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Endocrinology, doi:10.1210/en.2003-1424
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Endocrinology Vol. 145, No. 8 3731-3738
Copyright © 2004 by The Endocrine Society

The Role of Pituitary Ghrelin in Growth Hormone (GH) Secretion: GH-Releasing Hormone-Dependent Regulation of Pituitary Ghrelin Gene Expression and Peptide Content

Jun Kamegai, Hideki Tamura, Takako Shimizu, Shinya Ishii, Atsushi Tatsuguchi, Hitoshi Sugihara, Shinichi Oikawa and Rhonda D. Kineman

Department of Medicine (J.K., H.T., T.S., S.I., A.T., H.S., S.O.), Nippon Medical School, Tokyo 113-8603, Japan; and Department of Medicine (R.D.K.), University of Illinois at Chicago, Chicago, Illinois 60612

Address all correspondence and requests for reprints to: Jun Kamegai, M.D., Department of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-Ku, Tokyo 113-8603, Japan. E-mail: jkamegai{at}nms.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ghrelin is a GH-releasing peptide originally purified from the rat stomach. It has been demonstrated that ghrelin expression, within the gastroenteric system, is regulated by both the metabolic and GH milieu. Our laboratory and others have previously reported that ghrelin is also produced in the pituitary. Given that the receptor for ghrelin [GH secretagogue receptor (GHS-R)] is also expressed by the pituitary, the possibility exists that locally produced ghrelin plays an autocrine/paracrine role in regulating GH release. Because we have previously reported that GHRH infusion increases pituitary levels of ghrelin mRNA, we hypothesized that GHRH could be a key regulator of pituitary ghrelin expression. In this report, we demonstrate that 4-h GHRH infusion increased pituitary ghrelin peptide content. Interestingly, under experimental conditions in which hypothalamic GHRH expression is increased, e.g. GH deficiency due to GH gene mutation, glucocorticoid deficiency, and hypothyroidism, we observed that pituitary ghrelin expression (mRNA levels and peptide content) was also increased. Consistent with this positive correlation between GHRH and ghrelin, pituitary ghrelin expression (mRNA levels and peptide content) was found to be decreased in conditions in which hypothalamic GHRH expression is decreased, e.g. GH treatment, glucocorticoid excess, hyperthyroid state, and food deprivation. Collectively, these results suggest that pituitary ghrelin expression is GHRH dependent. We also conducted functional studies to examine whether the pituitary ghrelin/GHS-R system contributes to GH release after GHRH stimulation, by challenging pituitary cell cultures with GHRH in the presence of a GHS-R-specific inhibitor ([D-Lys-3]-GHRP-6). The GHS-R inhibitor did not affect GH release in the absence of GHRH, but significantly reduced GHRH-mediated GH release. This is the first report demonstrating that endogenous pituitary ghrelin can play a physiological role in GH release, by optimizing somatotroph responsiveness to GHRH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GHRELIN, AN ENDOGENOUS ligand for the GH secretagogue receptor (GHS-R), was originally purified from the rat stomach (1). Ghrelin is thought to be an important regulator of energy metabolism and GH secretion (2, 3, 4, 5, 6, 7, 8, 9). Ghrelin mRNA and peptide have been detected in the rat and human pituitary (10, 11), indicating that ghrelin is synthesized within the pituitary, where it may influence the release of GH in an autocrine and paracrine manner. Recently, it was reported that ghrelin is expressed in somatotrophs, lactotrophs, and thyrotrophs in the pituitary, and that ghrelin mRNA levels are influenced by experimental alterations in thyroid hormone and glucocorticoids levels, as well as reproductive status and age (12). We have also previously reported that the expression of the ghrelin gene in the pituitary is developmentally regulated (11). In addition, we have found that in vivo GHRH infusion increases pituitary levels of ghrelin mRNA (11). Therefore, we hypothesized that GHRH could be a key regulator of pituitary ghrelin expression. To clarify this hypothesis, we examined 1) whether GHRH-induced, pituitary ghrelin mRNA levels translate into an increase in ghrelin peptide production, in vivo; 2) whether pituitary ghrelin expression (mRNA and peptide levels) positively correlates with GHRH expression in experimental in vivo models that display changes in hypothalamic GHRH production; and 3) whether pituitary ghrelin is important for basal and GHRH-induced GH release in primary pituitary cell cultures, by blocking endogenous ghrelin action with the GHS-R antagonist ([D-Lys-3]-GHRP-6).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental paradigms
Effect of iv infusion of GHRH on pituitary ghrelin peptide content.
Male Sprague Dawley rats (250–280 g; Saitama Experimental Animal Supply Co. Ltd., Saitama, Japan) were housed in air-conditioned animal quarters, with lights on between 0800 and 2000 h, and given food and water ad libitum. Rats were anesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg) anesthesia, and iv cannula were inserted into the right jugular vein to the right atrium, 3 d before the experiment. To test the effect of GHRH on pituitary ghrelin peptide content, animals were infused for 4 h with either saline (vehicle; n = 6) or human GHRH (10 µg/h; n = 6; Sigma-Aldrich, Tokyo, Japan) under unanesthetized conditions. Immediately after the infusions, animals were killed by decapitation, and anterior pituitaries were collected and frozen at –70 C for analysis of ghrelin peptide levels by RIA. For this and all subsequent experiments, procedures were conducted according to the principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals with institutional animal care committee approval.

