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Neuropharmacology 63 (2012) 842e850

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Neuropharmacology

journal homepage: www.elsevier .com/locate/neuropharm

Involvement of neuropeptide Y Y1 receptor in the regulation ofamphetamine-mediated appetite suppression

Dong-Yih Kuo a, Pei-Ni Chen b, Ching-Han Yu a, Meng-Hsien Kuo c, Yih-Shou Hsieh b, Shu-Chen Chu d,*

aDepartment of Physiology, Chung Shan Medical University and Chung Shan Medical University Hospital, Taichung City 40201, Taiwan, ROCb Institute of Biochemistry and Biotechnology, Chung Shan Medical University and Chung Shan Medical University Hospital, Taichung City 40201, Taiwan, ROCcNational Taichung Second Senior High School, Taichung City 404, Taiwan, ROCdDepartment of Food Science, Central Taiwan University of Science and Technology, Taichung City 406, Taiwan, ROC

a r t i c l e i n f o

Article history:Received 22 January 2012Received in revised form14 May 2012Accepted 9 June 2012

Keywords:NPY-Y1 receptorMC3RAppetiteAmphetamine

Abbreviations: aCSF, artificial cerebrospinal fluid;AMPH, amphetamine; AP-1, activator protein-1; ARCylglycerol; i.c.v., intracerebroventricular; MC3R, meneuropeptide Y; IP, intraperitoneally; ODN, oligodeopiomelanocortin; S-ODN, phosphorothioate oligodeomedial hypothalamus; Y1R, NPY-Y1 receptor; Y5R, NP* Corresponding author. Tel.: þ886 4 24730022; fa

E-mail address: dykuo@csmu.edu.tw (D.-Y. Kuo).

0028-3908/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.neuropharm.2012.06.018

a b s t r a c t

Recently, we reported that an initial decrease followed by recovery of food intakewas observed during fourdays of amphetamine (AMPH) treatment and suggested that these changes in response were mediated bychanges in neuropeptide Y (NPY) and proopiomelanocortin (POMC). Here we investigated if Y1 receptor(Y1R) and/or Y5 receptor (Y5R) might be involved in this regulation. Rats were treated daily with AMPH forfour days. Changes in the expression levels of Y1R, Y5R, melanocortin receptor 3 (MC3R), and NPY wereassessed and compared. Results showed that Y1R and MC3R increased, with a maximal increase of about210% on Day 2 but with a restoration to the normal level on Day 4. In contrast, NPY decreasedwith a biggestreduction of about 45% on Day 2 and the pattern of expression during AMPH treatment was opposite tothose of Y1R andMC3R, while the expression of Y5Rwas not changed. Central inhibitions of NPY formationor Y1R activity modulated the anorectic response of AMPH and the reciprocal regulation of NPYandMC3R,revealing a crucial role of Y1R in this action. It is suggested that Y1R participates in the reciprocal regulationof NPY- and MC3R-containing neurons in the hypothalamus during the anorectic effect of AMPH. Theseresults may further the understanding of Y1R in the control of eating.

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1. Introduction

Neuropeptide Y (NPY) is a potent orexigenic peptide abundantin the brain. Hypothalamic NPYplays an essential role in the controlof food intake and body weight; therefore, an increase of NPY in thehypothalamus is directly relevant to the condition of energy defi-ciency and always correlates with hyperphagic behavior (Bradyet al., 1990; Chee and Colmers, 2008; Chao et al., 2011). Acuteintracerebroventricular injection of NPY can stimulate robustfeeding in a dose-dependent manner, while chronic injection ofNPY produces a sustained hyperphagia and increased body weightgain leading to obesity (Zarjevski et al., 1993). Moreover, hypotha-lamic NPY increases in ametabolic state of negative energy balance,such as in animal models of obesity and diabetes (Stephens et al.,1995; Sahu et al., 1997).

ANOVA, analysis of variance;, arcuate nucleus; DAG, diac-lanocortin receptor 3; NPY,xynucleotides; POMC, proo-xynucleotides; VMH, ventro-Y-Y5 receptor.x: þ886 4 23248119.

All rights reserved.

The mechanism underlying the appetite-suppressing effectof amphetamine (AMPH), a psychostimulant, is relevant to therelease of catecholamine. Therefore, local injections of catechol-aminergic antagonists into the lateral perifornical hypothalamuscan attenuate the anorexia induced by AMPH (Leibowitz andRossakis, 1978). Moreover, both subtypes of D1 and D2 receptorsand cerebral NPY are involved in the anorectic action of AMPH(Kuo, 2003). Furthermore, catecholamine may act directly orindirectly on hypothalamic NPY- and proopiomelanocortin(POMC)-containing neurons to suppress appetite (Hsieh et al.,2011; Kuo et al., 2011). POMC is a member of the anorexigenicsystem that is involved in the regulation of appetite suppressionand energy expenditure (Millington, 2007; Zheng et al., 2010).Melanocortin receptor 3 (MC3R), which belongs to POMC system,has been shown to produce a potent effect on food intake andenergy expenditure (Fan et al., 2000). During fasting, NPY geneexpression is up-regulated but POMC gene expression is down-regulated in the hypothalamus, suggesting that NPY- and POMC-containing neurons can act antagonistically to regulate foodintake and energy homeostasis (Roseberry et al., 2004; Valassiet al., 2008; Kuo et al., 2010).

