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Ghrelin Is Produced by and Directly Activates Corticotrope Cells from Adrenocorticotropin-Secreting Adenomas

Departments of Cell Biology, Physiology, and Immunology (A.J.M.-F., R.V.-M., M.D.-P., M.T.-S., M.M.M., J.P.C.) and Morphological Sciences (L.J.-R.), University of Córdoba, E-14014 Córdoba, Spain; Service of Endocrinology and Nutrition (J.M.-F., P.B.-L.) and Service of Neurosurgery (A.d.l.R.), Hospital Reina Sofía, E-14004 Córdoba, Spain; Department of Physiology (C.D.), University of Santiago de Compostela, E-15782 Santiago de Compostela, Spain; Department of Endocrinology (S.M.W.), Hospital Sant Pau, Autonomous University of Barcelona, E-08025 Barcelona, Spain; and Division of Endocrinology (A.P., A.L.-C.), Virgen del Rocio University Hospital, E-41013 Sevilla, Spain

Address all correspondence and requests for reprints to: Dr. Justo P. Castaño, Department of Cell Biology, Physiology, and Immunology, Edificio Severo Ochoa, Planta 3, Campus de Rabanales, University of Córdoba, E-14014 Córdoba, Spain. E-mail: justo{at}uco.es.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Context: In Cushing’s disease, ACTH hypersecretion by pituitary corticotrope adenoma cells and resulting hypercortisolism is accompanied by a severely blunted GH secretory response. Interestingly, in Cushing’s disease, ghrelin markedly increases plasma ACTH, whereas its stimulatory action on GH secretion is reduced. Although the reported expression of ghrelin receptors (GHS-R) in corticotrope tumors offers a potential mechanism for ghrelin-induced ACTH hypersecretion, studies on the direct effects of synthetic GH secretagogues on corticotropinoma cells offered contradictory results.

Objective and Design: To evaluate the direct action of ghrelin on corticotropinoma cells from two patients with Cushing’s disease, we measured its effect on free cytosolic calcium concentration ([Ca²⁺]_i). Additionally, expression of GHS-R and its ligand ghrelin was examined in these cells and in five additional corticotropinomas.

Results: Ghrelin (10^–6 M) induced a marked [Ca²⁺]_i increase in 89.5% (case 1; n = 19 cells) and 85% (case 2; n = 13 cells) of corticotropinoma cells. Moreover, RT-PCR showed that expression of GHS-R isoforms is accompanied by that of ghrelin in all seven corticotrope adenomas examined. Importantly, double immunogold electron microscopy revealed that ghrelin is costored within ACTH secretory vesicles in densely granulated adenomatous corticotropes.

Conclusions: These results constitute the first demonstration that ghrelin acts directly on corticotrope tumor cells derived from patients with Cushing’s disease. The presence of ghrelin and GHS-R suggests that pituitary ghrelin may play an autocrine/paracrine role in regulating ACTH release in Cushing’s disease. Our findings provide a plausible cellular basis for the exaggerated ACTH response to ghrelin in Cushing’s disease and suggest novel research strategies to develop medical treatments for this disease.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
CUSHING’S DISEASE IS most commonly caused by ACTH hypersecretion from a pituitary corticotrope adenoma, which accounts for approximately 70% of all cases of Cushing’s syndrome (1). The ideal curative treatment approach for Cushing’s disease is to remove the pituitary adenoma selectively (2), whereas medical therapy is adjunctive only and directed to reduce cortisol production by inhibiting the enzymes responsible for cortisol synthesis (3). Hitherto, there is a lack of curative medical treatment, which is mostly because of the unknown molecular pathology of this disease.

In pituitary-dependent Cushing’s disease, elevated ACTH and cortisol levels are accompanied by a severely blunted GH response (4). This GH blockade remains resistant to stimulation by diverse powerful pharmacological agents (5, 6, 7), yet it is reversible after curative surgery of ACTH-producing adenoma and normalization of cortisol levels (8). Interestingly, earlier studies revealed that patients with Cushing’s disease showed an increased ACTH response to the synthetic GH-releasing peptide (GHRP) hexarelin, which was hypothesized to result from direct action of this GHRP at the pituitary (9). Later on, it was reported that iv administration of ghrelin, the endogenous ligand for the receptor of GH secretagogues (GHS-R), to normal subjects induces a remarkable increase in plasma levels of GH, but it also stimulates ACTH and prolactin release (10, 11), whereas conversely, in conditions of chronic hypercortisolism such as Cushing’s disease, ghrelin caused an exaggerated stimulation in ACTH release but was unable to increase GH secretion (12, 13) (reviewed in Refs. 11 and 14). In line with this, recent studies have suggested that circulating ghrelin levels are altered in Cushing’s disease (13, 14, 15), although the possible causal relationship between ghrelin levels and excess cortisol and its potential involvement in the pathological response of pituitary ACTH/GH response in these patients is still under debate (13, 14, 15).