Effect of GH and IGF-I on the pituitary ghrelin mRNA levels.
For the experiments examining the impact of GH and IGF-I on pituitary ghrelin expression, we analyzed samples obtained from a previous study where we examined the hypothalamic-pituitary axis of the spontaneous dwarf rat (SDR; Ref. 13). SDRs were first identified in a Sprague Dawley colony and were found to lack GH as a consequence of a point mutation within the GH gene (14). Details of the experimental design for this series of studies were previously reported in detail (13) and are described in brief below.

Experiment 1: SDRs vs. normal rats.
Total RNA from pituitary extracts of male SDRs (3–5 months old; 90–110 g) and age-matched normal male Sprague Dawley rats (310–400 g; Harlan, Indianapolis, IN) were assessed for ghrelin mRNA levels by RT-PCR.

Experiment 2: Impact of GH replacement in SDRs.
Ghrelin mRNA levels were assessed in pituitary extracts from male SDRs (3–5 months old; 90–110 g) with or without GH replacement. Specifically, SDRs were anesthetized using ketamine/xylazine and osmotic minipumps (model 1003D; Alzet Co., Palo Alto, CA), containing rat GH (10 µg/µl; National Institute of Diabetes and Digestive and Kidney Diseases National Hormone and Pituitary Program) or vehicle (saline) were implanted sc (five animals per treatment group). The pumps released the hormone solution at a rate of 1 µl/h. Seventy-two hours after pump placement (1000–1200 h), rats were killed by decapitation, and pituitaries were removed and frozen at –70 C until analysis. This dose of GH (10 µg/h) raised circulating IGF-I levels of SDRs to levels comparable with that observed in normal male rats (13).

Experiment 3: Impact of IGF-I replacement in SDRs.
Ghrelin mRNA levels were assessed in pituitary extracts from male SDRs (3–5 months old; 90–110 g) with or without IGF-I replacement. Osmotic minipumps, containing recombinant human IGF-I (5 µg/µl; Genentech, Inc., South San Francisco, CA) or vehicle (saline), were implanted sc (five animals per treatment group). Seventy-two hours after pump placement (1000–1200 h), rats were killed by decapitation, and pituitaries were removed and frozen at –70 C until analysis. This dose of IGF-I raised circulating levels of IGF-I to within the range observed in normal rats (13).

Effect of hyper- and hypothyroidism on pituitary ghrelin mRNA and peptide levels and hypothalamic GHRH mRNA levels.
Adult male Sprague Dawley rats were treated with daily ip injections of T3 (50 µg/100 g body weight; Sigma-Aldrich), or methimazole (MMI; an inhibitor of thyroid hormone synthesis; 1.6 mg/100 g body weight; Janssen, Beerse, Belgium), or vehicle (5 mM NaOH) for 7 d (15, 16). Animals were decapitated 24 h after the final injection of T3, MMI, or vehicle. The pituitary and hypothalamus were removed, and stored at –70 C until subsequent analysis for ghrelin expression (mRNA and peptide) and GHRH mRNA, respectively.

Effect of exogenous glucocorticoid treatment and adrenalectomy on pituitary ghrelin mRNA and peptide levels and hypothalamic GHRH mRNA levels.
On d 1, adult male Sprague Dawley rats were bilaterally adrenalectomized or sham operated by the dorsal approach under ketamine (100 mg/kg) and xylazine (5 mg/kg) anesthesia between 1000 and 1200 h, as previously described (17). Animals received a daily sc injection of 200 µg dexamethasone (water soluble; Sigma-Aldrich) or 0.9% NaCl. Adrenalectomized rats were maintained on 0.9% NaCl in their drinking water. Animals were killed by decapitation between 1000 and 1200 h on d 8 of the experiment, approximately 4 h after the final dexamethasone injection. The pituitary and hypothalamus were removed, and stored at –70 C until subsequent analysis of ghrelin expression (mRNA and peptide) and GHRH mRNA, respectively.