NPY acts on at least five receptors, which include the Y1, Y2, Y4,Y5, and y6 subtypes (Michel et al., 1998). Of these NPY receptors, Y1

D.-Y. Kuo et al. / Neuropharmacology 63 (2012) 842e850 843

receptor (Y1R) and Y5 receptor (Y5R) have been suggested tomediate the effect of NPYon feeding (Gerald et al., 1996;Wyss et al.,1998; Antal-Zimanyi et al., 2008; Mashiko et al., 2009). Y1R iswidely expressed throughout the brain and involved in variousbrain functions, such as the modulation of stress response (Heilig,1995), ethanol consumption (Thiele et al., 2002), and metham-phetamine dependence and psychosis (Okahisa et al., 2009). Inthe study of feeding behavior, Y1R is strongly expressed in theventromedial hypothalamus (VMH) and activating Y1R inhibitsthe firing activity of VMH neurons, suggesting that stimulation offeeding behavior is in part attributed to the inhibitory actions onpostsynaptic Y1R within the VMH (Eva et al., 2006; Chee et al.,2010). In addition, the ARC-POMC neuron expresses functionalY1R and Y2R, which inhibit the POMC neuron when activated byNPY (Acuna-Goycolea and van den Pol, 2005). Y5 receptor immu-noreactivity has been reported in the ARC, and activating Y5R candepress ARC synaptic transmission (Campbell et al., 2001; Morinand Gehlert, 2006). Central administration of Y1R and Y5R antag-onists may abolish the inhibitory effects of 26RFa, a hypothalamicRFamide neuropeptide, on POMC expression (Lectez et al., 2009).

Recently, we reported that an initial decrease followed byrecovery of food intake was observed during a 4-day period ofAMPH treatment and suggested that these changes in responsewere mediated by changes in NPY and POMC (Hsieh et al., 2011;Kuo et al., 2012). To date, it is still unknown if Y1R and/or Y5R isinvolved in the reciprocal regulation of NPY and MC3R during theappetite-suppressing effect of AMPH. Previous evidence revealsthat Y1R antagonist can inhibit AMPH-induced behavioral effects,such as reduced anxiety or euphoria (Kask and Harro, 2000), andthat Y1R gene is involved in the control of methAMPH-inducedpsychosis (Okahisa et al., 2009). These results suggest that neuralsignaling via Y1R mediates central effects of AMPH. However,evidence also reveals that Y5R knockout mice can increase foodintake because Y5R is involved in the central feeding regulation ofPOMC (Higuchi et al., 2008). Thus, we predicted that Y1R and/orY5R would be involved in the reciprocal regulation of NPY andMC3R in AMPH-treated rats.

2. Materials and methods

2.1. Animals and drug treatments

Male Wistar rats (200e300 g, purchased from Animal Center of National ChengKung University Medical College) were housed individually in a cage, maintained at22 � 2 �C according to a 12 h light: 12 h dark cycle (light on at 6:00 am), andhabituated to frequent handling. Water and chow (5001 rodent diet, LabDiet com.,USA) were freely available throughout. This chow has been the standard ofbiomedical research for over four decades. Drug administration and food intakeassessment were performed daily at the beginning of dark phase (6:00 pm). Thisstudy has been carried out in accordance with the Guide for the Care and Use ofLaboratory Animals as adopted by the National Institutes of Health. This study hasbeen approved and reviewed by National Science Council in Taiwan, ROC.

2.2. Drug treatments

To examine the effect of daily D-amphetamine (AMPH) on feeding behavior andbody weight change, rats (n ¼ 6e8 for each group) were injected intraperitoneally(i.p.) with 0, 2 or 4mg/kg doses of AMPH once a day for 4 days. Moreover, to examinethe effect of haloperidol (Hal) pretreatment on AMPH-induced feeding behavior andbody weight change, rats were i.p. injected with 1 mg/kg Hal 40 min before 4 mg/kgAMPH treatment once a day for 4 days. AMPH-induced anorexia showed signifi-cantly changed only at the initial 0e6 h time interval in a 24-h test period asdescribed in our previous report (Kuo, 2005). Hal is a non-selective dopamine D1/D2receptor antagonist, which can block AMPH-induced anorexia during a 24-h testperiod (Chen et al., 2001). The first injection of AMPH was conducted at the end ofDay 0 (i.e., at 6:00 pm), which was regarded as the beginning of Day 1. The intakedata were calculated as the total amount of food during the previous day.

To assess the effect of daily AMPH on hypothalamic NPY, MC3R, Y1R, and Y5Rprotein levels, rats (n ¼ 6e8 each group) were injected with 2 mg/kg AMPH oncea day for 1, 2, 3 or 4 days depending on the group of rats. Rats received AMPH at40 min prior to being i.p. anesthetized with 30 mg/kg pentobarbital and decapitated

to remove hypothalamus from the brain immediately, which was then subjected todeterminations of protein levels or stored at �80 �C until further use.

