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Endocrinology Vol. 147, No. 11 5102-5109
Copyright © 2006 by The Endocrine Society

Ghrelin Increases Neuropeptide Y and Agouti-Related Peptide Gene Expression in the Arcuate Nucleus in Rat Hypothalamic Organotypic Cultures

Motomitsu Goto, Hiroshi Arima, Minemori Watanabe, Masayuki Hayashi, Ryouichi Banno, Ikuko Sato, Hiroshi Nagasaki and Yutaka Oiso

Department of Endocrinology and Diabetes, Field of Internal Medicine (M.G., H.A., M.W., M.H., R.B., I.S., Y.O.), and Department of Metabolic Medicine (H.N.), Nagoya University Graduate School of Medicine, Showa-ku, Nagoya 466-8550, Japan

Address all correspondence and requests for reprints to: Dr. H. Arima, Department of Endocrinology and Diabetes, Field of Internal Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: arima105{at}med.nagoya-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ghrelin, which was identified from the rat stomach, is a potent stimulant for food intake. Several lines of evidence suggest that the orexigenic action of ghrelin is mediated via the neuropeptide Y (NPY) neurons in the arcuate nucleus, although the detailed mechanisms by which ghrelin stimulates NPY neurons are not clear. In this study, we examined the gene regulation of NPY and agouti-related peptide (AGRP), another orexigenic peptide synthesized in the NPY neurons, in the arcuate nucleus by ghrelin in hypothalamic organotypic cultures. Incubation of the hypothalamic explants with ghrelin significantly increased NPY and AGRP mRNA expression in the presence, but not absence, of dexamethasone. Glucocorticoids were also necessary for ghrelin action in vivo because an intracerebroventricular injection of ghrelin significantly increased NPY and AGRP mRNA expression in the arcuate nucleus only in sham-operated, but not in adrenalectomized rats. The stimulatory effects of ghrelin on gene expression were not blocked by a sodium channel blocker tetrodotoxin in the organotypic cultures. Ghrelin also increased NPY heteronuclear (hn) RNA expression, the first transcript that has been used as an indicator for gene transcription. The stimulatory effects of ghrelin on NPY gene expression were abolished in the presence of cycloheximide, which blocks translation, suggesting that de novo protein synthesis is required for ghrelin action. These data suggest that ghrelin stimulates NPY and AGRP gene expression independently of action potentials only in the presence of glucocorticoids. Furthermore, our data demonstrate stimulatory action of ghrelin on NPY gene transcription, which requires de novo protein synthesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GHRELIN, A 28-AMINO-ACID peptide in which the serine 3 residue is n-octanoylated, was first identified from the rat stomach as an endogenous ligand for GH secretagogue receptor (GHS-R) (1). In addition to its GH-releasing action, it has been shown that peripheral injection of ghrelin increased food intake and body weight in humans and rodents (2, 3, 4). Furthermore, plasma ghrelin levels increase preprandially and decrease postprandially (5). These findings suggest that ghrelin synthesized in and released from stomach functions as an orexigenic hormone.

There are several lines of evidence suggesting that orexigenic action of ghrelin is mediated via neuropeptide Y (NPY) neurons in the arcuate nucleus, one of the most potent stimulants for food intake in the brain (6, 7, 8). NPY neurons in the arcuate nucleus reportedly express GHS-R (9), and peripheral as well as central injection of ghrelin increased the NPY mRNA expression (2, 10). The NPY neurons also express another orexigenic peptide agouti-related peptide (AGRP) (11), an endogenous antagonist for melanocortin receptors, and it is shown that administration of ghrelin increased AGRP expression as well (12). Furthermore, the orexigenic action of ghrelin was attenuated in rats injected with anti-NPY or anti-AGRP IgG (13). Although these data strongly suggest that ghrelin increases food intake by stimulating NPY neurons in the arcuate nucleus, it is not clear whether circulating ghrelin could directly affect the activities of NPY neurons in the arcuate nucleus. Date et al. (14) demonstrated that the orexigenic effects of ghrelin were diminished by vagotomy, suggesting that the action of ghrelin is mediated via vagal afferents. On the other hand, it has been shown that the arcuate nucleus could access the general circulation (15), and lower doses of ghrelin than in the plasma levels stimulated NPY neurons in isolated hypothalamic cells (16). It should be noted, however, that there exist hypothalamic neurons that express ghrelin and project to NPY neurons in the arcuate nucleus (17). Thus, it is possible that the stimulatory effects of ghrelin on NPY neurons shown in in vitro experiments might reflect the action of hypothalamic ghrelin.

