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Endocrinology, doi:10.1210/en.2005-1619
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Endocrinology Vol. 147, No. 9 4093-4103
Copyright © 2006 by The Endocrine Society

Growth Hormone-Releasing Hormone (GHRH) Neurons in the Arcuate Nucleus (Arc) of the Hypothalamus Are Decreased in Transgenic Rats Whose Expression of Ghrelin Receptor Is Attenuated: Evidence that Ghrelin Receptor Is Involved in the Up-Regulation of GHRH Expression in the Arc

Asuka Mano-Otagiri1, Takahiro Nemoto1, Azusa Sekino, Naoko Yamauchi, Yujin Shuto, Hitoshi Sugihara, Shinichi Oikawa and Tamotsu Shibasaki

Departments of Physiology (A.M.-O., T.N., A.S., N.Y., T.S.) and Medicine (Y.S., H.S., S.O.), Nippon Medical School, Bunkyo-ku, Tokyo 113-8602, Japan

Address all correspondence and requests for reprints to: Asuka Mano-Otagiri, Department of Physiology, Nippon Medical School 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan. E-mail: asuka{at}nms.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GH secretagogue (GHS)/ghrelin stimulates GH secretion by binding mainly to its receptor (GHS-R) on GHRH neurons in the arcuate nucleus (Arc) of the hypothalamus. GHRH, somatostatin, and neuropeptide Y (NPY) in the hypothalamus are involved in the regulatory mechanism of GH secretion. We previously created transgenic (Tg) rats whose GHS-R expression is reduced in the Arc, showing lower body weight and shorter nose-tail length. GH secretion is decreased in female Tg rats. To clarify how GHS-R affects GHRH expression in the Arc, we compared the numbers of GHS-R-positive, GHRH, and NPY neurons between Tg and wild-type rats. Immunohistochemical analysis showed that the numbers of GHS-R-positive neurons, GHRH neurons, and GHS-R-positive GHRH neurons were reduced in Tg rats, whereas the numbers of NPY neurons and GHS-R-positive NPY neurons did not differ between the two groups. The numbers of Fos-positive neurons and Fos-positive GHRH neurons in response to KP-102 were decreased in Tg rats. Competitive RT-PCR analysis of GHRH mRNA expression in the cultured hypothalamic neurons showed that KP-102 increased NPY mRNA expression level and that NPY decreased GHRH mRNA expression level. KP-102 increased GHRH mRNA expression level in the presence of anti-NPY IgG. GH increased somatostatin mRNA expression. Furthermore, GH and somatostatin decreased GHRH mRNA expression, whereas KP-102 showed no significant effect on somatostatin mRNA expression. These results suggest that GHS-R is involved in the up-regulation of GHRH and NPY expression and that NPY, somatostatin, and GH suppress GHRH expression. It is also suggested that the reduction of GHRH neurons of Tg rats is induced by a decrease in GHS-R expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
G H SECRETAGOGUES (GHSs), which were developed from the structure of met-enkephalin (1), strongly stimulate GH secretion by acting on GHRH neurons in the arcuate nucleus (Arc) of the hypothalamus, their main site of action, and on somatotrophs of the pituitary through the GHS receptor (GHS-R), a member of the G protein-coupled receptor superfamily (2). Ghrelin, an endogenous ligand for GHS-R, has been discovered in rat stomach extract (3). GHS-R mRNA is expressed in several hypothalamic nuclei, such as the Arc, paraventricular nucleus of the hypothalamus, ventromedial hypothalamic nucleus, supraoptic nucleus, suprachiasmatic nucleus, lateroanterior hypothalamic nucleus, and tuberomammillary nucleus, and other brain areas such as the hippocampus, substantia nigra, ventral tegmental area, and dorsal and median raphe nuclei (4). The systemic administration of GHSs induces c-fos mRNA expression in the neurons of Arc. Overall, of these c-fos mRNA-expressing neurons, 51% are neuropeptide Y (NPY) neurons, 23% are GHRH neurons, 11% are tyrosine hydroxylase (TH) neurons, 11% are proopiomelanocortin neurons, and 4% are somatostatin neurons (5, 6).

To clarify the physiological significance of GHS-R, we have created transgenic (Tg) rats expressing an antisense GHS-R mRNA that is designed to be specific for the region around the initiation codon of GHS-R, under the control of the promoter for TH (7). TH is a rate-limiting enzyme in catecholamine biosynthesis and is a marker for the dopaminergic neurons. TH is present in most neurons in the ventral portion of the Arc where GHRH neurons exist (8). TH mRNA-expressing neurons in the Arc of Tg rats have been shown to express antisense GHS-R mRNA (7). GHS-R mRNA is detected in NPY and GHRH neurons in the Arc (9, 10, 11). We have found that GHS-R is expressed in both GHRH and NPY neurons in the Arc of Tg rats and slc:SD wild-type (WT) rats and that GHS-R, as determined by Western blot analysis, in the Arc is reduced in Tg rats compared with WT rats (7). These Tg rats have lower body weight and less adipose tissue, suggesting that GHS-R plays a role in the regulation of adiposity (7). They also have lower GH responses to iv administered KP-102, one of the GHSs, than WT rats (7). Furthermore, GH secretion and plasma insulin-like growth factor I levels are significantly reduced in female Tg rats, suggesting that GHS-R is involved in the regulation of GH secretion and that GHS-R plays a more important role in the regulatory mechanism of GH secretion in female than in male rats (7). However, the details of GHRH, GHS-R, and NPY expression in the Arc of Tg rats have not yet been examined. The relation of the GHS-R expression level with the expression of GHRH and NPY in the Arc has also not yet been studied. Recently, it has been reported that Tg mice overexpressing GHS-R1A in the GHRH neurons show an increase in hypothalamic GHRH expression (12), suggesting that GHS/GHS-R may regulate GHRH expression.