To determine the effect of Hal pretreatment on the changes of NPY, Y1R, Y5R andMC3R levels in the hypothalamus in AMPH-treated rats on Day 1, rats (n ¼ 6e8 eachgroup) were injectedwith 1mg/kg Hal 40min before 4mg/kg AMPH treatment. Ratsreceived Hal and/or AMPH at 40 min prior to being i.p. anesthetized with 30 mg/kgpentobarbital and decapitated to remove hypothalamus from the brain immediately,which was then subjected to determinations of protein levels or stored at �80 �Cuntil further use.

To determine the effect of NPY antisense on anorectic response of AMPH, rats(n ¼ 6e8 for each group) were injected intracerebroventricularly (i.c.v.) withmissense (control group) or antisense (20 mg in a 10-ml vehicle) once a day at 1 hbefore daily treatment of 2 mg/kg AMPH for 4 days. Before AMPH treatment, ratswere injected (i.c.v.) with similar dose of missense (or antisense) daily for 2e3 daysuntil the response of feeding behavior was reduced slightly in antisense group. Thiswas due to the fact that either continuous or repeated injections of antisense mightbe necessary to maximize behavioral effect and especially to block the synthesis ofconstitutively active gene product (Zhang and Creese, 1993; Ogawa and Pfaff, 1998).

Another experiment was designed to determine the effect of NPY antisense (ormissense) on NPY, MC3R, and Y1R levels in AMPH-treated rats. Rats (n ¼ 6e8 foreach group) were infused daily with antisense or missense (20 mg in a 10-ml vehicle;i.c.v.) at 1 h before daily treatment with 2 mg/kg AMPH for 4 days. Before AMPHtreatment, rats were i.c.v. infused with similar dose of antisense (or missense) dailyfor 2e3 days until the response of feeding behavior was reduced slightly in antisensegroup. At 40 min after antisense (missense) and/or AMPH treatment, rats wereanesthetized and the hypothalamus of each rat was removed from the brain and itsNPY, MC3R, and Y1R levels were determined by Western Blot.

To determine the effect of Y1R antagonist on AMPH-induced anorexia and on thechanges of hypothalamic NPY and MC3R levels, rats (n ¼ 6e8 for each group) werepretreated with BIBP-3226 at 30 min before 4 mg/kg AMPH treatment. BIBP-3226 isdeveloped as an Y1R antagonist, which is known not to have any effect at the Y2, Y4,and Y5 receptors (Rudolf et al., 1994) and can significantly reduce NPY-inducedfeeding (O’Shea et al., 1997). We therefore studied the effect of BIBP-3226(80 nmole, i.c.v.; MW 473.6) on a 24-h AMPH-induced feeding response and onthe changes of NPY and MC3R levels. Rats received BIBP and/or AMPH at 40 minprior to being i.p. anesthetized with 30 mg/kg pentobarbital and decapitated toremove hypothalamus. BIBP-3226 had no significant effect on daily food intake(decrease about 10% compared to saline-treated group). It was for this reason thatwe allowed a 30-min period between injection of BIBP-3226 and the stimulant offeeding. The first injection of AMPH was conducted at the end of light period (i.e., at6:00 pm). The intake data were calculated as the total amount of food during theprevious day.

To examine whether the changes of NPY, MC3R and Y1R levels in 2 mg/kgAMPH-treated rats were due to the direct effect of AMPH treatment or due to thesecondary response of the decreased food intake, a pair-fed control group (whichwas fed with an amount of food equal to AMPH-treated group, n ¼ 6e8) wasnecessary. Their NPY, MC3R and Y1R levels in the hypothalamus would bemeasuredand comparedwith those of saline-treated control rats after 24 h of saline treatment.The amount of food intake per rats in pair-fed group was about 22 g/day, while thatin saline-control group was about 28 g/day.

2.3. Lateral ventricular cannulation

A surgery of rat was performed under anesthesia with 30 mg/kg pentobarbitalusing stereotaxic apparatus (Kopf Model 900, Tujunga, CA, USA). The target ofcannulation was close to the junction between the right lateral ventricle and thethird ventricle (coordinates: 0.8 mm posterior to Bregma, 1.5 mm from the midline,and 3.5e4.0 mm below the dura) (Paxinos and Watson, 1986). A 23-g stainless steelguide cannula was implanted and secured to the skull using stainless-steel screwsand dental cement. A correct placement was confirmed by observing a transient andrapid inflow of vehicle in PE tube connected with a 28-g injector cannula. Thecannula was then occluded with a 28-g stylet. For ICV infusion of antisense, thestylet was replaced with a 28-g injector cannula extending 0.5 mm below the tip ofguide cannula. Behavioral testing began at 1 week after the surgery. For all experi-ments, verification of cannula placement was done by the administration of 100 ng/rat angiotensin II and by the histological checking. Angiotensin II reliably inducedwater drinking in non-deprived rats when administered into the ventricles (Ritteret al., 1981). Only data from rats drinking more than 10 ml within 30 min wereincluded in this study.