To better understand the action of ghrelin on the NPY neurons in the arcuate nucleus, in the present study we examined the regulation of NPY and AGRP gene expression by ghrelin in hypothalamic organotypic cultures, which have been shown to maintain the intrinsic properties (18, 19).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Slice-explant culture procedure
Hypothalamic slice-explant cultures were performed as described previously (18, 19, 20). Sprague Dawley (SD) pups, 7–9 d old (Chubu Science Materials, Nagoya, Japan; lights on from 0900–2100 h), were killed by decapitation, and hypothalamic tissues were sectioned at 350 µm thickness on a Mcllwain tissue chopper (Mickle Laboratory Engineering Co., Surrey, UK). Three coronal slices containing arcuate nucleus were separated and placed in Hanks’ balanced salt solution (HBSS) (Invitrogen, Grand Island, NY) enriched with glucose. Selected sections were trimmed dorsally above the top of the third ventricle and laterally from the arcuate nucleus. Explants from individual rats were placed on 0.4 µm Millicell-CM filter inserts (pore size, 0.4 µm; diameter, 30 mm; Millipore, Billerica, MA), and each filter insert was placed in a Petri dish (35 mm) containing 1.1 ml of culture medium. Cultures were performed at 36.5 C in 5% CO2 enriched air under stationary conditions. The standard culture medium was composed of 50% Earle’s MEM (Invitrogen), 25% heat-inactivated horse serum (Invitrogen), HBSS, 25 U/ml penicillin/streptomycin (Invitrogen), 1 mM L-glutamine (Invitrogen), and 33 mM glucose. The serum-free medium was composed of 75% Earle’s MEM, 25% HBSS, 25 U/ml penicillin/streptomycin, 1 mM L-glutamine, and 5.5 mM glucose. Cultures were maintained in the standard medium for 15 d so that the slices became thin enough to perform in situ hybridization, and the medium was changed to defined serum-free medium for an additional 2 d before subjecting slices to different experimental conditions. The standard medium was changed three times a week, and the serum-free medium was changed every 24 h. All experiments were performed on d 17, and the slices were fixed with 4% formaldehyde in PBS for 30 min, washed twice in PBS, mounted on poly-L-lysine-coated slides, dried, and kept at –80 C until processed for in situ hybridization.

Effects of ghrelin on NPY and AGRP mRNA expression in organotypic cultures
To examine the possible interaction between ghrelin and glucocorticoids on the regulation of NPY and AGRP mRNA expression, slices were incubated with 10–7 M ghrelin or vehicle (H2O) in the presence or absence of 10 nM dexamethasone (DEX) for 24 h. To examine the time course effects of ghrelin on NPY and AGRP mRNA expression, slices were incubated with 10–7 M ghrelin for 6, 12, and 24 h, whereas 10 nM DEX was added for 24 h. Control slices were incubated with vehicle instead of ghrelin for 24 h. To examine the dose-dependent effects of ghrelin on NPY and AGRP mRNA expression, slices were incubated with 10–9 to 10–7 M ghrelin or vehicle for 24 h in the presence of 10 nM DEX. To assess whether the effects of ghrelin on NPY and AGRP mRNA expression were dependent on action potentials, slices were incubated with 10–7 M ghrelin and 10 nM DEX for 24 h in the presence of the sodium channel blocker tetrodotoxin (TTX; 1 µM, Sankyo, Tokyo, Japan).

Effects of ghrelin on NPY gene transcription and mRNA stability
To assess changes in NPY heteronuclear (hn)RNA levels, slices were incubated with 10–7 M ghrelin for 6, 12 and 24 h, whereas 10 nM DEX and 1 µM TTX were added to the medium for 24 h. Control slices were incubated with vehicle instead of ghrelin for 24 h. To see the changes in NPY mRNA stability, slices were incubated with 10–7 M ghrelin or vehicle together with 10 nM DEX and 1 µM TTX in the presence of 150 µM 5,6-dichloro-1-D-ribofuranosylbenzimidazole (DRB; Sigma, St. Louis, MO), which blocks gene transcription (21, 22), dissolved in 0.1% dimethyl sulfoxide for 24 h.