The cellular and molecular mechanisms underlying the augmented corticotrope response to ghrelin in Cushing’s disease are currently uncertain. Several studies have reported the expression of GHS-R in corticotrope tumors, thereby offering a possible cellular basis to explain the above findings (16, 17, 18). However, the effect of two different synthetic GHSs on corticotropinoma cells has yielded contradictory results, because GHRP-6 failed to elevate concentration of free cytosolic calcium ([Ca²⁺]_i) in single cells (19), whereas other authors found that the nonpeptidyl GHS MK-0677 stimulated ACTH secretion in cultured corticotrope tumor cells (17). Hence, there is a lack of unequivocal evidence on the direct action of ghrelin on these cells. In an attempt to ascertain this question, we have applied a multiple strategy to evaluate the response of corticotrope adenoma cells from two patients with Cushing’s disease to ghrelin. Specifically, the direct action of ghrelin on corticotrope cells was assessed by measuring its effect on [Ca²⁺]_i. Furthermore, we investigated the possible expression of GHS-R as well as its ligand ghrelin in these same cells and in additional pituitary corticotrope adenoma samples from patients with Cushing’s disease by RT-PCR.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
The study was carried out in accordance with the Declaration of Helsinki and approved by the Reina Sofia University Hospital Ethics Committee. Written informed consent was obtained from each patient.

Case 1

A 38-yr-old woman was referred in May 2003 with the clinical suspicion of Cushing’s syndrome. She had high urinary free cortisol (UFC) levels (1507 nmol/24 h; normal, 116–601 nmol/24 h), ACTH at 134.8 pg/ml (normal, 4.7–41 pg/ml), and clinical findings of hypercortisolism. The patient referred irregular menstruation, central obesity, and hirsutism. Analysis confirmed high UFC levels by chemiluminescence (725.5 µg/24 h). Initial study showed an elevated midnight serum cortisol (18.0 µg/dl, or 496.6 nmol/liter; normal, 2 µg/dl, or 54 nmol/liter) and more than 50% cortisol suppression by dexamethasone (8 mg) overnight (12.4 µg/dl, or 342.8 nmol/liter; normal, 3.4 µg/dl, or 94 nmol/liter). However, chest/abdominal computed tomography scans disclosed only bilateral hyperplasia of the adrenal glands. Pituitary magnetic resonance imaging (MRI) and whole-body OctreoScan ([¹¹¹In]-pentetreotide) were negative, so a petrosal sinus catheterization was carried out to evaluate ACTH basally and after stimulation with deamino--D-arginine vasopressin (DDAVP) (10 µg), which showed a central/peripheral ACTH gradient greater than 3 and left/right petrosal sinus gradient greater than 1.4. Hence, a transsphenoidal left hemihypophysectomy surgical approach was performed without complications.

The patient temporally improved after surgery (UFC, 302 nmol/24 h; serum cortisol, 9.4 µg/dl, or 253 nmol/liter), but 6 months later symptoms reappeared. Thus, a new complete biochemical/image study was performed that confirmed active Cushing’s disease: ACTH was 603.5 pg/ml (normal, 4.7–41 pg/ml) and after dexamethasone (8 mg), overnight suppression of cortisol remained at 18.6 µg/dl, or 513 nmol/liter (normal, 3.5 µg/dl, or 97 nmol/liter). Pituitary MRI detected a right pituitary microadenoma, although a second petrosal sinus catheterization repeated the left/right petrosal sinus ACTH gradient greater than 1.4. A second transsphenoidal total hypophysectomy was performed, after which all biochemical findings and clinical features of the disease disappeared (cortisol, 0.2 µg/dl, or 5.4 nmol/liter; ACTH not detectable).

Case 2

A 39-yr-old woman was admitted in March 2005 with hyperglycemic ketosis after a diagnosis of diabetes 4 months before, treated with diet and glimepiride; glycosylated hemoglobin was high (12.5%; normal, 4.5–6.5%). Past medical history revealed hypertension, diet-resistant obesity, and polycystic ovarian syndrome.