Therefore, in this study, we first compared the numbers of GHRH neurons, NPY neurons, GHS-R-positive neurons, GHS-R-positive GHRH neurons, and GHS-R-positive NPY neurons located in the Arc of Tg and WT rats to clarify whether the GHS-R expression levels affect GHRH and NPY expression. We then examined Fos expression of the neurons located in the Arc of Tg and WT rats in response to intracerebroventricular (ICV) injection of KP-102 to confirm that GHS-R is reduced in the Arc of Tg rats. We finally examined the effects of KP-102, NPY, somatostatin, and GH on the GHRH mRNA, NPY mRNA, or somatostatin mRNA expression level in primary cultured hypothalamic neurons of normal rats to clarify whether and how ghrelin/GHS-R affects the expression level of GHRH mRNA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Twelve-week-old random-cycling female Tg rats (7) and WT rats were used in this study. The WT rats used in the present study were not littermates of Tg rats, although we used WT rats, which were littermates of Tg rats, in our previous studies (7). WT rats, which we used for these 5 yr, always showed similar results. The Tg rats, which are homozygote for transgene, also showed unchanged phenotype. There were significant differences in body weights [Tg rats (n = 14), 199.6 ± 2.6 g vs. WT rats (n = 14), 215.6 ± 2.3 g, P < 0.001] and in nose-tail length [Tg rats (n = 14), 36.9 ± 0.2 cm vs. WT rats (n = 14), 37.5 ± 0.2 cm, P < 0.05]. All animals were housed under controlled temperature and illumination (0800–2000 h) and allowed ad libitum access to food and water. To administer samples ICV, a polyethylene cannula was implanted into the right lateral ventricle under sodium pentobarbital anesthesia (50 mg/kg body weight ip injection), as described previously, 5 d before the experiment was to be done (13). All experimental procedures were conducted in accordance with the guidelines on the use and care of laboratory animals approved by the Local Ethics Committee of Nippon Medical School, Japan.

Effect of ICV injection of KP-102 on Fos expression
On the day of the experiment, rats with ad libitum access to food and water were given vehicle (2 µl saline) or KP-102 (100 pmol/2 µl) ICV. Ninety minutes after the injection, the rat brain was fixed for immunohistochemistry.

Immunohistochemistry of GHS-R, GHRH, and NPY
To compare the numbers of GHRH neurons, NPY neurons, and GHS-R-positive neurons of Tg and WT rats, the rats were injected ICV with colchicine (100 µg/5 µl saline) through the cannula. Tg and WT rats were anesthetized with pentobarbital (50 mg/kg body weight ip injection) and perfused via an intracardiac cannula with PBS followed by 4% paraformaldehyde 48 h after the injection of colchicine. The brain was removed, left overnight in 4% paraformaldehyde and then transferred to 20% sucrose/PBS. Coronal sections (20 µm) were cut with a cryostat and mounted onto gelatinized slides. Successive sections were used for immunohistochemistry of GHS-R, GHRH, and NPY.

Immunohistochemistry was performed with the avidin-biotin-peroxidase method using specific antiserum against rat GHRH (14), mouse GHS-R (15), and rat/human NPY (16). Briefly, sections were incubated with specific polyclonal antiserum against GHRH (1:1000), GHS-R (1:500) (7), or NPY (1:1000) overnight at 4 C. The tissues were then rinsed in PBS and incubated in biotinylated goat antirabbit IgG (1:200; Vector Laboratories, Burlingame, CA) for 1 h at room temperature. This was followed by another 1-h incubation in avidin-biotin complex solution (Vectorstain ABC Elite kit, Vector Laboratories) at room temperature. The reaction product was visualized using a nickel-intensified diaminobenzidine reaction that gives a dark-brown precipitate. Briefly, sections were washed in 0.1 M sodium acetate buffer (pH 6.0) and incubated in the same buffer containing 2.5% nickel sulfate, 0.2% glucose, 0.04% ammonium chloride, 0.025% diaminobenzidine, and 30 U/ml glucose oxidase (Sigma-Aldrich, St. Louis, MO). The reaction was stopped by washing in 0.1 M acetate buffer under microscopic observation (17). The tissue was dehydrated through graded alcohols, cleared in xylenes, and then coverslipped with Vector Mount (Vector Laboratories). Preincubation of anti-GHRH serum with 1.0 µg GHRH, anti-GHS-R serum with 1.0 µg GHS-R, and anti-NPY serum with 1.0 µg NPY completely abolished the staining of GHRH, GHS-R, and NPY, respectively. Quantitative analysis of the number of GHS-R, GHRH, and NPY neurons was performed using an image analysis system (MCID Amersham Biosciences, Tokyo, Japan). Under light microscopy, at 200x magnification, the total number of positive neurons was counted in the Arc. Every three sections of 20 µm were used for the counting of GHS-R, GHRH, or NPY (eight sections for GHRH, GHS-R, and NPY, respectively, per rat).