2.4. Injection (i.c.v.) of NPY antisense

To determine the effect of pretreatment with NPY antisense oligonucleotide(ODN) on NPY gene expression, rats were i.c.v. infused with either NPY antisense ormissense at the dose of 10 mg in a 10-ml vehicle. An 18-mer NPY antisense near theinitiation codon encompassing bases 10e27 of the rat NPY mRNA sequence (Gen-bank access no. M15880) was selected (Hulsey et al., 1995; Kuo et al., 2001). Thesequence of the NPY antisense was 50-CCCCATTCGTTTGTTACC-30 , and the sequenceof missense was 50-TTATTCCCCCAGTTTGCC-30 . We used ODNs that were

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Fig. 1. Effects of daily pre-treatment with haloperidol (Hal) on AMPH-induced foodintake (upper panel) and body weight change (lower panel) over a 4-day period. 1 mg/kg Hal was i.p. administered to rats 40 min before 4 mg/kg/day AMPH once a day (at6:00 pm of each day) for 4 days. The first injection of AMPH was conducted at the endof Day 0. Each point represents the mean � SEM of 6e8 rats. *P < 0.05 vs. the controlgroup of each treatment day. #P < 0.05 vs. the treatment group on Day 2. i.p.,intraperitoneally.

D.-Y. Kuo et al. / Neuropharmacology 63 (2012) 842e850844

phosphorothioate-modified (S-ODN) only on the three terminal bases of both the 50

and 30 ends, because these S-ODNs had been shown to produce sequence-specificeffects without detectable toxicity in brain region and was regarded as a well-established agent in several vertebrate systems (Widnell et al., 1996; Ogawa andPfaff, 1998). Moreover, we selected a dose of 20 mg of antisense S-ODN becauseprevious studies had shown that i.c.v. injections of this amount of antisense opti-mally inhibited the expression of genes and the activity of feeding behavior(Schöbitz et al., 1997; Kuo et al., 2009). Both antisense and missense S-ODN weredissolved in artificial cerebrospinal fluid (aCSF) containing 140 mM NaCl, 3.35 mMKCl, 1.15 mM MgCl2, 1.26 mM CaCl2, 1.2 mM Na2HPO4, 0.3 mM NaH2PO4, pH 7.4.

2.5. Western blotting

Protein samples extracted from hypothalamus tissue were separated in a 12.5%polyacrylamide gel, transferred onto a nitrocellulose membrane and then incubatedseparately with specific antibodies against NPY, Y1R, Y5R, MC3R, and b-actin. Afterincubation with horseradish peroxidase goat anti-rabbit IgG, the color signal wasdeveloped by 4-chloro-1-napthol/3,30-diaminobenzidine, 0.9% (w/v) NaCl inTriseHCl. Relative photographic density was quantified by scanning the photo-graphic negative film on a Gel Documentation and Analysis System (AlphaImager2000, Alpha Innotech Corporation, San Leandro, CA, USA).

2.6. Statistical analysis

Data were presented as mean � SEM. Two-way or one-way ANOVA followed byDunnett’s test was used to detect significances among groups. P < 0.05 wasconsidered to be statistically significant.

2.7. Drugs, chemicals and reagents

Chow (LabDiet) was purchased from PMI Nutrition International (Brentwood,MO, USA). AMPH (D-amphetamine), BIBP-3226, TriseHCl solution, angiotensin II,ethidium bromide were purchased from Sigma-Aldrich (St. Louis, MO, USA); anti-body against NPY was purchased from Santa Cruz Biotechnology (Santa Cruz, CA,USA), while those against MC3R and b-actin were purchased from Gibco BRL, LifeTechnologies, Inc., (Rockville, MD, USA). Anti-NPY1R and anti-NPY5R polyclonalantibodies were purchased from Novus Biologicals, LLC (Littleton, CO, USA). TRIZOLreagent (Life Technologies, Inc., Grand Island, USA) was used in tissue homogeni-zation. Antisense or DNA primer was synthesized by Proligo Pty Ltd (Singapore).

3. Results

3.1. Effects of Hal pretreatment on changes of food intake in AMPH-treated rats

As shown in Fig. 1, daily AMPH for 4 days could dose-dependently decrease food intake (upper panel) and body weightchange (lower panel) compared to the control. Moreover, dailypretreatment with Hal could result in a restoration of food intakeand body weight change, revealing the involvement of D1/D2receptor in the regulation of AMPH-induced anorexia during a 4-day testing period. Statistical analysis by two-way ANOVArevealed significant dose-dependent [F(3,28) ¼ 11.43, P < 0.05] andtime-dependent effects [F(4,35) ¼ 4.86, P < 0.05]. Followed byDunnett’s test, it revealed that daily 2 mg/kg AMPH producedmarked decreases in food intake from Day1 to Day 2 (anorecticeffect) and a return to normal level (i.e., the development oftolerance to AMPH) on Day 3 and Day 4, but daily 4 mg/kg AMPHproduced a continuous anorectic response during a 4-day period oftime. The alterations of body weight change during AMPH treat-ment were in a way similar to that of feeding behavior.

The 2 mg/kg AMPH was therefore employed for most of thesubsequent studies since after two days there was a return tonormal level, while 4 mg/kg AMPH was used for co-administrationstudies of Hal/AMPH and BIBP-3226/AMPH since it can exerta more significant appetite-suppressing effect than 2 mg/kg AMPH.