Initial workup showed nonsuppressed cortisol levels after overnight dexamethasone (1 mg) (16.5 µg/dl, or 456.2 nmol/liter) and high UFC levels (1,296 µg/24 h, or 35,828.1 nmol/24 h). Serum cortisol after 2 mg dexamethasone was 18.5 µg/dl (511 nmol/liter) and suppressed more than 50% after 8 mg of dexamethasone overnight (4.4 µg/dl, or 118 nmol/liter). Pituitary MRI was positive for a right-sided microadenoma, which was removed by transsphenoidal surgery. Thereafter, normal cortisol levels and improvement of diabetes and hypertension were observed (serum cortisol, 2.5 µg/dl, or 67.5 nmol/liter; ACTH not detectable).

Additional tissue samples from pituitary corticotrope adenomas

To confirm the expression of GHS-R and ghrelin, five additional pituitary corticotrope adenomas were analyzed. Specifically, total RNA samples were extracted from pituitary tumor fragments obtained from one patient from Virgen del Rocio University Hospital of Sevilla, Spain, and four patients from Sant Pau Hospital, Autonomous University of Barcelona, Spain. In all cases, informed consent from the patients was obtained.

Laboratory methods

Serum cortisol and UFC levels were measured using a commercial chemiluminescence assay method (Abbott Axsym System, Chicago, IL). The inter- and intraassay coefficients of variation were less than 10%. Plasma ACTH levels were evaluated with a commercial ELISA (Euro-diagnostic, Apeldoorn, Holland). The sensitivity of the assay was 3 pg/ml. The inter- and intraassay coefficients of variation were less than 4%. Plasma ACTH after DDAVP administration was measured at times (expressed in minutes): –5, 0, +5, +10, and +15. We used 10 µg DDAVP to stimulate ACTH secretion as reported by Malerbi et al. (20). Blood samples were taken from cubital venous (peripheral) and left and right petrosal sinus (central). All dynamic tests were started between 0730 and 0800 h after overnight fasting and 30 min after venous cannulation and slow infusion of isotonic saline.

Cell dispersion and cell culture

Unless otherwise indicated, chemical products and tissue culture reagents were purchased from Sigma Chemical Co. (London, UK). Pituitary adenomas were removed by endoscopic transsphenoidal resection and transferred to sterile cold (4 C) culture medium (DMEM) containing 0.1% BSA, 0.01% L-glutamine, 100 U/ml penicillin, and 0.024 M HEPES. Under sterile conditions, the sample was minced into 1- to 2-mm³ pieces, and two to five of them were snap frozen in liquid nitrogen for posterior RNA isolation. Another two to six pieces were fixed and processed for subsequent electron microscopy studies as described below. Remaining tissue samples were dispersed after an enzymatic and mechanical dispersion procedure described elsewhere (21). Briefly, pieces were washed and incubated in culture medium supplemented with 0.2% collagenase and 0.3% trypsin. After 30 min of gentle shaking, the pieces were decanted by centrifugation. Incubation was continued for another 15 min in 2 mg/ml DNase and 0.1% trypsin inhibitor and again settled down by centrifugation. Finally, pieces were dissociated into individual cells by repeated aspiration into a smooth-tipped Pasteur pipette. Cells were washed once in medium, and cell number and viability were estimated by the trypan blue exclusion test in a Neubauer chamber. Viability was always higher than 92%.

For measurements of [Ca²⁺]_i, dispersed cells were plated at a density of 50,000 cells per coverslip. Coverslips were previously coated with poly-L-lysine to enhance cell adherence to the glass and subsequently processed as described below. Medium was replaced with a fresh one at 48 h.