Double-labeled immunohistochemistry for GHS-R with GHRH or NPY
Double-labeled immunofluorescence for GHS-R and GHRH or NPY coupled with confocal microscopic analysis was done using hypothalami of colchicine-treated rats. Coronal sections (10 µm) were mounted onto gelatinized slides. Sections were incubated in antiserum against GHS-R (1:500) overnight at 4 C. The tissues were then rinsed in PBS and incubated in fluorescein-conjugated goat antirabbit IgG (1:200, Vector Laboratories) for 3 h at room temperature. The sections were washed in PBS and subsequently incubated overnight at 4 C with the second antibody, GHRH or NPY. After washing in PBS, the tissues were incubated in Texas red-conjugated goat antirabbit IgG (1:200; Vector Laboratories) for 3 h at room temperature. The slides were coverslipped with VECTASHIELD Hard Set mounting medium (Vector Laboratories). Sections were examined using a Zeiss LSM 510 confocal microscope (Carl Zeiss Co. Ltd., Thornwood, NY). Immunofluorescence in tissue sections was visualized by using a Zeiss Axioplan photomicroscope with a multiband filter set for independent or simultaneous visualization of fluorescein (excitation range, 447–501 nm; emission range, 510–540 nm) and Texas red (excitation range, 560–596 nm; emission range, 610–655 nm) fluorophores. Double-labeled neurons for GHS-R and GHRH or NPY in the Arc were counted in five randomly selected sections under light microscopy at x400 magnification. All images were processed by using Adobe Photoshop software (Adobe Systems, San Jose, CA).

Immunohistochemistry for Fos
Twenty serial coronal sections (40 µm) were cut with a cryostat through the Arc. Immunohistochemistry was done using the free-floating method with the avidin-biotin-peroxidase method using Fos antibody (1:10000; rabbit polyclonal, Ab5; Oncogene, San Diego, CA). The antibody-peroxidase complex was visualized using diaminobenzidine (Vector DAB kit, Vector Laboratories).

Double-labeled immunohistochemistry for Fos and GHRH
Forty-micrometer sections were processed for immunohistochemical detection of Fos using the Vector DAB kit, and they were then incubated with GHRH antiserum (1:2000) overnight at room temperature. Immunostaining was visualized using the Vector SG substrate kit (Vector Laboratories), which gives a blue-gray precipitate. Quantitative analysis of the number of Fos-positive neurons was done using MCID image analysis system (MCID Amersham Biosciences). Under light microscopy at x200 magnification, the total number of Fos-positive neurons was counted in the Arc. In total, nine sections per Arc were used for counting. Double-labeled neurons for Fos and GHRH in the Arc were counted in five randomly selected sections under light microscopy at x400 magnification. Sections were viewed and photographed with an Olympus AX 80 microscope. All images were processed by using Adobe Photoshop software (Adobe Systems).

Primary culture of rat hypothalamic neurons
Newborn Wistar rats were killed by decapitation, and their hypothalami were removed under sterile conditions. Hypothalami were minced in a 1:1 mixture of DMEM and Ham’s nutrient mix F-12 containing 10% fetal calf serum, penicillin, and streptomycin (DMEM/F12) (Sigma-Aldrich) for suspension. The minced tissues were then washed twice in PBS. The mixture was incubated in PBS containing 0.047 g/liter MgCl2, 0.1 g/liter CaCl2, and 0.01% Dispase (Godoshusei, Tokyo, Japan) with constant stirring for 30 min at room temperature. After being washed three times with PBS, 1-ml aliquots of cell suspension containing 5.0 x 104 cells in a DMEM/F12 were placed in the wells of 24-well plates that were coated with poly-D-lysine (Sigma-Aldrich). The cells were subsequently allowed to attach to the bottom surface in a humidified 95% air-5% CO2 incubator for 6 d.

To test the effect of KP-102 on GHRH mRNA expression, cells were treated with KP-102 at concentrations ranging from 2–200 nM for 2 h, and cells were also treated with 20 nM KP-102 for 1–24 h to test the time course of the effect. To test the effect of KP-102 on NPY or somatostatin mRNA expression, cells were treated with 0.2, 2.0, or 20 nM KP-102 for 2 h or 20 nM KP-102 for 24 h.

To test the effect of NPY on GHRH mRNA expression, cells were treated with NPY at a concentration of 0.1 or 1.0 nM for 2 h. To delete the influence of NPY on KP-102-induced changes in GHRH mRNA expression in the culture system, anti-NPY IgG was used. To examine the binding capacity of anti-NPY IgG to NPY in the culture system, cells were treated for 2 h with 1 nM NPY plus anti-NPY IgG (3.6 µg/ml) or normal rabbit serum IgG (3.6 µg/ml) prepared from rabbit anti-NPY serum (16) or normal rabbit serum, respectively, using Protein A Sepharose 4FF (Pharmacia Biotech, Tokyo, Japan). Subsequently, the expression of GHRH mRNA was determined. To examine the effect of anti-NPY IgG on KP-102-induced changes in GHRH mRNA expression, cells were incubated with KP-102 plus anti-NPY IgG or normal rabbit serum IgG (3.6 µg/ml) for 2 h. To examine the effect of anti-NPY IgG on somatostatin-induced changes in GHRH mRNA expression, cells were incubated with 10 nM somatostatin plus anti-NPY IgG or normal rabbit serum IgG for 2 h.