Effects of blockade of translation on NPY hnRNA and mRNA
To determine whether the effects of ghrelin on NPY gene expression were mediated via de novo protein synthesis, slices were incubated with 10–7 M ghrelin, together with 10 nM DEX and 1 µM TTX for 24 h in the presence of 10 µM cycloheximide (Sigma), which blocks translation (23).

Regulation of NPY and AGRP gene expression by ghrelin in vivo
Eight-week-old male SD rats (body weight 250 g; Chubu Science Materials) were housed individually and habituated by handling every day under controlled conditions (23.0 ± 0.5 C, lights on from 0900–2100 h). The rats were anesthetized by ip injection of pentobarbital (50 mg/kg) for implantation of a 21-gauge stainless steel cannula stereotaxically into the lateral ventricle. The coordinates of intracerebroventricular operation were: 1.1 mm posterior to the bregma, 1.6 mm lateral to the midline, and 4.5 mm below the surface of the skull. Seven days after placement of cannula, adrenalectomy (ADX) or sham-adrenalectomy (Sham) was performed under pentobarbital (50 mg/kg) anesthesia. Through a dorsal midline incision, small incisions were made through the muscle layer below the rib cage on each flank. Adrenal glands from both sides were isolated by blunt dissection and removed in their capsules. After operation, rats were provided with 0.9% saline in place of water. Seven days after operation, ghrelin (0.2 µg, dissolved in 3 µl saline) or vehicle was injected into lateral ventricle at 0800, 1200, and 1600 h and food intake was monitored in both ADX and Sham rats, which were killed by decapitation at 2000 h. Blood sample were collected into chilled tubes and separated by centrifugation (3500 rpm, 4 C, 15 min), and serum was stored at –30 C until the corticosterone determination. To confirm the effectiveness of ADX, serum corticosterone levels were measured with RIA commercial kit (MP Biomedicals, Costa Mesa, CA). Rats that had plasma corticosterone concentrations of more than 25 ng/ml in ADX group were excluded from the study. All procedures were performed in accordance with the institutional guidelines for animal care at Nagoya University Graduate School of Medicine.

Probes for NPY and AGRP gene expression
A 733-bp fragment localized entirely within intron 1 of the rat NPY gene was subcloned into the pCRII-TOPO (Invitrogen). The plasmids containing the cDNA for rat preproNPY were kindly provided by Dr. S. L. Sabol (Laboratory of Biochemical Genetics, National Institute of Health, Bethesda, MD). The plasmids containing the cDNA for rat AGRP were kindly provided by Dr. K. L. Grove (Division of Neuroscience, Oregon Regional Primate Research Center, Oregon Health & Science University, Beaverton, OR). High specific RNA probes were synthesized using 55 µCi [35S]UTP and 171 µCi [35S]CTP (PerkinElmer Life Sciences, Natick, MA), Riboprobe Combination System (Promega), 15 U of RNasin, 1 µg of linearized template, and 15 U of T3, T7, or SP6 RNA polymerase. After 60 min of incubation at 42 C, the cDNA template was digested with deoxyribonuclease for 10 min at 37 C. Radiolabeled RNA products were purified using quick-spin columns (Roche Diagnostics, Indianapolis, IN), precipitated with ethanol, and resuspended in 100 µM of 10 mM Tris-HCl (pH 7.5), containing 20 mM dithiothreitol.