Effect of food deprivation on pituitary ghrelin mRNA and peptide levels and hypothalamic GHRH mRNA levels.
Adult male Sprague Dawley rats were fed or food deprived for 72 h, with water supplied ad libitum. Animals were decapitated after food deprivation. The pituitary and hypothalamus were removed, and stored at –70 C until analysis.

Effect of a GHS-R antagonist on basal and GHRH-induced GH release, in vitro.
Anterior pituitaries from adult male Sprague Dawley rats were enzymatically and mechanically dissociated into single cells, as previously described (13, 17). Cells were then washed and resuspended in {alpha}-MEM (Life Technologies, Inc., Grand Island, NY) supplemented with 0.1% BSA and antibiotics. Cell viability after dissociation was consistently greater than 95%, as assessed by the exclusion of trypan blue. Cells were plated at a density of 50,000 cells/well in 1 ml {alpha}-MEM supplemented with 10% horse serum, and placed in a humidified atmosphere containing 95% air-5% CO2. After 3 d of culture, cells were washed in serum-free medium and preincubated with or without [D-Lys-3]-GHRP-6, a GHS-R-specific inhibitor (1 h; 10–4 M; Bachem AG, San Carlos, CA) followed by a GHRH challenge (100 nM; 15 min) in the presence of the GHS-R antagonist. The amount of GH and ghrelin released into the medium was determined by RIA.

Measurement of pituitary ghrelin content, GH, and TSH by RIA
For determination of pituitary ghrelin peptide content, the pituitaries were heated at 100 C for 10 min in a 10-fold volume of water to inactivate intrinsic proteases. After cooling to 4 C, CH3COOH and HCl were added to the respective final concentrations of 1 M and 20 mM, after which the tissue was homogenized. The homogenate was centrifuged at 11,500 x g for 30 min. The supernatants were applied to Sep-Pak C18 cartridges (Waters, Milford, MA), and the peptides were eluted with 60% acetonitrile solution containing 0.1% trifluoroacetic acid. Rat ghrelin was measured with a commercial RIA kit (Phoenix Pharmaceuticals, Inc., Mountain View, CA), as previously described (18). In preliminary studies, it was determined that final recovery of ghrelin, which was added to the pituitary homogenate (10 and 100 pg) before heating and Sep-Pak purification, was greater than 95%. For assessment of the effectiveness of T3 and MMI in inducing hyper- and hypothyroid states, respectively, serum TSH concentrations were determined by RIA (Amersham, Arlington Heights, IL). For determination of GH released into the medium, in the in vitro experiments, medium concentrations of GH were determined using a double-antibody RIA, using materials supplied by National Hormone and Pituitary Program (Bethesda, MD), as previously described (9).

RT-PCR of pituitary ghrelin mRNA
Total pituitary RNA was extracted, and 1 µg was used as a template to generate cDNA by reverse transcription (RT) with random hexamer priming, as previously described (13). Pituitary ghrelin mRNA levels were assessed using RT-PCR, as previously described (11). Briefly, the RT products were amplified by the PCR using sense 5'-TCATCTGTCCTCACCACCAAGG-3' (corresponding to bases 7–28) and antisense 5'-GGCAGAAGCTGGATGTGAGTTC-3' (corresponding to bases 446–425) primers specific for the rat ghrelin cDNA sequence (GenBank accession no. AB029433). The PCR amplification was performed with the following cycle profile: 95 C for 3 min, annealing at 65 C for 1 min, and extension at 72 C for 2 min, followed by 26 cycles at 95 C for 40 sec, 65 C for 1 min, and 72 C for 2 min. The PCR products were gel electrophoresed, transferred to nylon membranes, and hybridized to specific radiolabeled cDNA probes for rat ghrelin. The ghrelin cDNA probe was synthesized by random oligonucleotide labeling of a PCR product generated by sense 5'-TGGCAGGTTCCAGCTTCTTGAG-3' (corresponding to bases 95–116) and antisense 5'-CTCCTGACAGCTTGATGCCAAC-3' (corresponding to bases 295–274) primers located internal to the PCR primers used to amplify cDNA obtained from RT of pituitary total RNA extracts. Membranes were washed using high-stringency conditions and exposed to a phosphorscreen for 1 h. The hybridization signals were analyzed with the BAS 2000 system (Fujix, Tokyo, Japan). The ghrelin signal was adjusted by the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal.