To test the effect of somatostatin on GHRH mRNA expression, cells were treated with somatostatin at a concentration of 1, 10, or 100 nM for 2 h. To delete the influence of somatostatin on the KP-102-induced change in the GHRH mRNA expression level in the culture system, antisomatostatin IgG was used. Antisomatostatin serum was obtained from female New Zealand white rabbits by immunizing them with synthetic rat somatostatin-14 coupled with porcine thyroglobulin through water-soluble carbodiimide hydrochloride. The antiserum was used in the RIA at a final concentration of 1/380,000 and showed no cross-reactivity with GHRH, NPY, or corticotropin-releasing factor. The antisomatostatin IgG fraction was prepared using Protein A Sepharose 4FF (Pharmacia Biotech). To test the binding capacity of antisomatostatin IgG to somatostatin in the culture system, cells were treated with somatostatin (10 nM) plus antisomatostatin IgG (3.6 µg/ml) or normal rabbit serum IgG (3.6 µg/ml) for 2 h, and then the level of GHRH mRNA expression was determined. To examine the effect of antisomatostatin IgG on KP-102-induced changes in GHRH mRNA expression, cells were incubated with KP-102 plus antisomatostatin IgG or normal rabbit serum IgG (3.6 µg/ml) for 2 h. To examine the effect of somatostatin on NPY mRNA expression, cells were treated with somatostatin at a concentration of 10 nM for 2 h.

To test the effect of GH on GHRH, NPY, or somatostatin mRNA expression, cells were treated with human recombinant GH (ProSpec-Tany TechnoGene LTD, Rehovot, Israel) at concentrations ranging from 1–500 ng/ml for 2 h.

To test the effect of KP-102, NPY, or somatostatin on GHRH synthesis and release, cells were treated with KP-102 at concentrations ranging from 2–200 nM, NPY, or somatostatin at concentrations of 1 and 10 nM for 4–24 h.

RT-PCR
Total RNA was extracted from cells using Isogen according to the manufacturer’s instructions (Takara, Shiga, Japan). To avoid false-positive results caused by DNA contamination, a deoxyribonuclease (DNAse) treatment for 60 min at 37 C using ribonuclease-free DNase (Takara) was done. First strand cDNA was synthesized using 1 µg denatured total RNA under conditions of 42 C for 30 min, 99 C for 5 min, and 5 C for 5 min using RT-PCR kit (Takara). PCR was carried out under conditions of denaturation at 94 C for 10 sec, annealing at 50 C for 5 sec, and extension at 72 C for 60 sec for 30 cycles, using specific primers for GHRH, NPY, and somatostatin (Table 1Go). After amplification, the PCR products were subjected to 2% agarose gel electrophoresis, stained with 0.5 µg/ml ethidium bromide, and were then visualized under UV illumination. All PCR-amplified DNAs were sequenced for purposes of confirmation.


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  		TABLE 1. Primer sequences of the studied genes

 
Competitor construction
Homologous competitive internal standards that shared the same primer binding sites but contained a shortened internal sequence with respect to the endogenous target RNA for GHRH, NPY, or somatostatin were prepared as follows. The products resulting from PCR were subcloned into the pGEM-T vector (Promega, Madison, WI) and sequenced. The cloned pGEM-T vector was linearized by NcoI restriction digestion and transcribed into the cRNA template by SP6 RNA Polymerase (Promega). The DNA template was removed after transcription, and the cRNA product was quantified and used as an internal standard in RT-PCR for GHRH, NPY, and somatostatin gene expression.

Competitive RT-PCR
After DNase treatment, the amount of mRNA present in the samples was normalized using ß-actin primers as an internal reference standard (Table 1Go). To test for possible pseudogene or genomic DNA contamination, either the RT enzyme or RNA was omitted from the reaction tube. To confirm that RNA competitor is not contaminated with DNA, we performed RT-PCR using only RNA competitor (109 copies) at the maximum amount. The reaction mixture and RNA competitor were added to each tube. RT reaction was carried out under conditions of 42 C for 30 min, 99 C for 5 min, and 5 C for 5 min. Then, PCR mixture was dispensed into each tube, which contained RT reactant. PCR was carried out under the following conditions: denaturation at 94 C for 10 sec, annealing at 60 C for 5 sec, and primer extension at 72 C for 60 sec for 30 cycles. PCR products were separated on 2% agarose gel and visualized with ethidium bromide. The intensities of the bands of the PCR products of GHRH, NPY, and somatostatin were quantified using National Institutes of Health image software. The ratio of internal standard to endogenous area was plotted as a function of the competitor concentration added to each PCR. The concentrations of GHRH, NPY, and somatostatin mRNA were determined at the point where the ratio of the internal standard and the endogenous area of each gene were equal to 1 (the equivalence point). Experiments of same protocol were repeated twice or three times, and the results were combined for statistical analysis.

RIA for GHRH
RIA for GHRH was performed as described previously (14). In short, synthetic rat GHRH was iodinated using the chloramine-T method and purified on a column of Sephadex G-50. PBS [0.1 M (pH 7.5)] containing 0.01% Nonidet P-40 (Nacalai Inc., Kyoto, Japan), 5 mM EDTA-Na, and 0.02% NaN3 was used for RIA. Standard synthetic rat GHRH or sample was incubated with antirat GHRH antiserum in 3-ml plastic tubes for 24 h at 4 C. 125I-labeled GHRH was then added to each tube and incubated for another 24 h. Goat antirabbit IgG was used to separate tracer bound to antiserum from free tracer. The anti-GHRH antiserum was used for RIA at a final concentration of 1:400,000 to yield a maximum binding of approximately 30%.