In situ hybridization
In in vivo experiments, brains were removed immediately after decapitation, frozen on dry ice, and stored at –80 C until sectioning for in situ hybridization. Coronal sections (12 µm) at 2.8 mm caudal from bregma, according to the brain atlas of Paxinos and Watson (24), were cut on a cryostat, mounted onto four sets of poly-L-lysine-coated slides (three brain sections per slide), and then stored at –80 C until in situ hybridization. Prehybridization, hybridization, and posthybridization procedures were performed as described previously (19, 25). In brief, after thawing at room temperature, sections were fixed in 4% formaldehyde in PBS for 5 min and acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine and 0.9% NaCl (pH 8), for 10 min at room temperature. Sections were then dehydrated in 70, 80, 95, and 100% ethanol, delipidated in chloroform, and hybridized overnight at 55 C with 2 x 10–6 cpm of 35S-labeled probes in 90 µl of hybridization buffer (50% formamide, 200 mM NaCl, 2.5 mM EDTA, 10% dextran sulfate, 250 µg/ml yeast tRNA, 50 mM dithiothreitol, and Denhardt’s solution). At the end of incubation, sections were subjected to consecutive washes in 4x standard saline citrate (SSC) for 15 min at room temperature and 50% formamide/250 mM NaCl containing dithiothreitol for 15 min at 60 C. After treatment with ribonuclease A (20 µg/ml) for 30 min at 37 C, sections were washed with 2x SSC, 1x SSC, and 0.5x SSC for 5 min at room temperature, followed by washes with 0.1x SSC containing dithiothreitol for 15 min at 50 C, 0.1x SSC to cool at room temperature, and 70% ethanol for 15 sec. After the final washes, sections were air-dried and exposed to Kodak BioMax MR films (Eastman Kodak, Rochester, NY) for various periods yielding appropriate signal intensities. Sections hybridized with antisense or sense probes for NPY hnRNA were dipped in nuclear Kodak NTB2 emulsion (Kodak, Rochester, NY) and exposed for 3 wk. To assist cellular localization of the hybridized signals, emulsion-dipped sections were stained with cresyl violet.

Quantification and statistical analyses
The ODs of the autoradiograph were quantified using a computer image analysis system (Imaging Research, St. Catharines, Ontario, Canada) and the public domain NIH Image program. Changes in gene expression were quantified by measurements of the integrated OD (OD x area) of the film images. For adult rats, the anatomically best-matched section among the three sections from each rat was chosen for the analysis. In the hypothalamic cultures, the total sum of OD signals in the bilateral arcuate nuclei in three explants from each rat was used for the analysis. In each culture, control explants, which were incubated with 10 nM DEX for 24 h, were involved, and the expression levels were expressed as 100. The statistical analyses were performed with ANOVA followed by Fisher’s protected least significant difference test. Results are expressed as means ± SE (n = 10), and differences were considered statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of ghrelin on NPY and AGRP mRNA expression in organotypic cultures
As reported previously (26), NPY mRNA expression in the arcuate nucleus in the hypothalamic organotypic cultures was significantly increased with the incubation of 10 nM DEX for 24 h (Fig. 1AGo). AGRP mRNA was also expressed in the arcuate nucleus in the hypothalamic cultures (Fig. 2EGo), and the incubation with 10 nM DEX for 24 h significantly increased AGRP mRNA expression (Fig. 2AGo). The incubation with 10–7 M ghrelin for 24 h significantly increased both NPY and AGRP mRNA expression in the presence, but not absence, of 10 nM DEX (Figs. 1AGo and 2AGo). Therefore, all the following experiments were performed in the presence of 10 nM DEX. The time course study showed that 10–7 M ghrelin did not significantly increase NPY or AGRP mRNA expression until 24 h (Figs. 1BGo and 2BGo), and the dose response study demonstrated that ghrelin significantly increased both NPY and AGRP mRNA expression at the concentrations of 10–8 and 10–7 M (Figs. 1CGo and 2CGo). As shown in Figs. 1DGo and 2DGo, incubation with 10–7 M ghrelin significantly increased NPY and AGRP mRNA expression even in the presence of 1 µM TTX, which has been shown to abolish action potentials in the hypothalamic organotypic cultures (27), indicating that the action of ghrelin on NPY and AGRP mRNA expression was independent of action potentials. Representative photographs showing the effects of ghrelin on NPY and AGRP mRNA expression are presented in Figs. 1EGo and 2EGo. The following experiments were all performed in the presence of TTX.