3.2. Effects of AMPH on NPY, MC3R, Y1R, and Y5R expression levels

Results shown in Fig. 2 revealed that daily 2 mg/kg AMPHresulted in a significant decrease of NPY during AMPH treatment,which was in accordance with the response of feeding response.

However, daily AMPH resulted in increases of MC3R and Y1R levels,while Y5R level was not changed. Statistical analysis with one-wayANOVA indicated a decrease of NPY contents [F(4,25) ¼ 4.62,P < 0.05] from Day 1 to Day 3 with a biggest decrease of about 45%on Day 2. However, it revealed a significant increase of MC3R[F(4,25) ¼ 4.62, P < 0.05] from Day 1 to Day 3 with a maximumincrease of about 210% on Day 2, and a significant increase of Y1R[F(4,25) ¼ 4.58, P < 0.05] from Day 1 to Day 3 with a maximumincrease of about 200% on Day 2 if compared to the control group.These results revealed that NPY was decreased and expressed inamanner reciprocal to that of MC3R and Y1R, whichwere increasedduring AMPH treatment.

3.3. The effect of Hal pretreatment on changes of NPY, Y1R, Y5Rand MC3R

Results shown in Table 1 revealed that pretreatment with Hal inAMPH-treated rats could result in a restoration of NPY, Y1R andMC3R levels on the first day of drug treatment (Day 1). Statisticalanalysis with one-way ANOVA indicated a decrease of NPY level[F(3,24) ¼ 3.62, P < 0.05] and increases of Y1R level [F(3,24) ¼ 2.82,P< 0.05] andMC3R level [F(3,24)¼ 3.13, P< 0.05] in AMPH-treatedgroup compared to the control (Day 0). Moreover, results alsoindicated the effect of Hal on the restoration of NPY, Y1R and MC3Rlevels in Hal/AMPH-treated group compared to the AMPH-treatedgroup, while Y5R level remained unchanged in all drug-treatedgroups. This result revealed that Hal treatment could modulatethe effects of AMPH on NPY, Y1R and MC3R protein levels.

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Fig. 2. Effects of daily AMPH on hypothalamic NPY, Y1R, Y5R and MC3R expression levels over a 4-day period. Upper panel: the results of western blot analyzing NPY, Y1R, Y5R andMC3R levels. Lower panel: relative densitometric values for proteins in AMPH- and saline-treated groups. Content of NPY, Y1R, Y5R and MC3R in 2 mg/kg AMPH-treated group wasindicated as the percentage of the control (Day 0). Bars were mean � SEM. N ¼ 6e8 each group. *P < 0.05 vs. control.

Table 1The effect of haloperidol (Hal) pretreatment on the changes of NPY, Y1R, Y5R andMC3R levels in the hypothalamus in AMPH-treated rats on Day 1.

NPY Y1R Y5R MC3R

Control (Day 0) 100 � 8 100 � 10 100 � 11 100 � 9Hal (1 mg/kg, i.p.) 98 � 10 98 � 11 105 � 9 108 � 11AMPH (4 mg/kg, i.p.) 60 � 10* 180 � 18* 101 � 12 156 � 15*Hal þ AMPH 90 � 12# 110 � 12# 98 � 15 105 � 13#

Contents of each protein in drug-treated groups were indicated as the percentage ofthe control group (Day 0). Each densitometric value represents the mean � SEM of6e8 rats. *P < 0.05 vs. the control group. #P < 0.05 vs. the AMPH-treated group.

D.-Y. Kuo et al. / Neuropharmacology 63 (2012) 842e850 845

3.4. Effects of ICV injection of NPY antisense on AMPH anorexia

As shown in Fig. 3, a pretreatment with NPY antisense couldcontinuously enhance the decreasing effect of AMPH on food intakeand body weight for 4 days. Using two-way ANOVA to analyze theeffect of NPYantisense from Day 1 to Day 4, it revealed a significantdose-dependent [F(3,24) ¼ 4.56, p < 0.05] and time-dependenteffects [F(4,30) ¼ 5.16, p < 0.05]. Comparing the food intakebetween antisense/AMPH-treated and missense/AMPH-treatedrats in every day, significant effect was seen from Day 1 to Day 4.Furthermore, significant effect was also seen from Day 1 to Day 4by comparing between antisense/AMPH-treated and control(missense alone) rats. The feeding response in missense alone-treated rats was similar to that in saline-treated rats (shown in

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Daily AMPH Treatment

Fig. 3. The effect of NPY antisense (or missense) pretreatment on AMPH-inducedfeeding behavior over a 4-day test period. Missense or antisense treatment (20 mg/10 ml/day, i.c.v.) was administered 1 h before daily 2 mg/kg AMPH treatment. *P < 0.05vs. the missense groups of each treatment day. #P < 0.05 vs. the AMPH-treated groupsof each treatment day. Bars are mean � SEM. N ¼ 6e8 per group.