Image analysis
Image analysis was performed with an Olympus AX-80 microscope and a digital camera (DP50, Olympus, Tokyo, Japan). Images were assembled using Lumina Vision (Mitani Corp., Tokyo, Japan).

Statistical analysis
Data are expressed as mean ± SEM. The statistical analysis was completed using StatView 4.5 (Abacus Concepts, Inc., Berkeley, CA); for in vivo study, one-way ANOVA followed by a post hoc Fisher’s test was performed, whereas for the in vitro study, ANOVA followed by Fisher’s PLSD was done. P < 0.05 considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distribution and number of GHS-R-positive, GHRH, and NPY neurons in the Arc
GHS-R-positive neurons were widely distributed in the Arc of the WT and Tg rats (Fig. 1Go, A and B). The mean number of GHS-R-positive neurons was significantly less in Tg rats than in WT rats (Tg rats, 927 ± 57 vs. WT rats, 1409 ± 199, P < 0.05) (Fig. 1GGo). GHRH neurons were distributed in the ventral and lateral part of the Arc of the WT and Tg rats (Fig. 1Go, C and D). The mean number of GHRH neurons was significantly less in Tg rats than that in WT rats (Tg rats, 206 ± 33 vs. WT rats, 326 ± 59, P < 0.05) (Fig. 1GGo). NPY neurons were distributed in the portion medial to that of the GHRH neurons in the Arc (Fig. 1Go, E and F). The mean number of NPY neurons did not differ between Tg and WT rats (Tg rats, 468 ± 49 vs. WT rats, 478 ± 49) (Fig. 1GGo).


Figure 1
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  		FIG. 1. Localization GHS-R-positive neurons, GHRH neurons, and NPY neurons in the Arc of female WT and Tg rats. GHS-R-positive neurons of female WT and Tg rats are shown in A and B, respectively, GHRH neurons of female WT and Tg rats in C and D, respectively, and NPY neurons of female WT and Tg rats in E and F, respectively. Scale bars, 100 µm. The numbers of GHS-R-positive neurons, GHRH neurons, and NPY neurons in the Arc are shown in G. *, P < 0.05 vs. WT rats. The number of rats of each group was five.

 
Coexpression of GHS-R with GHRH or NPY in the Arc
GHS-R-positive neurons (green), GHRH neurons (red), and GHS-R-positive GHRH neurons (yellow) in the Arc of WT rats (A–C) and Tg rats (D–F) are shown in Fig. 2Go. The numbers of GHRH neurons and GHS-R-positive GHRH neurons in Tg rats were significantly reduced than those in WT rats, respectively (GHRH neurons, 41 ± 5, GHS-R-positive GHRH neurons, 20 ± 3 in Tg rats; GHRH neurons, 82 ± 5, GHS-R-positive GHRH neurons, 54 ± 7 in WT rats, P < 0.05). GHS-R-positive neurons (green), NPY neurons (red), and GHS-R-positive NPY neurons (yellow) in the Arc of WT rats (A–C) and Tg rats (D–F) are shown in Fig. 3Go. There was no difference in the number of NPY neurons and GHS-R-positive NPY neurons between Tg rats (NPY neurons, 322 ± 27, GHS-R-positive NPY neurons, 315 ± 28) and WT rats (NPY neurons, 311 ± 18, GHS-R-positive NPY neurons, 309 ± 18). The number of GHRH neurons in Fig. 2Go or NPY neurons in Fig. 3Go was lower than that in Fig. 1Go. These differences are explained by the methodological difference between glucose oxidase diaminobenzidine nickel intensify method used in Fig. 1Go and double-labeled immunofluorescence used in Figs. 2Go and 3Go, the former is more sensitive than the latter.


Figure 2
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  		FIG. 2. Double-labeled immunohistochemistry of GHS-R and GHRH in the Arc of female WT and Tg rats. Confocal images of GHS-R (A), GHRH (B), and both (C) of WT rats and those of GHS-R (D), GHRH (E), and both (F) of Tg rats. Arrows, Positive neurons. G, Numbers of GHRH and GHS-R-positive GHRH neurons in the Arc. Scale bars, 10 µm. *, P < 0.05 vs. WT rats. The number of rats of each group was seven.

 

Figure 3
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  		FIG. 3. Double-labeled immunohistochemistry of GHS-R and NPY in the Arc of female WT and Tg rats. Confocal images of GHS-R (A), NPY (B), and GHS-R-positive NPY neurons (C) of WT rats and GHS-R (D), NPY (E), and GHS-R-positive NPY neurons (F) of Tg rats. Arrows, Positive neurons. The numbers of NPY and GHS-R-positive NPY neurons in the Arc are shown in G. Scale bars, 10 µm. The number of rats of each group was seven.

 
Fos expression in response to KP-102 in the Arc
Fos-positive neurons in the Arc of WT and Tg rats in response to vehicle are shown in Fig. 4Go, A and C, respectively. In response to ICV administration of KP-102, Fos-positive neurons distributed widely in the Arc of WT and Tg rats are shown in Fig. 4Go, B and D, respectively. The mean number of Fos-positive neurons after ICV administration of KP-102 was significantly reduced to 46% of that seen in WT rats (Fig. 4EGo).