Figure 1
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  		FIG. 1. Effects of ghrelin on NPY mRNA expression in organotypic cultures. A, NPY mRNA expression was significantly increased with incubation of 10–7 M ghrelin for 24 h in the presence, but not absence, of 10 nM DEX. B, Time-course effects of ghrelin on NPY mRNA expression. C, Dose-response effects of ghrelin on NPY mRNA expression. D, TTX treatment did not affect the stimulatory effects of ghrelin on NPY mRNA expression in the presence of DEX. E, Representative autoradiographs showing the effects of ghrelin on NPY mRNA expression. Mean NPY mRNA expression levels with DEX treatment are expressed as 100. Results are expressed as mean ± SE (n = 10). n.s., Not significant. *, P < 0.05 vs. values without ghrelin or DEX (A), at time 0 (B), or without ghrelin (C).

 

Figure 2
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  		FIG. 2. Effects of ghrelin on AGRP mRNA expression in organotypic cultures. A, AGRP mRNA expression was significantly increased with incubation of 10–7 M ghrelin for 24 h in the presence, but not absence, of 10 nM DEX. B, Time-course effects of ghrelin on AGRP mRNA expression. C, Dose-response effects of ghrelin on AGRP mRNA expression. D, TTX treatment did not affect the stimulatory effects of ghrelin on AGRP mRNA expression in the presence of DEX. E, Representative autoradiographs showing the effects of ghrelin on AGRP mRNA expression. Mean AGRP mRNA expression levels with DEX treatment are expressed as 100. Results are expressed as mean ± SE (n = 10). n.s., Not significant. *, P < 0.05 vs. values without ghrelin or DEX (A), at time 0 (B), or without ghrelin (C).

 
NPY hnRNA expression in the arcuate nucleus in vivo
In situ hybridization with antisense probes demonstrated that NPY hnRNA was expressed in the arcuate nucleus (Fig. 3AGo), whereas no visible signals were detected with sense probes (Fig. 3BGo). Analysis of the emulsion-dipped slides showed that the transcripts were confined to the nucleus (Fig. 3CGo), as was characteristic of hnRNA expression shown for other genes such as vasopressin and CRH (28, 29).


Figure 3
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  		FIG. 3. NPY hnRNA expression in the arcuate nucleus of 8-wk-old SD rats. NPY hnRNA expression was detected with antisense probes in the arcuate nucleus in the hypothalamus (A), whereas no visible signals were detected with sense probes (B). C, NPY hnRNA expression was largely confined to the nucleus. Scale bar, 20 µm.

 
Effects of ghrelin on NPY gene transcription and NPY mRNA stability
Incubation with 10–7 M ghrelin did not increase NPY hnRNA expression significantly until 12 h (Fig. 4AGo). Incubation with DRB for 24 h completely abolished NPY hnRNA expression in the arcuate nucleus stimulated by ghrelin (data not shown), indicating that DRB blocked gene transcription in the organotypic cultures. Incubation with DRB for 24 h significantly decreased NPY mRNA levels, and ghrelin did not affect NPY mRNA levels significantly in the presence of DRB (Fig. 4BGo). Representative autoradiographs showing the effects of ghrelin on NPY hnRNA expression are demonstrated in Fig. 4CGo.


Figure 4
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  		FIG. 4. Effects of ghrelin on NPY gene transcription (A and C) and NPY mRNA stability (B) in the organotypic cultures. A, Time-course effects of ghrelin on NPY hnRNA expression. B, Treatments with 150 µM DRB significantly decreased NPY mRNA expression and abolished stimulatory effects of ghrelin on NPY mRNA expression. C, Representative autoradiographs showing effects of ghrelin on NPY hnRNA expression. Mean NPY hnRNA or mRNA expression levels in control are expressed as 100. Results are expressed as mean ± SE (n = 10). *, P < 0.05 vs. values at time 0 (A) or without ghrelin or DRB (B). n.s., Not significant.

 
Effects of blockade of protein synthesis on NPY hnRNA and mRNA
Incubation with cycloheximide for 12 h did not affect NPY hnRNA levels significantly by itself, but it abolished the stimulatory effects of ghrelin on NPY hnRNA expression (Fig. 5AGo). Likewise, incubation with cycloheximide for 24 h did not affect NPY mRNA levels significantly by itself, but it abolished the stimulatory effects of ghrelin on NPY mRNA expression (Fig. 5BGo).