D.-Y. Kuo et al. / Neuropharmacology 63 (2012) 842e850846

Fig. 1) during a 4-day period of treatment. Moreover, the anorecticresponse in missense/AMPH-treated rats was not changed signifi-cantly when compared to that in AMPH-treated rats. These resultsrevealed the noninterference of missense treatment in this study.Taken together, results revealed that NPY knock-down couldabolish the development of tolerance to AMPH on feeding responseand body weight change.

Fig. 4. Effects of NPY antisense pretreatment on hypothalamic NPY, Y1R, and MC3Rcontents in missense/AMPH-treated rats over a 4-day test period. Content of NPY, Y1R,and MC3R in drugs-treated rats was indicated as the percentage of control. Bars aremean � SEM. N ¼ 6e8 per group. *P < 0.05 vs. control (missense alone).

3.5. Effects of NPY antisense pretreatment on NPY, Y1R andMC3R levels

As shown in Fig. 4, NPY antisense by itself could reduce NPYlevel but showed slight but no significant effect on Y1R and MC3Rlevels compared to the control (missense-treated) group, revealinga specific effect of NPY antisense on NPY expression. Moreover,a pretreatment with NPY antisense in AMPH-treated rats resultedin continuous reductions of NPY and continuous increases of Y1Rand MC3R from Day 1 to Day 4, which was consistent with theresponse of feeding behavior. Using b-actin as the internal stan-dard, the protein ratio of NPY, MC3R, or Y1R over b-actin in eachgroup was calculated and compared. By one-way ANOVA followedby Dunnett’s test (P < 0.05), it revealed that NPY expression levelswere decreased about 20 � 5% in antisense-treated, 40 � 6% inAMPH-treated and 50 � 7% in antisense/AMPH-treated ratscompared to the control group [F(6,35) ¼ 3.85, P < 0.05]. Bycontrast, Y1R expression levels were increased about 210 � 20% inboth AMPH-treated and antisense/AMPH-treated groups comparedto the control group [F(6,35) ¼ 4.15, P < 0.05]. Similarly, MC3Rcontents were increased about 205 � 18% in both AMPH-treated

and antisense/AMPH-treated rats compared to the control group[F(6,35) ¼ 3.96, P < 0.05].

3.6. Effects of BIBP-3226 on feeding, NPY and MC3R levels inAMPH-treated rats

As shown in part (A) of Fig. 5, it reveal that pretreatment withBIBP-3226 before 4mg/kg AMPH could attenuate an AMPH-inducedanorectic response. Statistical analysis with one-way ANOVArevealed a significant effect [F(3,28) ¼ 8.51, P < 0.05]. AMPH coulddecrease the food intake about 50% compared to the control andpretreatment with BIBP-3226 before AMPH could reverse foodintake about 50% compared to AMPH-treated group. The food intakein control (vehicle-treated or aCSF-treated) rats was similar to that

A

B

Fig. 5. (A) The effect of Y1R inhibitor (BIBP-3226) on AMPH-induced appetitesuppression during the first day of drug treatment. AMPH-induced appetite suppres-sion could be modulated by prior administration of BIBP-3226 (80 nmol, i.c.v.). (B) Theeffect of BIBP-3226 on the changes of hypothalamic NPY and MC3R in 4 mg/kg AMPH-treated rats during the first day of drug treatment. AMPH-induced changes of NPY andMC3R could be modulated by prior administration of BIBP-3226. Upper panel: resultsof western blot. Lower panel: relative densitometric values for protein contents.Contents of NPY and MC3R in BIBP- and/or AMPH-treated group were indicated as thepercentage of controls. *P < 0.05 vs. the control (vehicle-treated, i.c.v.) group. Thevehicle is an aCSF solution. #P < 0.05 vs. the vehicle/AMPH-treated groups of eachtreatment day. Bars are mean � SEM. N ¼ 6e8 per group.

Food

Inta

ke (g

/day

)

10

20

30

40

50

*

SC group PF group

NPY

MC3R

SC PF

Y1R

β-actin

SC PF

Fig. 6. A comparison of the expression levels of NPY, MC3R and Y1R between the pair-fed (PF) and saline-control (SC) groups during a 24-h period of food intake. Each of theprotein levels was measured by western blot. Rats in PF group were fed with anamount of food equal to rats in 2 mg/kg AMPH-treated group. *P < 0.05 vs. the controlgroup. Bars are mean � SEM. N ¼ 6e8 per group.

D.-Y. Kuo et al. / Neuropharmacology 63 (2012) 842e850 847

in saline-treated rats, revealing the noninterference of vehicle in thisstudy. Moreover, the expression of feeding in BIBP-3226-treated ratswas slightly but not significantly reduced (decreased about 10%food intake) compared to that in vehicle-treated rats, revealing that

BIBP-3226 had no significant effect on basal food intake in a 24-htesting period.

