Immunohistochemical and genomic evidence for the involvement of hypothalamic neuropeptide Y (NPY) in phenylpropranolamine-mediated appetite suppression

Immunohistochemical and genomic evidence for the involvement of hypothalamic neuropeptide Y (NPY) in phenylpropranolamine-mediated appetite suppression

Peptides 25 (2004) 2155–2161 Immunohistochemical and genomic evidence for the involvement of hypothalamic neuropeptide Y (NPY) in phenylpropranolamin...

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Peptides 25 (2004) 2155–2161

Immunohistochemical and genomic evidence for the involvement of hypothalamic neuropeptide Y (NPY) in phenylpropranolamine-mediated appetite suppression Yih-Shou Hsieha , Jeng-Dong Hsub , Shun-Fa Yanga , Dong-Yih Kuoc,∗ a b c

Institute of Biochemistry, Chung Shan Medical University, Taichung City 402, Taiwan, ROC Department of Pathology, Chung Shan Medical University, Taichung City 402, Taiwan, ROC Department of Physiology, Chung Shan Medical University, Taichung City 402, Taiwan, ROC Received 10 June 2004; received in revised form 14 August 2004; accepted 20 August 2004 Available online 25 September 2004

Abstract Phenylpropanolamine (PPA) is an appetite suppressant. The mechanism for the anorectic effect of PPA has been attributed to its action on the site of hypothalamic paraventriculum. Neuropeptide Y (NPY) is an appetite stimulant that is widely distributed in the site of hypothalamus. It is not clear whether hypothalamic NPY is involved in the anorectic action of PPA. This study was aimed to investigate the mechanism underlying the involvement of NPY gene in the anorectic action of PPA. Results revealed that PPA treatment in rats could decrease both NPY content and mRNA level in the hypothalamus. In addition, the expression of NPY immunoreactivity following PPA treatment was decreased in areas of hypothalamic arcuate nucleus, paraventricular nucleus and periventricular area using immunohistochemical staining, suggesting an involvement of NPYergic pathway in the action of PPA anorexia. Our results provided immunohistochemical and genomic evidence to suggest that PPA might reduce feeding by altering NPY gene expression. © 2004 Elsevier Inc. All rights reserved. Keywords: Anorectic agent; Orexigenic peptide; Food intake; Metabolism; Immunohistochemistry; RT-PCR; Hypothalamus

1. Introduction Phenylpropanolamine (PPA) is an appetite suppressant and is clinically used as an over-the-counter weight loss pill in some countries [33,37]. In animal study, evidence has revealed that administration of PPA can induce a dosedependent reduction in feeding behavior [26]. Pharmacologically, PPA is regarded as a catecholamine (CA) agonist that can exert its anorectic action predominantly on ␣-1 adrenoceptors within the site of hypothalamic paraventricular nucleus (PVN) [24,28,32,35]. Hypothalamic PVN functions as a nexus for many systems including CA-producing and neuropeptide Y (NPY)-producing systems known to control appetite [3]. ∗

Corresponding author. Tel.: +886 424 730 022; fax: +886 424 739 030. E-mail address: [email protected] (D.-Y. Kuo).

0196-9781/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2004.08.015

PPA is also regarded as a sympathomimetic agent, which is structurally and functionally related to amphetamine-like anorectic drugs. However, PPA has a better safety profile than amphetamine due to the fact that a repeated administration of PPA is not subject to abuse [20,36]. Although some casecontrol studies have limited the use of PPA in diets due to the risk of hemorrhagic stroke [13,16,25], PPA is still sold recently as an over-the-counter appetite suppressant in the study of anti-obesity in the United States [4]. NPY is an appetite stimulant, which is widely distributed in the central nervous system and found at high concentration in hypothalamus [10,15]. Hypothalamic NPY plays an important role in the regulation of feeding behavior since an infusion of NPY into the brain of satiated rats may elicit a ravenous food intake and repeated infusions may lead to obesity [29,30]. Hypothalamic NPYergic neurons, which project from the arcuate nuclei (ARC) to PVN or perifornical area