Immunocytochemical detection of ghrelin by electron microscopy

Freshly isolated tumor adenomatous pituitary pieces were processed for electron microscopy as described previously with minor modifications (22). In brief, pituitary pieces were immersed in a fixative solution containing 2% paraformaldehyde and 1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 2 h at 4 C. Subsequently, and after 1 h postfixation in 1% osmium tetroxide and dehydration in graded ethanol and propylene oxide, pieces were embedded in Araldite Luft 502 (Polysciences, Eppelheim, Germany). Ultrathin sections (approximately 100 nm) were obtained with an ultramicrotome and collected on 300-mesh nickel grids. After etching with a saturated aqueous solution of sodium metaperiodate, a single or double immunogold labeling was performed. Single immunolabeling for ghrelin detection was carried out using, as primary antiserum, 1:100 dilution in 0.1 M Tris buffer of polyclonal goat antighrelin (Santa Cruz Biotechnology, Santa Cruz, CA), an antibody directed against an internal region of human ghrelin, which therefore recognizes total, acylated, and des-acyl-ghrelin. Thereafter, cells were identified on ultrathin sections by immunogold-labeled rabbit antigoat secondary antiserum (10 nm in diameter). Double immunocytochemistry for colocalization of ghrelin and ACTH was performed by sequentially staining the two sides of the grid. Specifically, on one side, a rabbit antihuman ACTH (1:1000; Dako Diagnostic, Barcelona, Spain) was used as the primary antiserum, followed by 10-nm immunogold-labeled antirabbit secondary antiserum; on the other side, primary antiserum was 1:100 polyclonal rabbit antighrelin (-Diagnostic, San Antonio, TX) directed against a 13-amino-acid peptide, n-octanoylated at Ser3, sequence from the N terminus of mature human ghrelin conjugated to keyhole limpet hemocyanin, followed by 20-nm immunogold-labeled goat antirabbit secondary antiserum. After immunolabeling, grids were counterstained with uranile acetate and lead citrate. Samples were examined in a Philips CM 10 electron microscope. Specificity of the immunoreaction was examined by replacing the specific antiserum with normal goat or rabbit serum.

Immunofluorescence

After dispersion of adenomatous tissue, cells were plated at a density of 50,000 cells per coverslip and fixed in Bouin’s solution for 30 min, rinsed in PBS, and incubated in the same buffer containing 1% BSA and 0.3% Triton X-100 for 10 min. Thereafter, cells were incubated overnight at 4 C in a humid atmosphere with antihuman-ACTH (1/1000; National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD) diluted in PBS containing 0.3% Triton X-100 and 1% BSA. Cells were then rinsed in PBS and incubated for 2 h at room temperature with secondary antibody Alexa594-conjugated (1/100; Molecular Probes, Eugene, OR). Finally, cells were rinsed in PBS and mounted with PBS/glycerol (vol/vol). To study the specificity of the immunoreaction, the following controls were performed: 1) omission of the specific antiserum; and 2) incubation with nonimmune rabbit serum instead of the primary antiserum.

PCR amplification

Total RNA was isolated using Trizol reagent (Invitrogen, Barcelona, Spain) as recommended by the manufacturer’s instructions. RNA (3.6 µg) was reverse transcribed to cDNA by using PowerScript (BD Bioscience, Erembodegem, Belgium) reverse transcriptase following the manufacturer’s manual. PCR analysis was applied to assess the expression of ghrelin (GenBank accession no. NM_016362) and its two receptor variants, the functional, long-form GHS-R1a (GenBank accession no. NM_198407) and the truncated isoform GHS-R1b (GenBank accession no. NM_004122) as well as for the ACTH precursor proopiomelanocortin (POMC) (GenBank accession no. NM_000939). PCRs were performed in 25 µl final volume using an iCycler IQ (Bio-Rad, Madrid, Spain) thermocycler by adding 1 µl of 1/10 diluted RT product (cDNA), 0.2 µM reverse and forward primers, 2 mM dNTPs, and 1.25 U EcoTaq polymerase (Ecogen, Barcelona, Spain). Temperature profiles for GHS-R1a, GHS-R1b, and POMC were as follows: 94 C for 15 sec, 68 C for 15 sec, and 72 C for 15 sec for 35 cycles, and the temperature profile for ghrelin amplification was 94 C for 15 sec, 60 C for 15 sec, and 72 C for 15 sec for 37 cycles. For all different primer pairs used, a negative control with an identical amount of non-retrotranscribed total RNA was performed. PCR products were electrophoresed in a 2% agarose gel containing ethidium bromide and extracted using the QiaQuick gel extraction kit (QIAGEN GmBH, Hilden, Germany). Identities of amplicons were confirmed by sequencing.

Measurement of free cytosolic calcium concentration ([Ca²⁺]_i) in single cells

Cells were incubated for 30 min at 37 C with 2.5 µM fura-2 AM (Molecular Probes, Eugene, OR) in phenol red-free DMEM containing 20 mM NaHCO₃ (pH 7.4). Coverslips were washed with phenol red-free DMEM and mounted on the stage of a Nikon (Eclipse TE2000-E) microscope with attached back thinned-CCD cooled digital camera (ORCA II BT; Hamamatsu Photonics, Hamamatsu, Japan). Cells were examined under a x40 oil immersion objective during exposure to alternating 340- and 380-nm light beams, and the intensity of light emission at 505 nm was measured every 5 sec. Changes in [Ca²⁺]_i were recorded as ratios of the corresponding excitation wavelengths (F340/F380) using MetaFluor Sofware (Imaging Corp., West Chester, PA).