As shown in part (B) of Fig. 5, BIBP-3226 treatment alone didn’taffect the expression levels of NPY and MC3R compared to thecontrol group. However, a pretreatment with BIBP-3226 in AMPH-treated rats resulted in partial restorations of NPY and MC3R levelstoward normal level. Using b-actin as the internal standard, theprotein ratio of NPY or MC4R over b-actin in each group wascalculated and compared. By one-way ANOVA followed by Dun-nett’s test (P < 0.05), it revealed that significant decrease of NPYcontent was observed in AMPH-treated and BIBP-3226/AMPH-treated groups compared to the control group [F(3,28) ¼ 3.45,P < 0.05]. Moreover, BIBP-3226 could partially restore NPYexpression about 52% toward normal level compared to the AMPH-treated group. However, MC3R contents were increased in AMPH-treated and BIBP-3226/AMPH-treated groups compared to thecontrol group [F(3,28) ¼ 4.01, P < 0.05]. Moreover, BIBP-3226 couldpartially restore MC3R expression about 58% toward normal levelcompared to the AMPH-treated group.

3.7. The expression of NPY, MC3R and Y1R in saline-control andpair-fed groups

Results shown in Fig. 6 revealed that the expression levels forNPY, MC3R and Y1R were unchanged in pair-fed groups comparedto the saline-control group (t-test). This result revealed that thedecrease of food intake in pair-fed group during a 24-h testingperiod didn’t affect each of the NPY, MC3R and Y1R levels, con-firming that that the changes of protein levels during AMPHtreatment were due to the effect of drug treatment but not due tothe decrease of food intake.

4. Discussion

Current results showed that Y1R alterations, but not Y5R wereassociated with the susceptibility to AMPH-induced anorexia andthat NPY was expressed in a manner opposite to Y1R or MC3Rduring AMPH treatment. Moreover, modulation of NPY expressionin the brain could modify feeding behavior and expression levels ofY1R and MC3R in AMPH-treated rats. Furthermore, inhibition ofY1R activity could modify food intake and expression levels of NPYand MC3R. These results suggest that hypothalamic Y1R is involvedin the opposite regulation of NPY- and MC3R-mediated appetite

D.-Y. Kuo et al. / Neuropharmacology 63 (2012) 842e850848

suppression in AMPH-treated rats. Consistent with these findings,a recent report indicated that NPY can inhibit leptin-excitedanorexigenic neurons in hypothalamus, which is mediated by Y1receptor (Chee et al., 2010).

The present results showed that daily treatment with AMPHcould markedly reduce food intake and NPY expression during Day1 and Day 2 (anorectic effect), followed by a gradual reversion tonormal level (i.e., the development of tolerance to AMPH). Thus,NPY participated in both the anorectic response of AMPH, whichwas due to a decrease in NPYexpression, and in tolerance to AMPH,which was due to the restoration of NPY expression (Hsieh et al.,2007). Moreover, Y1R and MC3R expression levels were elevatedwith the maximum increases on Day 2 during AMPH treatment,which were almost opposite to both feeding response and NPYexpression with the maximum decreases on Day 2. These resultsimplied that Y1R participated in regulating AMPH-inducedanorexia and that Y1R- and MC3R-containing neurons mightfunction in a reciprocal manner against NPY-containing neurons inregulating appetite-suppressing effect of AMPH. Daily pre-treatment with Hal could prevent the response of AMPH-inducedanorexia for four days, in which case dopamine D1/D2 receptorswere involved in AMPH-induced anorexia.

In the present study, rats treated with AMPHwere initially in ananorectic state during the first two days of drug treatment, whichwas associated with the reduction of NPY expression. On subse-quent days, a gradual reversion of food intake was observed, whichwas associated with the restoration of NPYexpression. Moreover, incontrast to NPY expression, Y1R and MC3R expression levels wereincreased during the anorectic response but reversed gradually tonormal during the development of tolerance to AMPH. The presentdata suggest that changes of food intakes during the initialanorectic effect and later the development of tolerance to AMPHwere associated with changes in hypothalamic NPY, Y1R, andMC3Rexpression levels.

To further investigate the role of Y1R in NPY-mediated anorecticresponse to AMPH, the following two experiments were per-formed. Using pre-treatment with NPY antisense (i.c.v.) or Y1Rinhibitor (BIBP-3226), which aimed to reduce NPY or inhibit Y1Ractivity, respectively, we found that pre-treatment with NPY anti-sense in AMPH-treated rats could reduce the expression of NPY onDay 1 and Day 2 and continue to reduce NPY expression on Day 3and Day 4, i.e., prevent tolerance to AMPH. In contrast, NPY anti-sense could continuously increase the expression levels of Y1R andMC3R from Day 1 to Day 4 in AMPH-treated rats. These resultssuggested that the increases of Y1R andMC3Rwere associated withthe decrease of NPY, and that MC3R and NPY may function recip-rocally in regulating AMPH anorexia. Results in Y1R inhibitorexperiment showed that BIBP-3226 could block AMPH-inducedanorexia by about 50% and partly restore NPY expression byabout 52% and MC3R expression by about 58% toward normal. Thisresult showed that Y1R was involved in the reciprocal regulation ofNPY and MC3R expression during AMPH treatment.