Figure 4
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  		FIG. 4. Changes of Fos-expressing neurons in the Arc of female WT and Tg rats after KP-102 administration. Fos-positive neurons in the Arc 90 min after ICV administration of 2 µl saline (A and C) and 100 pmol KP-102 (B and D) were immunohistochemically determined in WT (A and B) and Tg (C and D) rats. Scale bars, 200 µm. E, Statistical analysis of the Fos-positive neurons in the Arc of WT and Tg rats. *, P < 0.01 vs. WT rats treated with saline; #, P < 0.01 vs. WT rats treated with KP-102. The number of rats of each group was seven.

 
The distributions of Fos-positive GHRH neurons of the Arc in response to saline or KP-102 in the WT and Tg rats are shown in Fig. 5Go. The distribution of Fos-positive GHRH neurons in response to saline in WT rats is shown in Fig. 5AGo and that of Fos-positive GHRH neurons in response to KP-102 in WT rats is in Fig. 5BGo. The distribution of Fos-positive GHRH neurons in response to saline in Tg rats is shown in Fig. 5CGo, whereas that of Fos-positive GHRH neurons in response to KP-102 in Tg rats is in Fig. 5DGo. The mean number of Fos-positive GHRH neurons in response to KP-102 was significantly less in Tg rats than in WT rats (Fig. 5EGo).


Figure 5
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  		FIG. 5. Distribution and number of Fos-positive GHRH neurons in the Arc in response to saline or KP-102 in female WT and Tg rats. A, Fos-positive GHRH neurons in the Arc 90 min after ICV administration of 2 µl saline (A and C) and 100 pmol KP-102 (B and D) were immunohistochemically determined in WT (A and B) and Tg (C and D) rats. Scale bars, 100 µm. E, Statistical analysis of the Fos-positive GHRH neurons in the Arc of WT and Tg rats. *, P < 0.05 vs. WT rats treated with saline. #, P < 0.05 vs. WT rats treated with KP-102. The number of rats of each group was eight.

 
GHRH mRNA expression in primary cultured hypothalamic neurons
The results of a typical RT-PCR analysis of GHRH, NPY, and somatostatin mRNA levels in cultured hypothalamic neurons are shown in Fig. 6Go. Treatment of cultured neurons with KP-102 at concentrations ranging from 2–200 nM for 2 h did not significantly affect the level of GHRH mRNA expression (Fig. 7AGo). KP-102 at 20 nM did not significantly affect the GHRH mRNA expression level during 1, 2, 8, and 24 h, although with 2-h incubation, there was a trend to increased GHRH mRNA expression (Fig. 7BGo). Treatment of cultured neurons with KP-102 for 2 h did not significantly increase the level of NPY or somatostatin mRNA expression at a concentration of 0.2, 2.0, or 20 nM (Fig. 7Go, C and D). However, treatment of cultured neurons with KP-102 at a concentration of 20 nM for 24 h but not 2 h significantly increased the NPY mRNA expression level (Fig. 7EGo). The level of somatostatin mRNA expression was not affected by KP-102 given at a concentration of 20 nM for either 2 or 24 h (Fig. 7FGo).


Figure 6
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  		FIG. 6. Competitive RT-PCR assay for GHRH (A), NPY (B), and somatostatin (C) of cultured hypothalamic neurons. Extracted total RNAs were coamplified with serially diluted competitors. The competitor copy numbers were expressed as copies per microliter = OD260 x 40 (ng/µl) x 10–9 x 6 x 1023/(length x 345). The ratio of the intensities of the target gene to the competitor was plotted against the concentration of the competitor on a log scale.

 

Figure 7
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  		FIG. 7. Effects of KP-102 on GHRH, NPY, and somatostatin mRNA expression in cultured rat hypothalamic neurons. A, Dose-response effect of KP-102 on GHRH mRNA expression. B, Effect of duration of treatment with KP-102 on GHRH mRNA expression. C, Effect of KP-102 at concentrations of 0.2, 2.0, and 20 nM on NPY mRNA expression for 2 h. D, Effect of KP-102 on somatostatin mRNA expression at concentrations of 0.2, 2.0, and 20 nM for 2 h. E, Effect of KP-102 at a concentration of 20 nM on NPY mRNA expression for 2 or 24 h. F, Effect of KP-102 at a concentration of 20 nM on somatostatin mRNA expression for 2 or 24 h. The numbers of wells in each experimental group (A–F) were 12, 12, 9, 9, 8, and 8, respectively. *, P < 0.05.

 
NPY at a concentration of 1 nM but not 0.1 nM significantly decreased the GHRH mRNA expression level (Fig. 8AGo). The inhibitory effect of 1 nM NPY on GHRH mRNA expression was completely blocked by anti-NPY IgG (3.6 µg/ml) (Fig. 8BGo). Anti-NPY IgG itself did not affect the level of GHRH mRNA expression. KP-102 significantly increased the level of GHRH mRNA expression approximately 2-fold in the presence of anti-NPY IgG during 2-h incubation, but it did not induce significant change in the expression level when given with normal rabbit serum IgG or without IgG (Fig. 8CGo).