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Validation/clinical features

Patients improved their clinical Cushing’s symptoms after transsphenoidal surgery. Postoperative normalization of both ACTH and cortisol levels was consistent with the diagnosis of Cushing’s disease.

ACTH immunofluorescence and immunocytochemical detection of ghrelin by electron microscopy

Immunocytochemical staining of individual adenomatous pituitary-derived cells using fluorescent secondary antibodies showed that the vast majority of cells (>99%) were ACTH positive (Fig. 1), thus identifying the tissues as corticotrope adenomas.

View larger version (56K): [in this window] [in a new window]   FIG. 1. Fluorescence ACTH immunostaining (right) and corresponding bright-field image (left) of dispersed corticotropinoma cells from a patient with Cushing’s disease (case 1). These representative images illustrate that the vast majority of cells derived from pituitary corticotrope adenomas were ACTH immunoreactive. Inset, Detailed area revealing the cytosolic localization of ACTH immunofluorescence.

View larger version (188K): [in this window] [in a new window]   FIG. 2. Electron micrograph of ultrathin sections obtained from a pituitary adenoma from a patient with Cushing’s disease. The main image illustrates the typical ultrastructural features of a densely granulated corticotrope adenoma. a, Magnified area disclosing the presence of immunoreactive ghrelin (10-nm gold particles) within secretory granules; b, detailed area of a representative corticotrope tumor cell demonstrating colocalization of ACTH and ghrelin, as shown by the presence of both immunoreactive ACTH (10-nm gold particles) and ghrelin (20-nm gold particles) within the same secretory granules. Bar, 5 µm.

[Ca²⁺]_i was measured in 32 single cells obtained from the two corticotropinomas. In case 1, 89.5% of 19 examined cells exhibited a marked increase in [Ca²⁺]_i in response to treatment with 1 µM ghrelin. Similarly, 85% of cells in case 2 (n = 13) exhibited a clear elevation in [Ca²⁺]_i after ghrelin administration. A detailed analysis of [Ca²⁺]_i recordings from responsive cells showed that ghrelin caused a biphasic increase in [Ca²⁺]_i in virtually all studied corticotrope adenoma cells from both cases (Fig. 3). This response was characterized by a rapid and prominent Ca²⁺ spike followed by a sustained plateau phase that gradually declined to near-basal levels (Fig. 3).

View larger version (20K): [in this window] [in a new window]   FIG. 3. Representative profiles of changes in the concentration of [Ca2+]i in pituitary corticotropinoma cells derived from two patients with Cushing’s disease in response to ghrelin administration (arrow). Upper and lower panels represent the [Ca2+]i kinetics observed in response to treatment with 1 µM ghrelin in two single adenomatous corticotropes obtained from case 1 (top) and case 2 (bottom).

Analysis of total RNA obtained from adenomatous pituitaries after transsphenoidal surgery was performed by PCR using gene-specific primers based on the sequences of human ghrelin, GHS-R1a, GHS-R1b, POMC, and 18 S (Fig. 4). PCR amplification of ghrelin gave the expected fragment size of 190 bp in all tumors tested (Fig. 4, line 3). DNA sequencing of the purified bands confirmed the identity of the amplified transcripts, thus demonstrating that ghrelin is expressed in corticotrope adenomas from the two patients with Cushing’s disease whose cells were functionally analyzed in detail (lanes 1 and 2) as well as in the additional five corticotrope pituitary adenoma samples examined (lanes 3–7). Furthermore, GHS-R1a and GHS-R1b transcripts were coexpressed with ghrelin in corticotrope adenomatous tissue (Fig. 4, lines 1 and 2, respectively). The amplified products presented the expected size for GHS-R1a (300 bp) and GHS-R1b (130 bp), and the nucleotide sequences matched the published sequences for the respective human GHS-Rs. Finally, strong expression of POMC (Fig. 4, line 4) confirmed the nature of the adenomatous tissue under study as corticotrope tumors. Different band intensities reflect differences in the levels of expression of ghrelin and its receptors among different patients.

