allow us to discriminate its action at the Y1 receptor from its action at a novel Y1-like receptor and additional experimentation is needed to resolve this issue. We conclude that 1229U91 is a potent Y1 antagonist with a moderate agonistic activity for the Y5 receptor.

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Deduced from the effects of 1229U91 on feeding behavior in rodents, the Y1 receptor or novel Y1-like receptor, sensitive to 1229U91, seems to play a key role in NPYinduced food intake.

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