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2. Materials and methods

were sacrificed at 40 min later by decapitation under the anesthesia of pentobarbital. For the assay of NPY contents, the hypothalamus was removed rapidly and placed separately in 2N acetic acid and sonicated for 20 s at 4 ◦ C. Then, an aliquot (50 ␮l) of the sonicated tissue was dissolved in 1N NaOH and assayed for their protein contents [23], the rest of tissue samples were immediately boiled for 5 min, cooled on ice and centrifuged for 20 min at 3000 rpm. The supernatants were stored at −20 ◦ C until further assay. Each sample was then measured for hypothalamic NPY contents by the RIA technique using commercial kits purchased from Peninsula Labs Inc. (Belmont, CA, USA). All procedures were performed as described previously [1]. To examine whether the decrease of NPY content was due to the direct effect of PPA treatment or due to the secondary response of the decreased food intake, a pair-fed control group (which was fed with an amount of food equal to PPA-treated group) was necessary. Their NPY contents would be measured and compared with those of saline-treated or PPA-treated rats.

2.1. Animal treatments

2.3. Immunohistochemistry (IHC)

Male Wistar rats, weighing 200–250 g, were obtained from the Animal Center of National Cheng Kung University Medical College. They were housed individually in a metal cage, maintained at 22 ± 2 ◦ C in a room with a 12-h light–dark cycle (light on at 6:00 a.m.), and habituated to frequent handling. Water and chow were freely available throughout. Rats were treated with PPA (0, 40, 80, or 120 mg/kg; i.p.; n = 6–8 each group) at the beginning of the dark period (at 6:00 p.m.) and the feeding behavior was examined at 24 h after drugs treatment. Meanwhile, other groups of rats were treated with a similar dose of PPA (0, 40, 80, or 120 mg/kg; i.p.; n = 6–8 each group) but the injection time was changed to be at the beginning of the light period (at 6:00 a.m.). This was aimed to compare the effect of PPA-induced anorexia between light and dark period. Rats treated with PPA were sacrificed and the hypothalamus was removed from their brain to determine the contents of hypothalamic NPY at 24 h following drugs treatment. Similarly, rats treated with PPA (0 and 80 mg/kg; i.p.; n = 6–8 per group) were sacrificed at 24 h later to examine hypothalamic NPY immunoreactivity or NPY mRNA level. At 40 min before sacrifice, rats received a treatment of 80 mg/kg PPA to enhance the effect of drug. The present study has been carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health.

Rats were sacrificed under ether anesthesia by a transaortic perfusion with 50 ml heparinized saline, and then treated with 250 ml of fixative (4% paraformaldehyde in 0.1 M phosphate buffer; pH 7.4). Brains were removed and immersed immediately in the fixative for 90 min, washed in 0.1 M phosphate buffer saline (PBS), and then placed in 0.1 M PBS containing 30% sucrose at 4 ◦ C for 48 h. Coronal sections (10 ␮m thick) prepared by cryostat were collected in gelatin-coated slides and stored at –80 ◦ C until further use. Brain sections were processed for IHC by using the indirect immunoperoxidase procedure as described previously [30]. Sections were washed in 0.01% PBS, incubated with 0.2% hydrogen peroxidase (20 min), washed in 0.01% PBS, and then incubated with blocking solutions (Vector Laboratories, Inc., Berlingame, CA) at a 1:100 dilution for 30 min. Sections were incubated with rabbit NPY antiserum (Industrial Boulevard, Stillwater, USA; 1:3000 dilution) at 4 ◦ C for 24 h. Then, tissues were washed in cold 0.01% PBS, incubated with biotinylated goat antirabbit IgG (Vectastain ABC kit; 1:200 dilution) for 1 h at 22 ◦ C, then washed in 0.01% PBS and incubated with avidin-biotin complex coupled to horseradish peroxidase (1:100 dilution) for 1 h at 22 ◦ C. The reaction signal was visualized and enhanced with 0.05% glucose-nickel-3,3-diaminobenzidine tetrahydrochloride and 0.02% H2 O2 , dissolved in 0.01% PBS. The reaction was terminated by three consecutive distilled-water washes, then the sections were stained with cresyl violet (Nissl stain) for the cytoarchitectonics of tissue and washed with running water for 10 min. Finally, the sections were treated with graded alcohol and xylene, and then coverslipped with Permount. To examine the specific efficiency of primary antibody (NPY), brain sections without NPY antibody were compared