Northern blot analysis for hypothalamic GHRH mRNA
The hypothalamus was dissected, total tissue RNA was extracted with the TRIzol reagent (Life Technologies), and mRNA levels for GHRH and the housekeeping gene ß-actin were determined by Northern blot analysis, as previously described (13, 18). In brief, samples (20 µg RNA/lane) were electrophoresed through a 1.5% agarose gel containing 2.2 M formaldehyde, and then transferred by capillary blotting onto a nylon membrane (Hybond-N+; Amersham). Membranes were prehybridized in 1 M NaPO4, 20% sodium dodecyl sulfate (SDS), and 0.1% BSA for 3 h at 65 C. 32P-Labeled specific riboprobes for GHRH and ß-actin were added, and the membranes were hybridized overnight at 65 C. The riboprobes for GHRH and ß-actin were synthesized as previously described (13, 18). The membranes were then washed in 2x standard saline citrate (SSC) for 30 min, 2x SSC with 0.1% SDS for 30 min, and 0.5x SSC with 0.1% SDS for 30 min at 65 C, and then exposed to Kodak (Rochester, NY) XAR film for 72 h. The hybridization signal was determined from the autoradiograms using a MCID image analysis system (Imaging Research, Inc., St. Catherines, Ontario, Canada).

Data analysis
All comparisons were made between cDNA samples amplified in the same PCR and electrophoresed on the same gel. Differences in pituitary ghrelin peptide content after GHRH infusion in vivo, between normal and SDRs and between fed and fasted rats, were determined by Student’s t test. The effects of hyper- and hypothyroidism, exogenous glucocorticoid treatment and adrenalectomy, and changes in GH secretion in vitro in response to GHS-R antagonist/GHRH treatment were assessed by ANOVA using Duncan’s New Multiple Range test. A value of P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of iv infusion of GHRH on pituitary ghrelin peptide content
We have previously reported that the GHRH infusion increases pituitary levels of ghrelin mRNA (11). In this experiment, we examined whether the GHRH infusion increases pituitary peptide content of ghrelin. The GHRH infusion resulted in a 1.6-fold increase in the levels of ghrelin protein relative to the control rats (Fig. 1Go).



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  		FIG. 1. Effect of GHRH infusion on pituitary ghrelin peptide contents in adult male rats. Freely moving male Sprague Dawley rats were infused for 4 h with human GHRH (10 µg/h) or vehicle and subsequently killed, and pituitaries were collected and extracted for determination of ghrelin peptide content by RIA. The data represent the mean ± SEM (n = 6 animals/group). *, P < 0.05.

 
Effect of GH and IGF-I on the pituitary ghrelin mRNA levels
We used RT-PCR to examine whether GH could regulate pituitary ghrelin gene expression in SDRs. In the absence of GH feedback regulation, SDR pituitary ghrelin mRNA levels were 35.7 ± 6.4% greater (n = 5/group; P < 0.05) than those observed in normal controls (Fig. 2AGo). Three-day sc infusion of rat GH by osmotic pump reduced SDR pituitary ghrelin mRNA levels to 84.6 ± 4.1% of vehicle-treated controls (n = 5/group; P < 0.05; Fig. 2BGo). To test whether the changes in ghrelin mRNA levels observed after GH treatment were due to elevation of circulating IGF-I concentrations, SDRs were infused with recombinant human IGF-I. Replacement of IGF-I did not significantly alter pituitary ghrelin mRNA levels (Fig. 2CGo).



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  		FIG. 2. RT-PCR analysis of pituitary ghrelin mRNA of male SDR and normal controls (A) and effect of GH (B) and IGF-I (C) replacement on SDR pituitary ghrelin mRNA levels. A, RT-PCR analysis of pituitary ghrelin mRNA of male SDR and normal controls. To correct for variations in total RNA used in the RT reaction, GAPDH was amplified from a separate aliquot of the same RT reaction used to generate the ghrelin PCR product. The top panels show Southern blots of pituitary ghrelin and GAPDH cDNA from individual normal (N) and SDRs (D). Data are expressed as percentage of values in normal rats (100%). B, Effect of GH replacement on SDR pituitary ghrelin mRNA levels. SDRs were implanted with osmotic minipumps containing vehicle (saline) or rat GH (delivery rate of 10 µg/h). Animals were killed at 72 h, and pituitary ghrelin mRNA levels were determined by RT-PCR. Data are expressed as percentage of vehicle-treated values. C, Effect of IGF-I replacement on SDR pituitary ghrelin mRNA levels. SDRs were implanted with osmotic minipumps containing vehicle (saline) or recombinant human IGF-I (delivery rate of 5 µg/h). Animals were killed at 72 h, and pituitary ghrelin mRNA levels were determined by RT-PCR. Values shown are the mean ± SEM (n = 5 animals/group). *, P < 0.05.