Figure 8
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  		FIG. 8. Effects of NPY and somatostatin on GHRH mRNA expression and the KP-102-induced changes in GHRH mRNA expression level. A, Inhibitory effect of NPY on GHRH mRNA expression. B, Effect of anti-NPY IgG on NPY-induced inhibition of GHRH mRNA expression. C, Effect of anti-NPY IgG on KP-102-induced changes in GHRH mRNA expression. Cells were treated with vehicle (white bar) or 20 nM KP-102 (black bar) for 2 h in the presence of NRS or anti-NPY IgG. D, Inhibitory effect of somatostatin on GHRH mRNA expression. E, Effect of antisomatostatin IgG on somatostatin-induced inhibition of GHRH mRNA expression. F, Effect of antisomatostatin IgG on KP-102-induced changes of GHRH mRNA expression. Cells were treated with vehicle (white bar) or 20 nM KP-102 (black bar) for 2 h in the presence of NRS or antisomatostatin IgG. The number of wells in each experimental group (A–F) were 8, 9, 12, 8, 8, and 8, respectively. NRS, Normal rabbit serum IgG (3.6 µg/ml); Anti-NPY, anti-NPY IgG (3.6 µg/ml); Anti-SS, antisomatostatin IgG (3.6 µg/ml). *, P < 0.05.

 
Somatostatin significantly decreased the GHRH mRNA expression level at concentrations of 10 and 100 nM during the 2-h incubation period (Fig. 8DGo). The 10 nM somatostatin-induced suppression of GHRH mRNA expression level was completely reversed in the presence of antisomatostatin IgG (3.6 µg/ml) but not in the presence of normal rabbit serum IgG (Fig. 8EGo). The antisomatostatin IgG did not affect the level of GHRH mRNA expression in the presence of KP-102 (Fig. 8FGo). Treatment of neurons with somatostatin at a concentration of 10 nM for 2 h induced no significant change in NPY mRNA expression (Fig. 9AGo). The somatostatin-induced suppression of GHRH mRNA expression was partially reversed by anti-NPY IgG (Fig. 9BGo). GH at concentrations ranging from 10–500 ng/ml for 2 h significantly increased somatostatin mRNA expression level and suppressed GHRH mRNA expression level without influence on NPY mRNA expression (Fig. 10Go).


Figure 9
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  		FIG. 9. Effect of somatostatin on NPY mRNA expression and effect of anti-NPY IgG on somatostatin-induced inhibition of GHRH mRNA. A, Effect of 10 nM somatostatin for 2 h on NPY mRNA expression. B, Effect of anti-NPY IgG on somatostatin-induced inhibition of GHRH mRNA expression. NRS, Normal rabbit serum IgG (3.6 µg/ml); Anti-NPY, anti-NPY IgG (3.6 µg/ml). The numbers of wells in each experimental group of A and B were nine. *, P < 0.05.

 

Figure 10
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  		FIG. 10. Effects of GH on GHRH, NPY, and somatostatin mRNA expression in cultured rat hypothalamic neurons. The number of wells in each treatment group was six. *, P < 0.05.

 
Concentrations of GHRH in cultured neurons
Although the basal secretion of GHRH from cultured hypothalamic neurons was undetectable level by RIA, the cellular concentrations of GHRH after 4-h treatment with KP-102 at concentrations of 2.0 and 20 nM were significantly increased (control, 567.5 ± 11.7 pg/well, 2.0 nM KP-102, 602.0 ± 13.6 pg/well, P < 0.001, vs. control, 20 nM KP-102, 651.0 ± 10.9 pg/well, P < 0.001 vs. control n = 6). The cellular concentrations of GHRH after 4-h treatment with somatostatin at a concentration of 1.0 and 10 nM did not show any significant changes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously created Tg rats expressing an antisense GHS-R mRNA under the control of the promoter for TH and have reported that the concentrations of GHS-R protein in the Arc determined by Western blot analysis are lower in Tg rats than in WT rats (7). In the present study, we found that the numbers of GHS-R-positive neurons, GHRH neurons, and GHS-R-positive GHRH neurons were significantly lower in Tg rats compared with WT rats, whereas the number of NPY neurons or GHS-R-positive NPY neurons did not differ between the two groups. The expression level of NPY mRNA in the Arc of the Tg rats is thought not to be affected by the induction of GHS-R antisense due to the absence of TH in most of the NPY neurons located in the Arc (18, 19). The present study shows that, in response to the ICV injection of KP-102, one of the GHSs, Fos-positive neurons, and Fos-positive GHRH neurons in the Arc were reduced in Tg rats, reflecting the reduced expression of GHS-R in neurons including GHRH neurons in the Arc of Tg rats. These results suggest that the ghrelin/GHS-R system plays a role in up-regulating GHRH mRNA expression. This hypothesis is supported by a previous study, which found that Tg mice that constitutively overexpress GHS-R in the GHRH neurons show an increase in hypothalamic GHRH expression (12). However, unexpectedly, the ICV administration of ghrelin has been reported to induce no changes in the levels of GHRH mRNA expression levels, although it increases the levels of NPY and agouti-related peptide mRNA expression in the Arc (20, 21). Therefore, it seems that some mechanism masks the stimulatory effect of exogenous ghrelin on GHRH mRNA expression level in in vivo experiments.

Because GH, NPY, and somatostatin are involved in the feedback mechanism of the regulation of GH secretion, these hormones are the likely candidates that mask the stimulatory effect of ghrelin on GHRH mRNA expression. ICV administration of ghrelin increases GH secretion in nonanesthetized and anesthetized rats (22, 23). It is reported that iv administration of GH induces the expression of c-fos mRNA in NPY neurons of the Arc (24), where the GH receptor is expressed (25, 26). However, the present study showed that GH did not affect NPY mRNA expression. The difference in the effects of GH on NPY neurons may be explained by the difference of GH signal transport between in vivo study and in vitro study. Although these experiments do not show how GH modifies the release of NPY, the results of our study do not suggest a direct action of GH on NPY neurons.