(PFA), have been postulated to be the major controller for feeding behavior and energy metabolism in rodent animals [3,17,22]. Thus, the ARC-PVN NPY pathway should be activated in response to starvation and body weight loss, as well as in genetic models of obesity and diabetes mellitus [38]. Several behavioral and pharmacological studies have indicated that CAergic pathway played a role in suppressing NPY-elicited feeding behavior and that treatment of CAergic drug might decrease the NPY contents in several brain regions [7,14,19,39]. However, until recently, little is known about the genomic mechanism underlying the involvement of NPY in the appetite suppressing effect of PPA. Therefore, the present study was aimed to investigate whether hypothalamic NPY neurons were involved in the anorectic action of PPA using in situ immunohistochemical staining. In addition, we also investigated whether the content of NPY mRNA was changed following a PPA treatment in rats.

2.2. Radioimmunoassay (RIA) Following the confirmation of appetite suppressing effect of PPA at 24 h after drug treatment, rats were treated with similar dose of PPA (0, 40, 80 and 120 mg/kg, i.p.; n = 6–8 per group) again to enhance the drug effect. PPA-treated rats

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to that labeled with NPY antibody in the area of hypothalamus.

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58 ◦ C for 1 min (annealing), and 72 ◦ C for 30 s (extension) for 25 cycles followed by a final elongation step at 72 ◦ C for 7 min, and finally the PCR products were soaked at 16 ◦ C.

2.4. RNA extraction 2.6. Gel electrophoresis Hypothalamic NPY mRNA levels were measured in a block of mediobasal hypothalamic tissue as described previously [27]. In brief, total RNA was isolated from this block using the modified guanidinium thiocyanate–phenol–chloroform method [9]. Each hypothalamic block was homogenized in 1 ml of TRIZOL reagent (Life Technologies, Inc., Grand Island, USA) using an Ultrasonic Processor (Vibra Cell, Model CV17; Sonics & Materials Inc., Danbury, Connecticut, USA). After an incubation at 22 ◦ C for 5 min, each sample was added with 0.2 ml of chloroform, shaken vigorously for 15 s, incubated at 22 ◦ C for 3 min, then centrifuged at 12,000 × g for 15 min under 4 ◦ C. After removal of aqueous phase and precipitation with 0.5 ml isopropanol, samples were incubated at 22 ◦ C for 10 min and centrifuged at 12,000 × g for 15 min under 4 ◦ C. The gel-like RNA pellets were washed with 75% ethanol by vortexing and centrifugation at 7,500 × g for 5 min under 4 ◦ C. Thereafter, RNA pellets were dried briefly, dissolved in RNase-free water, and stored at −80 ◦ C. The content of RNA was determined spectrophotometrically at 260 nm. 2.5. Reverse transcription-polymerase chain reaction (RT-PCR) Using the 1st Strand cDNA Synthesis Kit (Boehringer Mannheim GmbH, Germany), RNA was reversely transcribed into single-stranded cDNA. For each sample, 8 ␮l of sterile DEPC water containing 2 ␮g of RNA were added to oligo-p(dT)15 primer (0.8 ␮g/␮l) followed by being denatured at 65 ◦ C for 15 min, cooled at 25 ◦ C for 10 min, then added to a reaction mixture consisting of 10× reaction buffer (100 mM Tris, 500 mM KCl; pH 8.3), deoxynucleotide mix (10 mM each), MgCl2 (25 mM), RNase inhibitor (40 U/␮l), and AMV reverse transcriptase (25 U/␮l). Reaction mixtures were incubated at 42 ◦ C for 2 h and then brought to 95 ◦ C for 5 min to terminate the reaction followed by soaking at 16 ◦ C. PCR was subsequently carried out by mixing 3 ␮l of cDNA product with mastermix solution consisting of DEPC water, 10× reaction buffer, MgCl2 (25 mM), deoxynucleotide mix (10 mM each), P1 and P2 primers (1 ␮g/␮l each), and Taq polymerase (5 U/␮l). An 18-mer oligonucleotide near the initiation codon, encompassing bases 25–42 of rat NPY, was selected as P1 primer (5 -GGGCTGTGTGGACTGACC), and that near the termination codon, inversely complementary to bases 271–288, was selected as P2 primer (5 -GGAAGGGTCTTCAAGCCT). GAPDH was used as the internal standard calibrator. The sequences of GAPDH primers were 5 -TCCCTCAAGATTGTCAGCAA-3 and 5 AGATCCACAACGGATACATT-3 . PCR reactions were carried out on a PCR thermocycler (Perkin-Elmer GeneAmp 9600) with the following steps: 91 ◦ C for 1 min (denaturing),