 
Effect of hyper- and hypothyroidism on pituitary ghrelin mRNA and peptide levels and hypothalamic GHRH mRNA levels
T3 treatment decreased plasma TSH levels (3.8 ± 0.2 ng/ml for T3 vs. 17.0 ± 0.6 ng/ml for control; P < 0.05; n = 5/group), whereas MMI treatment increased plasma TSH levels (47.4 ± 7.1 ng/ml for MMI; P < 0.01; n = 5/group), thus confirming the effectiveness of treatment on thyroid status. Hyperthyroidism, as a result of T3 treatment, decreased pituitary ghrelin mRNA levels, pituitary ghrelin peptide content, and hypothalamic GHRH mRNA levels (P < 0.05; Fig. 3Go). Hypothyroidism, as a result of MMI treatment, increased pituitary ghrelin mRNA levels, pituitary ghrelin peptide content, and hypothalamic GHRH mRNA levels (P < 0.05; Fig. 3Go).



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  		FIG. 3. Effects of treatment with T3 or MMI on pituitary ghrelin mRNA levels (A), pituitary ghrelin peptide content (B), and hypothalamic GHRH mRNA levels (C). Male Sprague Dawley rats were treated with daily ip injections of T3 (50 µg/100 g body weight), MMI (1.6 mg/100 g body weight), or vehicle (5 mM NaOH) for 7 d. Animals were decapitated 24 h after the final injection of T3, MMI, or vehicle. A, Ghrelin mRNA levels were assessed by RT-PCR. The top panel shows a representative Southern blot of pituitary ghrelin and GAPDH. The lower panel is a summary of the data for ghrelin mRNA levels, which are expressed as a percentage of the control group. The values for ghrelin mRNA levels were corrected for GAPDH mRNA levels. B, Pituitary ghrelin peptide content was assessed by RIA. Data are expressed as picograms per milligram of wet pituitary tissue. C, Hypothalamic GHRH mRNA levels were assessed by Northern blot analysis. The values for GHRH mRNA levels were corrected for ß-actin mRNA levels. The data represent the mean ± SEM (n = 5 animals/group). *, P < 0.05, compared with the control group.

 
Effect of exogenous glucocorticoid treatment and adrenalectomy on pituitary ghrelin mRNA and peptide levels and hypothalamic GHRH mRNA levels
Bilateral adrenalectomy increased pituitary ghrelin mRNA levels and pituitary ghrelin peptide content compared with sham-operated animals (P < 0.05; Fig. 4Go). Hypothalamic GHRH mRNA levels were also increased by adrenalectomy; however, this difference did not reach statistical significance (P > 0.05; Fig. 4Go). This effect was reversed by exogenous dexamethasone treatment. In addition, dexamethasone treatment of rats with intact adrenals decreased pituitary ghrelin mRNA levels, pituitary ghrelin peptide content, and hypothalamic GHRH mRNA levels compared with vehicle-treated animals (P < 0.05; n = 5/group).



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  		FIG. 4. Effects of adrenalectomy or dexamethasone treatment on pituitary ghrelin mRNA levels (A), pituitary ghrelin peptide content (B), and hypothalamic GHRH mRNA levels (C). Adult male rats were bilaterally adrenalectomized or sham operated under ketamine/xylazine anesthesia on d 1 of the experiment. The animals received daily sc injections of 200 µg dexamethasone or 0.9% NaCl (vehicle). Animals were killed by decapitation on d 8 of the experiment. A, Pituitary ghrelin mRNA quantified by RT-PCR. The top panel shows a representative Southern blot of pituitary ghrelin and GAPDH cDNA from sham-operated and adrenalectomized rats treated with vehicle or dexamethasone (Dex). The bottom panel is a summary of the data that are expressed as percentage of sham-operated, vehicle-treated controls (mean ± SEM; n = 5 animals/group). B, Pituitary ghrelin peptide content was assessed by RIA. Data are expressed as picograms per milligram of wet pituitary tissue. The data represent the mean ± SEM (n = 5 animals/group). C, Hypothalamic GHRH mRNA levels were assessed by Northern blot analysis. The values for GHRH mRNA levels were corrected for ß-actin mRNA levels. The data are expressed as a percentage of the sham-operated, vehicle-treated controls (mean ± SEM; n = 5 animals/group). Letters (a, b, c) represent values that are significantly different from each other (P < 0.05).