When it is administered ICV, NPY inhibits GH secretion in rats (27, 28) at least in part through somatostatin release because NPY stimulates somatostatin release from the hypothalamus in vitro (29, 30). NPY neurons in the Arc project to the periventricular nucleus (PeV) (31), and synaptic connections between NPY axons and somatostatin neurons have been demonstrated in the PeV (32). These findings suggest that GH secreted in response to ghrelin may act on somatostatin neurons because GH receptor mRNA is present in somatostatin neurons in the PeV (33), and the iv administration of GH induces c-fos mRNA expression in somatostatin neurons in the PeV and Arc (24). The results of the present study actually showed that GH directly stimulated somatostatin mRNA expression and inhibited GHRH mRNA expression, suggesting the inhibitory role of GH in the GHRH expression.

Furthermore, NPY and somatostatin neurons activated by ghrelin directly may inhibit GHRH expression because somatostatin and NPY inhibit GHRH release from the hypothalamus in vitro (14, 30, 34). Histological connections between these neurons support this possibility; GHRH neurons are present in the ventrolateral part of the Arc where NPY fibers are concentrated (8, 35). GHRH neurons are innervated by somatostatin fibers, and somatostatin receptors are present on GHRH neurons (36, 37, 38, 39). However, deleting the influence of GH, the present study showed that KP-102 significantly stimulated NPY mRNA expression and did not affect somatostatin mRNA expression, although both NPY and somatostatin inhibited GHRH mRNA expression. Therefore, NPY but not somatostatin activated by ghrelin seems to inhibit the activity of GHRH neurons. Furthermore, the inhibitory effect of somatostatin on GHRH mRNA expression was partially blocked by anti-NPY IgG, although somatostatin did not significantly affect NPY mRNA expression in the present study. Therefore, NPY released in response to somatostatin may also be involved in the inhibitory mechanism of GHRH mRNA expression by somatostatin in addition to direct action of somatostatin on GHRH neurons.

In contrast to our results, several reports showed that ICV administration of somatostatin stimulates GH release (40, 41, 42) and that ICV administration of antisense oligonucleotides against somatostatin 1 receptor suppresses GH tone in rats (43). Furthermore, somatostatin stimulates in vitro GHRH release in rat hypothalamic perfusion system (44). These reports suggest that somatostatin has a dual effect on GH secretion, although we found only inhibitory action of somatostatin on GHRH mRNA expression. The mechanism by which somatostatin stimulates GH secretion still remains unclear. Further studies are needed to clarify the complex mechanism.

We have found that KP-102 significantly increased the GHRH mRNA expression level only in the presence of anti-NPY rabbit IgG. Although the concentrations of NPY in the culture media were not measured in the present study, ghrelin has already been shown to stimulate in vitro NPY release from the rat hypothalamus (45). Therefore, these results suggest that KP-102 not only up-regulates GHRH mRNA expression but also stimulates NPY release in the Arc and that the NPY that is released by KP-102 attenuates the stimulatory effect of KP-102 on GHRH mRNA expression. The results of the present study are in agreement with a report that fasting-induced inhibition of GHRH mRNA expression in WT mice is abolished in npy null mice (46).

The present study showed that somatostatin inhibited the GHRH mRNA expression level. The results of the present study also showed that the effect of KP-102 on the GHRH mRNA expression level was not influenced by antisomatostatin IgG and that KP-102 did not significantly affect somatostatin mRNA expression, suggesting that somatostatin does not play a significant role downstream of the action of KP-102 on the expression of GHRH mRNA expression. These results are in agreement with other studies showing that ghrelin and GHSs have no effect on in vitro release of somatostatin from rat hypothalamus (30, 45).

In summary, the present study has shown that the number of GHS-R-positive GHRH neurons is reduced in Tg rats whose GHS-R expression is attenuated. It has also been shown that KP-102, one of the GHSs, stimulates the NPY mRNA expression level of cultured rat hypothalamic neurons and that NPY reduces the GHRH mRNA expression level. The present study also demonstrated that KP-102 stimulates the level of GHRH mRNA expression when NPY action is deleted. Furthermore, GH and somatostatin inhibit GHRH mRNA expression. These results indicate that GHS-R is involved in the up-regulation of GHRH and NPY expression in the Arc and that NPY as well as GH and somatostatin down-regulate GHRH mRNA expression. It is also suggested that the reduction of GHRH neurons in the Arc of Tg rats is induced by the decrease in GHS-R expression.


   Acknowledgments
 
We thank Ms. M. Iketani and S. Inada for technical assistance.


   Footnotes
 
This work was supported by grants from the Ministry of Health, Labor, and Welfare, by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, and by a grant from the Foundation for Growth Science of Japan.

All authors have nothing to declare.

First Published Online May 25, 2006

1 A.M.-O. and T.N. contributed equally to this work. Back

Abbreviations: Arc, Arcuate nucleus; DNase, deoxyribonuclease; GHS, GH secretagogue; GHS-R, GH secretagogue receptor; ICV, intracerebroventricular; NPY, neuropeptide Y; PeV, periventricular nucleus; Tg, transgenic; TH, tyrosine hydroxylase; WT, wild type.

Received December 20, 2005.

Accepted for publication May 11, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
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