After RT-PCR, 8 ␮l of each PCR product was subsequently separated by flat-bed gel electrophoresis on a 3% agarose gel. Gels stained by ethidium bromide (0.5 ␮g/ml) were visualized under UV light, photographed, and then scanned densitometrically. Ratio of NPY mRNA and GAPDH mRNA was calculated to determine mRNA level. 2.7. Statistical analysis Data are presented as the mean ± S.E.M. The t-test or oneway ANOVA followed by Dunnett’s test was used to detect significances among groups. P < 0.05 was considered to be statistically significant.

3. Results 3.1. The effect of PPA on food intake The results of food intake in rats receiving PPA were shown in Fig. 1. Rats receiving a PPA injection at 6:00 p.m. showed a decrease of food intake. Analysis with one-way ANOVA [F(3,28) = 51.2, P < 0.01] followed by Dunnett’s test (P < 0.05) indicated the decrease of PPA in food intake was dose-dependent as compared with the control. Only PPA injections at doses higher than 80 mg/kg could induce a significant effect. However, rats receiving a similar dose pattern (0, 40, 80 and 120 mg/kg) of PPA showed no change of feeding

Fig. 1. The effect of phenylpropanolamine (PPA) on the decrease of food intake. Rats were intraperitoneally given with PPA at the beginning of dark period (6:00 p.m.) and were tested for a period of 24 h. Each point represents the mean ± S.E.M. of six to eight animals. ∗ P < 0.05 vs. control group.

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Table 1 The effect of phenylpropanolamine (PPA) treatment on the change of hypothalamic NPY level Groups Normal-fed control Pair-fed control PPA-treated

Doses (mg/kg; i.p.) 0 0 40 80 120

Hypothalamic NPY contents (pg/ml) 63.5 ± 4.5 62.4 ± 3.6 56.4 ± 3.8 49.6 ± 3.1a 44.5 ± 2.8a

NPY contents were measured using radioimmunoassay (RIA). Each data were expressed as mean ± S.E.M. of six rats for each group. a P < 0.05 compared to control group.

behavior when the injection time was changed to be at 6:00 a.m. 3.2. The effect of PPA on NPY content The results shown in Table 1 revealed that PPA treatment at doses of 80 and 120 mg/kg resulted in a significant decrease in concentrations of hypothalamic NPY (P < 0.05) as compared with both the normal-fed and pair-fed control groups. Analysis with one-way ANOVA [F(3,20) = 7.8, P < 0.01] followed by Dunnett’s test (P < 0.05) indicated a dose-dependent

decrease as compared with the control group. There was no difference of NPY content between normal-fed and pair-fed control groups (Fig. 2). 3.3. The effect of PPA on NPY immunoreactivity NPY-immunoreactive (IR) particles were detected in regions of ARC, PVN, PVA and PFA in normal rats while no such signal was detected in control section (data not shown). NPY-IR particles appeared to be less in the regions of PVN, ARC, and periventricular area (PVA), but showed no change in perifornical area (PFA), in rats receiving 80 mg/kg PPA (Fig. 3) as compared with that of saline-treated control (Fig. 2). The method used to define down-regulation of NPYIR particles was by a restricted area with overall reduced expression measured by digital densitometry. The relative ratios of NPY-IR particle measured by digital densitometry between PPA-treated and saline-treated groups were 45 ± 6% in PVN, 55 ± 8% in PVA and 62 ± 7% in ARC. 3.4. The effect of PPA on NPY mRNA level The level of hypothalamic NPY mRNA in PPA-treated rats was significantly decreased (P < 0.01, t-test) compared to that