 
Effect of food deprivation on pituitary ghrelin mRNA levels, pituitary ghrelin peptide content, and hypothalamic GHRH mRNA levels
Seventy-two hours of food deprivation decreased pituitary ghrelin mRNA levels, pituitary ghrelin peptide content, and hypothalamic GHRH mRNA levels compared with ad libitum-fed rats (Fig. 5Go; P < 0.05; n = 5/group).



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  		FIG. 5. Effect of food deprivation on pituitary ghrelin mRNA levels (A), pituitary ghrelin peptide content (B), and hypothalamic GHRH mRNA levels (C). Adult male Sprague Dawley rats were food deprived for 72 h. Animals were killed at 72 h. A, Pituitary ghrelin mRNA levels determined by RT-PCR. The top panel shows a representative Southern blot of pituitary ghrelin and GAPDH cDNA from food deprived (FD) and control (C) rats. The bottom panel is a summary of the data, expressed as percentage of controls (mean ± SEM; n = 5 animals/group). B, Pituitary ghrelin peptide content was assessed by RIA. Data are expressed as picograms per milligram of wet pituitary tissue. The data represent the mean ± SEM (n = 5 animals/group). C, Hypothalamic GHRH mRNA levels were assessed by Northern blot analysis. The values for GHRH mRNA levels were corrected for ß-actin mRNA levels. The data are expressed as a percentage of the control group and represent the mean ± SEM (n = 5 animals/group). *, P < 0.05, compared with the control group.

 
Effect of a GHS-R antagonist on basal and GHRH-induced GH secretion, in vitro
To examine whether the pituitary ghrelin/GHS-R system regulates GH release under basal and stimulated conditions, primary cell cultures from normal rat pituitaries were preincubated with or without [D-Lys-3]-GHRP-6, a GHS-R- specific inhibitor, followed by a GHRH challenge. The GHS-R inhibitor did not significantly affect GH release in the absence of GHRH (39.8 ± 2.4 ng/ml·15 min for vehicle treated vs. 59.0 ± 10.4 ng/ml·15 min for inhibitor treated; P = 0.20; n = 3 independent experiments with six wells/experiment; Fig. 6Go). However, the GHS-R antagonist suppressed GHRH-induced GH secretion to 84.0 ± 3.9% of vehicle-treated controls (227.3 ± 14.7 ng/ml·15 min for vehicle treated vs. 191.0 ± 9.1 ng/ml·15 min for inhibitor treated; P = 0.02; Fig. 6Go). Immunoreactive ghrelin was not detectable in the culture medium by RIA.



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  		FIG. 6. Effect of a GHS-R-specific inhibitor, [D-Lys-3]-GHRP-6, on baseline and GHRH-induced GH secretion from the rat pituitary cells in vitro. Adult male rat pituitaries were enzymatically dispersed and plated at 50,000 cells/well in {alpha}-MEM/10% horse serum. After 3 d of culture, cells were washed in serum-free medium and preincubated with or without [D-Lys-3]-GHRP-6 (1 h; 10–4 M) followed by a GHRH challenge (100 nM; 15 min) in the presence or absence of the inhibitor. The amount of GH released into the medium was determined by RIA. The data are the mean ± SEM (n = 6 wells/treatment group). These results are representative of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This report extends our previous observations (11) and demonstrates that GHRH-induced pituitary ghrelin mRNA levels translate into an increase in pituitary ghrelin peptide content. A physiological role of GHRH in stimulating pituitary ghrelin expression is suggested by the positive correlation of pituitary ghrelin mRNA levels with physiological states known to display enhanced hypothalamic GHRH expression. In the SDR, in which hypothalamic GHRH mRNA levels are elevated, due to the absence of GH negative feedback (13), pituitary ghrelin mRNA levels were also elevated. Replacing GH in SDRs, which was previously shown to suppress hypothalamic GHRH mRNA (13), significantly reduced ghrelin mRNA levels. However, treatment of SDRs with IGF-I, which failed to alter hypothalamic GHRH mRNA levels (13), did not affect pituitary ghrelin expression.