The partial blocking effect of BIBP-3226 on the anorecticresponse of AMPH and the expression levels of MC3R and NPYshowed that Y1R was not the only mediator to regulate AMPH-induced anorexia. Comparing the blocking effect between Hal andBIBP-3226 showed that Hal could restore food intake, NPY andMC3R by about 90% (shown in Fig. 1 and Table 1), while BIBP-3226could restore food intake, NPYandMC3R only by about 50% (shownin Fig. 5) compared to the control group. These result showed that,in addition to Y1R, other mediators which were down-stream toD1/D2 receptors might be involved in the regulation of AMPH-mediated anorexia. It has been documented that GABA andagouti-related protein, which can be co-released from NPY-containing neurons, can block the anorexigenic POMC system in

the ARC and PVN through different receptors (Horvath et al., 1997;Pu et al., 1999; Cowley et al., 1999; Wu and Palmiter, 2011).

Although the present results showed that the increase in Y1Rfollowing AMPH treatment was the cause of NPY decrease, blockingY1R using antagonist did not completely reverse NPY and MC3Rchanges. It is possible that at least part of Y1R increasemight be dueto reduced NPY signaling (rather than the other way around). It isplausible that some Y1R-expressing neurons could up-regulate Y1Rto maintain a specific level of NPY signaling as NPY levels werereduced. As Y1R levels increase at least some neurons must thusstill be responding to NPY signaling, even if NPY levels werereduced. It is likely that by the second day changes were reversedbecause some critical tipping point was reached, where Y1R levelshad increased sufficiently for some target cell population tobecome adequately responsive to NPY.

Several previous reports have supported our view that Y1R wasinvolved in the reciprocal regulation of NPY and MC3R duringAMPH treatment. It has been reported that POMC neurons in thearea of arcuate nucleus (ARC) (i.e., ARC-POMC) can increase firingduring positive energy balance (or in satiety), while NPY neurons inthe ARC (i.e., ARC-NPY) can increase firing during negative energybalance (or in hunger) (Fioramonti et al., 2007). In the presentstudy, the initial anorectic state of rats during AMPH treatment wassimilar to that in satiety, and thus MC3R expression was increasedbut NPY expression was decreased. In contrast, the feeding stateduring the development of tolerance to AMPHwas similar to that inhunger, and thus MC3R expression was gradually decreased butNPY expression was gradually increased toward a normal level. Arecent report indicated that the Y1R in the VMH may participate inthe reciprocal regulation of ARC-POMC and ARC-NPY neurons inenergy homeostasis (King, 2006; Chee et al., 2010). Under satiatedconditions, ARC-POMC neurons are activated due to the decreasedinhibition of ARC-NPY neurons on VMH, which is via the decreasedactivation of postsynaptic Y1R in VMH, resulting in the increasedactivity in glutamatergic VMN efferent neurons that innervate inARC-POMC neurons. Moreover, in extracellular recordings madein vitro, NPY reduced the firing activity of VMN neurons by acti-vating Y1R (Kumarnsit et al., 2003). Furthermore, the Y1R antago-nist could inhibit AMPH- and apomorphine-induced behavioraleffects (Kask et al., 1998; Kask and Harro, 2000), suggesting thatneural signaling via Y1R mediates central effects of AMPH.

Current results showed that hypothalamic Y5R was not involvedin the regulation of AMPH-mediated appetite suppression. Thismight be related to the source of NPY, i.e., whether the NPY isexogenous or endogenous. Previous reports (Gerald et al., 1996;Kask et al., 1998) reveal that the stimulatory effect of exogenousNPY is probably mediated through an NPY receptor subtype that isnot identical with the Y1R subtype, but is possibly via the action onthe Y5R subtype. Moreover, the Y1R subtype may mediate theeffect of endogenous NPY in conditions of increased energydemand or during the intake of highly palatable diets. In thepresent study, AMPH treatment could decrease the release ofendogenous NPY in the hypothalamus, and thus could reduce foodintake via the action of the Y1R subtype.

Although a previous report indicated that substantial fooddeprivation might lead to increased NPY gene expression (Richard,1995), we ruled out the possibility that the change in 24-h NPYlevel following AMPH treatment was simply secondary to reducedfeeding, rather than the rapid action of AMPH on hypothalamicNPY. This is because NPY is increased during food restriction(Richard, 1995), while the present results showed reduced NPY andreduced intake during AMPH treatment. In the former case NPYincreases because food is limited, while here the reduction in intakeis likely due to the reduced NPYexpression itself. Moreover, AMPH-induced anorexia following drug treatment occurred only at the

D.-Y. Kuo et al. / Neuropharmacology 63 (2012) 842e850 849

0e6 h time interval, but showed no change at other intervals(6e12 h, 12e18 h, 18e24 h) in a 24-h test period in normal rats asdescribed in our previous report (Kuo, 2005). Moreover, the presentresults showed that even if the food intakes were different betweenpair-fed group and the saline-control group, hypothalamic NPY,MC3R and Y1R contents remained unchanged.

Despite the present findings in AMPH-induced anorexia, wemust acknowledge theweakness of the experimental design in thatall drug interactions were based on single doses. No doseeresponsefunctions were obtained.

5. Conclusions

In summary, the present results showed that Y1R participates inthe reciprocal regulation of NPY- and MC3R-containing neurons inthe hypothalamus, which are involved in the regulation of AMPH-mediated appetite suppression.

Acknowledgments

This study was supported by a grant from the National ScienceCouncil (NSC- 101-2320-B-040-006-MY3) and a grant from theChung Shan Medical University (CSMU 101-OM-A-133).

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