Fig. 2. NPY immunoreactivity (IR) in the hypothalamus of rats treated with saline (regarded as control). NPY expression was down regulated in regions of (A) paraventricular nuclei (PVN), (B) the magnification (×2.5) of PVN observed in A (asterisk), (C) periventricular area and (D) arcuate nucleus. Frontal sections through the PVN level were obtained. Arrowhead indicated the NPY-IR particles that might appear from light to deep dark. Square indicated a selective area measured by digital densitometry. 3rd V means the third ventricle. Scale bars: 50 ␮m.

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Fig. 3. NPY immunoreactivity in the hypothalamus of rats treated with phenylpropanolamine (PPA). NPY expression was down-regulated in regions of (A) paraventricular nuclei (PVN), (B) the magnification (×2.5) of PVN observed in A (asterisk), (C) periventricular area, and (D) arcuate nucleus. Frontal sections through the PVN level were obtained. Arrowhead indicated the NPY-IR particles that might appear from light to deep dark. Square indicated a selective area measured by digital densitometry. 3rd V means the third ventricle. Scale bars: 50 ␮m.

in saline-treated rats (Fig. 4). Using GAPDH as the internal standard, the ratio of NPY mRNA over GAPDH mRNA in PPA-treated rats was markedly decreased as compared with that in saline-treated rats. The ratio of NPY/GAPDH mRNA measured by digital densitometry was 88 ± 3% in salinetreated group, and 60 ± 4% in PPA-treated group. The relative ratio between PPA-treated and saline-treated group was 68 ± 5%.

4. Discussion PPA has been reported to elicit an anorectic response in the site of hypothalamic PVN [35]. Nevertheless, it is still unclear whether hypothalamic NPY gene is involved in the anorectic effect of PPA. The present study provided genomic evidence to suggest a functional role of NPY gene in regulating the anorectic response of PPA. At first, we explored the treating dosage used in this study. It was documented that an infusion (via miniosmotic pump, Alzet model 2002) of PPA at doses higher than 90 mg/kg per day was necessary to decrease a 24-h pattern of food consumption, and that termination of a continuous PPA infusion at 90 mg/kg per day PPA for 2 weeks would not produce hyperphagia or tolerance in rats [37]. In our previous study, we had found that a PPA treatment at a dose higher than 80 mg/kg

per day was necessary to suppress appetite during a 24-h testing period [8]. We ruled out the possibility that the anorectic effect of PPA was due to the depressing effect (i.e. decrease of locomotor activity) caused by PPA that might nonspecifically disrupt the appetite of an animal [12]. It was because the decrease of locomotor activity induced by PPA was only observed at the initial 20 min of a 24-h testing period (data not shown). This result revealed the potential role of PPA in the induction of appetite suppression during a 24-h testing period. In PPA-treated rats, the decrease of NPY content was consistent with the decrease of feeding behavior, suggesting that NPY was involved in PPA-induced feeding suppression. We ruled out the possibility that the changes in NPY level were simply secondary to the feeding reduction rather than the direct action of PPA on hypothalamic NPY, because pair-fed animals showed no change in NPY level if compared to the control. A subpopulation of NPY-producing neurons originated in the ARC has been shown to project along the medial site of hypothalamus (i.e., the PVA) into the site of PVN [2]. As the expression of NPY-IR following PPA administration was reduced in ARC, PVA, and PVN in the present study, we suggested that ARC-PVA-PVN NPYergic pathway might involve in the anorectic action of PPA. This result provided direct morphological evidence to suggest that hy-