In the present study, hypothalamic GHRH and pituitary ghrelin were positively correlated with changes in thyroid status. Thyroid-mediated changes in GHRH expression have been previously reported by others (19, 20, 21, 22). Propylthiouracil-induced hypothyroidism in rats increases hypothalamic GHRH mRNA expression (21), whereas hyperthyroidism due to exogenous T3 administration decreases GHRH mRNA levels (19, 22). These changes in GHRH are hypothesized to be mediated by thyroid hormone modulation of GH secretion, resulting in changes in the magnitude of GH negative feedback (20, 21, 22). In the present study, induction of hypothyroidism using MMI augmented hypothalamic GHRH expression as well as pituitary ghrelin mRNA levels and peptide content. Conversely, T3 treatment, sufficient to induce a hyperthyroid state, decreased hypothalamic GHRH and pituitary ghrelin expression, once again demonstrating that pituitary ghrelin expression parallels hypothalamic GHRH expression.

In this study, we also demonstrated that excess glucocorticoids decreased, whereas glucocorticoid deficiency, due to bilateral adrenalectomy, increased pituitary ghrelin mRNA and peptide content. Glucocorticoids are known to regulate GH secretion by influencing both hypothalamic and pituitary function (23, 24). Pathophysiological glucocorticoid excess inhibits GH secretion and the GH responses to several stimuli (23, 24). Glucocorticoid exerts these negative effects by acting at the level of the hypothalamus to inhibit GHRH and stimulate somatostatin synthesis and release (25, 26, 27). In contrast, glucocorticoids act at the level of the pituitary to stimulate GH (28, 29) and GHRH receptor (GHRH-R) (30, 31) gene expression. In this report, pathophysiological glucocorticoid excess suppressed hypothalamic GHRH input, and this decrease in GHRH tone could negatively impact on pituitary ghrelin expression. Although the magnitude of suppression of pituitary ghrelin mRNA and hypothalamic GHRH mRNA were comparable in dexamethasone-treated rats, it should be noted that the magnitude of pituitary ghrelin peptide suppression after treatment was far less than the magnitude of ghrelin mRNA suppression. It is possible that this disparity is due to the high dose of dexamethasone used, which may have altered the rate of ghrelin peptide degradation to the nonimmunoreactive form or vascular clearance from the pituitary. Pituitary ghrelin expression was suppressed after 72-h food deprivation, a condition that is also associated with elevated glucocorticoids levels and suppressed hypothalamic GHRH mRNA levels (32, 33, 34). Although the argument of GHRH being the primary mediator of pituitary ghrelin levels in these animal models is compelling, we cannot rule out the possibility that glucocorticoids act directly at the pituitary level to suppress ghrelin gene expression. However, in the stomach, several laboratories report that food deprivation leads to an increase in ghrelin gene expression that is suppressed with refeeding (2, 35, 36). The sharp contrast in the effects of starvation on pituitary and stomach ghrelin expression lessens the possibility of a direct effect of glucocorticoids on ghrelin gene expression.

Although a dominant source of circulating ghrelin appears to be the stomach (37) and exogenous ghrelin treatment increases circulating GH levels (9, 38), no significant correlation exists between endogenous circulating GH levels and ghrelin in the systemic circulation (39, 40). Also, iv injection of antisera, directed against bioactive ghrelin, did not affect pulsatile GH secretion of adult male rats (40). Taken together, these results suggest that circulating ghrelin is not a key regulator of pituitary GH release. This argument is strengthened by the fact that plasma bioactive acyl-ghrelin concentrations are at most 0.07 pmol/ml under physiological conditions (36); in contrast, exogenous delivery of ghrelin (10 µg/kg to adult human or 10 µg to an adult rat) adequate to enhance GH release, leads to increased plasma ghrelin concentration to more than 61- to 100-fold of baseline values (39, 41). Another source of ghrelin is the hypothalamus in which ghrelin immunoreactivities have been observed in a previously uncharacterized group of neurons adjacent to the third ventricle between the hypothalamic arcuate nucleus, ventromedial, dorsomedial, and paraventricular nuclei (42). This network of neurons sends efferents to neuropeptide Y/agouti-related protein, pro-opiomelanocortin, and corticotropin-releasing factor neurons that are located in the hypothalamic regions that are important to metabolic regulation (42). However, it remains to be demonstrated that ghrelin-immunopositive neurons project to the median eminence, or to the GHRH and somatostatin neurons that are important in regulating pituitary GH release. Recently, we reported that adult male rats expressing a GHS-R antisense transgene within the arcuate nucleus of the hypothalamus, have severe attenuation of GHS-R expression but display typical high bursts of pulsatile GH secretion with normal plasma IGF-I levels (8). Taken together, these reports suggest that ghrelin derived from the hypothalamus does not play a major role in the regulation of GH secretion. However, a recent report showed GHS-R knockout mice have lower body weights and decreased serum IGF-I levels compared with their wild-type counterparts (43), suggesting that endogenous ghrelin contributes to physiological GH secretion.