Figure 5
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  		FIG. 5. Effects of blockade of protein synthesis on NPY hnRNA (A) and mRNA (B). A, Incubation with cycloheximide for 12 h did not affect NPY hnRNA levels significantly by itself, but it abolished the stimulatory effects of ghrelin on NPY hnRNA expression. B, Incubation with cycloheximide for 24 h did not affect NPY mRNA levels significantly by itself, but it abolished the stimulatory effects of ghrelin on NPY mRNA expression. Mean NPY hnRNA or mRNA expression levels in control are expressed as 100. Results are expressed as mean ± SE (n = 10). n.s., Not significant.

 
Effects of adrenalectomy on NPY and AGRP gene expression stimulated by intracerebroventricular injection of ghrelin
To see whether glucocorticoids are necessary for ghrelin action to increase NPY and AGRP gene expression also in vivo, effects of intracerebroventricular injection of ghrelin on gene expression were examined in both ADX and Sham rats. BW was significantly less in ADX rats than in Sham rats 7 d after operation (ADX, 255 ± 6.4 g, Sham, 277 ± 7.2 g, P < 0.05). Although food consumption after injection of either ghrelin or vehicle was significantly less in ADX than in Sham rats, ghrelin injection increased food intake significantly in both Sham and ADX rats (Sham, vehicle 2.00 ± 0.45 g, ghrelin 3.24 ± 0.45 g, P < 0.05; ADX, vehicle 0.95 ± 0.37 g, ghrelin 2.44 ± 0.47 g, P < 0.05). However, ghrelin injection increased both NPY and AGRP mRNA expression levels only in Sham rats, but not in ADX rats (Fig. 6Go).


Figure 6
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  		FIG. 6. Effects of adrenalectomy on NPY and AGRP gene expression stimulated by intracerebroventricular injection of ghrelin. Ghrelin injection increased both NPY (A) and AGRP (B) mRNA expression levels only in Sham rats, but not in ADX rats. C, Representative autoradiographs showing effects of ghrelin on NPY and AGRP mRNA expression. Mean NPY mRNA or AGRP mRNA expression levels in control are expressed as 100. Results are expressed as mean ± SE (n = 10). n.s., Not significant.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we examined the regulation of NPY and AGRP gene expression in the arcuate nucleus by ghrelin in hypothalamic organotypic cultures. Our data demonstrated that 1) ghrelin increased NPY and AGRP mRNA expression in the presence, but not the absence, of DEX; 2) action of ghrelin on NPY neurons was independent of action potentials; 3) ghrelin increased NPY gene transcription, but not mRNA stability, and 4) de novo protein synthesis is required for ghrelin action on NPY gene expression. It is also demonstrated that glucocorticoids are required for regulation of NPY neurons by ghrelin in vivo because ghrelin increased NPY and AGRP gene expression only in Sham rats but not in ADX rats.

NPY neurons express glucocorticoid receptors (GR) (30), and we have shown in a previous study that DEX significantly increased NPY mRNA expression in the arcuate nucleus in organotypic cultures (26). In the present study, we have shown that DEX also increased AGRP mRNA expression significantly, and that ghrelin increased NPY and AGRP mRNA expression only in the presence of DEX. DEX is a synthetic glucocorticoid, and the dose (10 nM) employed in the present study is similar to that measured in humans after injection of a clinically used amount of DEX (31). Similar to the changes in plasma levels of ghrelin (5, 32, 33), the plasma levels of glucocorticoids are elevated after a fast (34, 35) when NPY and AGRP gene expression is increased (36, 37, 38). The in vivo experiments in the present study also demonstrated that ghrelin did not increase NPY or AGRP gene expression in ADX rats. These data suggest that glucocorticoid receptor-mediated signaling is prerequisite for ghrelin action on NPY and AGRP gene expression in the arcuate nucleus. In a previous paper, we showed that insulin inhibited NPY mRNA expression only in the presence of DEX in the hypothalamic organotypic cultures (26). Thus, it is likely that glucocorticoids play a permissive role in the regulation of NPY neurons that are affected by signals reflecting energy balance. On the other hand, although DEX significantly increased NPY and AGRP gene expression in organotypic cultures, the expression levels of NPY and AGRP mRNA were not significantly affected by ADX. Because BW was significantly less in ADX than in Sham rats, the discrepancy between in vivo and in vitro experiments might suggest that other than glucocorticoids signals, which reflect changes in energy balance, might stimulate NPY and AGRP gene expression in vivo. Our data also showed that ghrelin increased food intake even in ADX rats, consistent with a previous paper (39). GHSR is expressed widely in the brain (40), and it is shown that ghrelin could act on other than NPY neurons including ventral tegmental area (41) and orexin neurons in the lateral hypothalamic area (42), which are involved in the regulation of food intake (43). Our data suggest that stimulatory effects of centrally injected ghrelin on food intake are mediated via not only NPY neurons but also other neurons, where glucocorticoids are not required for ghrelin action.