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Fig. 4. Effects of phenylpropanolamine (80 mg/kg, i.p.; PPA) treatments on hypothalamic NPY mRNA levels. (A) The results of RT-PCR analyzing hypothalamic mRNA for NPY and GAPDH in stained ethidium bromide gels. (B) Relative densitometric values between PPA- and saline-treated groups for RT-PCR products of hypothalamic NPY mRNA. Content of NPY mRNA in PPA-treated group was indicated as the percentage of control. Bars are mean ± S.E.M. n = 6 for each group. ∗ P < 0.01 vs. control (t-test).

pothalamic NPY neurons were involved in the anorectic action of PPA. However, NPY immunoreactivity in the region of PFA was not changed despite the changes in regions of ARC, PVA and PVN. This result was consistent with the previous pharmacological evidence, which indicated that PPA could suppress appetite by direct injection into the site of PVN, but produced no effect if injected into the site of PFA [33,35]. In PPA-treated rats, the current result showed that NPY mRNA level decreased following PPA treatment, suggesting that NPY gene was involved in the anorectic action of PPA. This result was different from that in serotonin-treated rats, which indicated that NPY gene was not involved in serotonin-mediated anorexia. Serotonin was also regarded as an appetite-inhibitory neuromodulator in the brain. It was documented that central injection of p-chlorophenylalanine, a serotonin synthesis inhibitor, could decrease NPY content in the area of PVN, but the level of NPY mRNA in hypothalamus and NPY content in the area of ARC were not changed [11]. Another evidence revealed that an antiobesity drug sibutramine, which was regarded as a serotonin/norepinephrine reuptake inhibitor, could induce an

anorectic effect that was independent of the action on hypothalamic NPY neurons [5,6]. Thus, CA agonists might be more effective than serotonin agonists in the induction of appetite suppression due to a more effective effect on NPY gene expression. Interestingly, the attempt to induce PPA anorexia was failed when the injection time was changed to the beginning of light phase (6:00 a.m.). The reason was unknown but might be relevant to the short-term anorectic effect of PPA. It was documented that PPA had an elimination half-life of approximately 1 h and therefore might lead to a temporary anorectic effect [31]. Another reason might be relevant to the circadian change of NPY concentrations, which appeared to be higher at the time near the beginning of dark-on phase, but lower at the time near the beginning of light-on phase in a 12-h light–dark cycle [21]. Moreover, the circadian change of feeding behavior was also related. In rodent animals, feeding behavior was shown more prominent in the dark phase than in the light phase, and NPY activity was responsible for this behavioral difference [18]. Thus, for the purpose of inducing appetite suppression, PPA should be injected at the beginning of dark phase (6:00 p.m.) due to the prominent activity of NPY and feeding behavior of animals in this phase. The present data revealed that injection of PPA once a day (at 6:00 p.m.) could reduce NPY content. For SKF 38393 (a selective dopamine D1 agonist; SKF) or quinpirole (a selective D2 agonist; QNP), however, injections twice a day (at 6:00 p.m. and 12:00 p.m.) were needed to reduce NPY content [18]. This difference implied that PPA was more effective than SKF or QNP in exerting an inhibitory effect on NPY neurons. The reason might be relevant to the simultaneous co-activation of CA receptor subtypes by PPA. Recently, we found that co-activation of ␣1 adrenoceptor and D1 receptor was involved in the anorectic action of PPA [8], which was different from a previous report indicating that only ␣1 adrenoceptor was involved [34]. PPA might thus induce a stronger inhibitory effect on NPY neurons than did SKF or QNP, which induced only a single activation of D1 or D2 receptor subtype, respectively. Together with our previous findings, which revealed that co-activation of D1 and D2 receptor subtypes additively decreased food intake and hypothalamic NPY levels in experimental animals [18], we believed that PPA had a stronger inhibitory effect on NPY neurons than that of SKF or QNP alone due to the co-activation of ␣1 and D1 receptor subtypes induced by PPA. In summary, this study provided immunohistochemical and genomic evidence to suggest that PPA might suppress feeding by altering NPY gene expression. Acknowledgements The authors express the gratitude to Dr. Juei-Tang Cheng, the Professor of the National Cheng Kung University, for the invaluable suggestions in this study. This study was supported by a grant from Chung Shan Medical University (CSMU 92OM-B-023).

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