We suggest that the ghrelin important to GH regulation is located in the pituitary, because we observed pituitary ghrelin content well within a range sufficient to augment GH release (0.76–1.2 pg/mg wet weight, which would be equivalent to >500 ng/ml in a tissue culture system) and the fact that a GHS-R antagonist could partially inhibit GHRH-stimulated GH release in pituitary cultures. Recently, it has been reported that there is a direct interaction between the GHRH-Rs and the GHS-Rs in the pituitary somatotroph (44). In cells cotransfected with GHRH-R and GHS-R, activation of the GHS-R alone had no effect on cAMP production; however, coactivation of the GHS-R and GHRH-R produced a cAMP response approximately twice that observed after activation of the GHRH-R alone. Thus, pituitary ghrelin could enhance the GH-releasing action of GHRH through modifying its signal transduction in an autocrine and paracrine manner. However, it should be noted that there is evidence indicating that the GHS-R could act independent of the ligand. Specifically, GHS-R-transfected COS-7 and human embryonic 293 cell lines were shown to display high constitutive signaling of the ghrelin receptor (45). It should also be noted that, in this same report, a putative GHS-R antagonist, [D-Arg-1,D-Phe-5,D-Trp-7,-9,Leu-11]-substance P, was shown to display agonist activities (45). Although the GHS-R antagonist used in the current report, [D-Lys-3]-GHRP-6, has not been tested for agonist activity in an artificial transfection system, as described above, it has been reported to display consistent antagonist activities in primary cultures (46). In the present study, there was a slight but insignificant rise in GH release in cultures treated with the GHS-R antagonist alone when data were pooled from three independent experiments. This slight increase could be interpreted as indicative of [D-Lys-3]-GHRP-6 having an agonist activity. However, we feel that this is not the case, in that two of the three independent experiments did not show an increase in GH release in the presence of the GHS-R antagonist, but all showed a consistent reduction in GHRH-induced GH release in the presence of the antagonist. Therefore, our in vitro data indicate that the endogenous ghrelin/GHS-R system can play a significant modulatory role in GHRH-initiated GH release.

In this study, we used a ghrelin RIA kit that equally recognizes des-acylated and acylated forms of ghrelin, where the acylated variant is considered the biologically relevant GHS-R ligand (1). It should be noted that acylated ghrelin is estimated at less than 1% of total ghrelin in the pituitary (47). However, it has been very recently reported that des-acylated ghrelin also has some physiological role in bone marrow (48), heart (49, 50), and prostate carcinoma cells (51). Therefore, further studies are necessary to distinguish the roles of acylated and des-acylated ghrelin in the pituitary, and to understand the mechanisms and regulation of acylation of the ghrelin peptide.

In conclusion, we have shown that pituitary ghrelin expression is regulated under various hormonal conditions, with a consistent positive association between pituitary ghrelin and hypothalamic GHRH expression. We also demonstrated that pituitary ghrelin/GHS-R interaction is essential for optimal GHRH-stimulated GH release in vitro. Based on these findings, it is suggested that pituitary ghrelin acts as a key modulator of biological function of GHRH by augmenting its GH-releasing activity. This is the first report that supports a physiological significance of endogenous ghrelin in the modulation of GH secretion.


   Acknowledgments
 
We thank Ms. Masayo Asizawa for technical assistance.


   Footnotes
 
This work was supported by a Grant-in-Aid for Scientific Research (C; KAKENHI 13671165) from the Japanese Ministry of Education, Culture, Sports, Science and Technology (to J.K.), a grant from Hakujikai, Institute of Gerontology (to J.K.), and National Institutes of Health Grant DK-30667 (to R.D.K).

Results from this work were presented in part at the 84th Annual Meeting of The Endocrine Society, San Francisco, 2002.

Abbreviations: GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; GHRH-R, GHRH receptor; GHS-R, GH secretagogue receptor; MMI, methimazole; RT, reverse transcription; SDR, spontaneous dwarf rat; SDS, sodium dodecyl sulfate; SSC, standard saline citrate.

Received October 22, 2003.

Accepted for publication April 6, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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