The concentrations of ghrelin that significantly increased NPY and AGRP mRNA expression in the present study were 10–8 to 10–7 M. Although it is reported that 10–10 M ghrelin increased cytosolic Ca2+ concentration in isolated NPY neurons (16), our results are similar to those in a previous study, in which 10–7 M ghrelin stimulated NPY release in hypothalamic explants (44). Serum concentrations of ghrelin in rats are reported to be 10–9 to 10 –8 M (5, 32, 33), and thus it is likely that higher doses of ghrelin than in the plasma levels are required to stimulate NPY release and synthesis in the arcuate nucleus. Ghrelin immunoreactive neurons are localized in the hypothalamus and innervate NPY neurons (17). A previous paper showed that ghrelin mRNA and protein contents within the hypothalamus varied in terms of the energy balance (45). It is also reported that ghrelin mRNA is expressed in the hypothalamus at the age of 5 and 10 d in rats (46), and given that hypothalamic explants could maintain in vivo intrinsic properties (18, 19), our data might suggest the action of hypothalamic ghrelin rather than that of peripheral ghrelin. However, it is also demonstrated that ghrelin in the blood could pass the blood brain barrier in a saturable manner, suggesting there exist transporters for ghrelin (47). Because transport systems of ghrelin in the arcuate nucleus have not been fully elucidated, we could not exclude the possibility that peripheral ghrelin could directly affect NPY gene expression.

To clarify the action of ghrelin on NPY gene expression in more detail, we examined changes in gene transcription and mRNA stability using intronic probes to detect hnRNA and DRB to block gene transcription, respectively. Heteronuclear RNA is the first transcript that has been used as a sensitive indicator of gene transcription for other hypothalamic neuropeptides such as vasopressin and CRH (28, 29, 48, 49). Our data clearly showed that ghrelin increased NPY mRNA expression by increasing gene transcription without affecting mRNA stability. Because ghrelin increased NPY gene transcription in the presence of TTX, it is indicated that the action of ghrelin is independent of action potentials. However, ghrelin did not increase NPY hnRNA until 12 h after starting incubation. This time course is in marked contrast to rapid changes in hnRNA expression of vasopressin and CRH, which were examined not only in vivo (28, 29, 48) but also in hypothalamic organotypic cultures (18, 20). Therefore, we examined the effects of ghrelin on NPY gene expression in the presence of cycloheximide, which blocks translation (23), and demonstrated that de novo synthesis of protein is required for ghrelin action. Because ghrelin increases NPY release in a much shorter time course (16, 44), it is suggested that NPY release and gene transcription were differentially regulated by ghrelin.

In conclusion, we showed that ghrelin could increase NPY and AGRP gene expression only in the presence of glucocorticoids in the arcuate nucleus. Furthermore, our data demonstrated that ghrelin action on NPY neurons was independent of action potentials but requires de novo protein synthesis.


   Footnotes
 
This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Health, Labor, and Welfare of Japan.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 3, 2006

Abbreviations: ADX, Adrenalectomy; AGRP, agouti-related peptide; DEX, dexamethasone; DRB, 5,6-dichloro-1-D-ribofuranosylbenzimidazole; GHS-R, GH secretagogue receptor; HBSS, Hanks’ balanced salt solution; hn, heteronuclear; NPY, neuropeptide Y; SD, Sprague Dawley; SSC, standard saline citrate; Sham, sham-adrenalectomy; TTX, tetrodotoxin.

Received January 25, 2006.

Accepted for publication July 26, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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