View larger version (47K): [in this window] [in a new window]   FIG. 4. Expression of ghrelin and its receptors GHS-R1a and -1b in corticotrope tumors. Agarose gels were loaded with RT-PCR products obtained using specific primers for GHS-R1a (line 1), GHS-R1b (line 2), ghrelin (line 3), POMC (line 4), and 18 S (line 5) from pituitary adenoma samples derived from seven patients with Cushing’s disease. The amplified fragments were of the expected size: 300 bp for GHS-R1a, 130 bp for GHS-R1b, 190 bp for ghrelin, 225 bp for POMC, and 137 bp for 18 S. Lanes 1–7 correspond to case 1 (lane 1), case 2 (lane 2), and five different pituitary corticotrope adenoma samples (lanes 3–7); M is for a 100-bp -DNA ladder.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

Activation of GHS-R by its natural ligand, ghrelin, or by synthetic GHS stimulates not only GH but also ACTH and prolactin secretion in healthy human subjects (9, 10, 11, 23). In fact, ghrelin is more effective than synthetic GHS in eliciting ACTH release and causes a marked effect in the hypothalamus-pituitary-adrenal axis, which compares well with that induced by administration of naloxone, vasopressin, and even CRH (reviewed in Ref. 11). In normal subjects, ghrelin-induced ACTH release is thought to be totally dependent on a central nervous system-mediated action (11). On the other hand, ghrelin and GHS administration to pituitary-dependent Cushing’s disease patients causes an exaggerated ACTH release, which has been proposed to be mediated via a direct action of this peptide on corticotropes (9, 10, 11, 23). In fact, before the discovery of ghrelin, it was already postulated that the potent ACTH-releasing effects caused by a synthetic GHRP, hexarelin, in patients with Cushing’s disease were affected directly at the pituitary level (9). However, in vitro studies on the effects of synthetic GHSs at the cellular level have rendered conflicting results (17, 19). Our present study provides strong evidence that the endogenous GHS-R ligand ghrelin does act directly on corticotrope tumor cells by elevating a pivotal second messenger, Ca²⁺, which is directly linked to hormone release. Furthermore, such a direct action of ghrelin on corticotrope cells from two pituitary adenomas is consistent with the expression of its known receptor, GHS-R, in these cells. Indeed, PCR analysis detected the presence in these two tumors, as well as in extracts from five additional corticotropinoma tumors, of significant transcript levels for its two subtypes, GHS-R1a, the one considered to be functional in mediating ghrelin action, and a shorter isoform, GHS-R1b, whose precise functional significance is still unclear (11). These data are well in agreement with previous studies that report the presence of GHS-R in diverse pituitary tumors, including corticotrope adenomas (Refs. 16 , 17 , and 24 ; reviewed in Ref. 11). In this scenario, our present findings support the notion that the exaggerated ACTH/cortisol response found in vivo after ghrelin administration in patients with ACTH-dependent Cushing’s disease (10, 12, 13, 14) can be exerted by this peptide directly at the pituitary level. Furthermore, given the reported ability of ghrelin to stimulate proliferation in a number of cell types, including the rat pituitary somatotroph cell line GH3 (25), it seems conceivable that locally produced ghrelin could also affect proliferation of corticotrope tumor cells and thereby influence tumor progression, a possibility that will certainly merit analysis in future studies.

There is increasing evidence that ghrelin plasma levels are dysregulated in patients with Cushing’s syndrome compared with healthy subjects, although there are contradictory reports on the precise action of cortisol excess on circulating ghrelin. Specifically, whereas some authors have reported plasma ghrelin levels to be decreased under endogenously or exogenously induced hypercortisolism (15), other authors did not find cortisol excess to directly affect ghrelin levels in patients with Cushing’s disease (26). More recently, a detailed study on this subject indicated that circulating ghrelin was not reduced in Cushing’s disease despite the typical features accompanying this disease (i.e. increased body mass index and insulin and glucose levels) (14), which are commonly associated with a reduction in circulating ghrelin levels, as is clearly the case in obese patients (27). Thus, additional work will be required to elucidate the possible contribution of circulating ghrelin to the exaggerated ACTH secretory response to this peptide in Cushing’s disease. On the other hand, our current data showing local production of ghrelin by corticotrope adenomas causing Cushing’s disease and, most importantly, its accumulation within secretory granules in ACTH-secreting tumor cells, coupled to the presence of GHS-R, point to an autocrine loop in these tumor cells, whereby locally produced ghrelin would stimulate ACTH secretion in a positive feedback mechanism and may also enhance cell proliferation (25). In support of this notion, previous studies have shown that ghrelin is present in human pituitary corticotrope tumors (24). Although ghrelin expression was found to be lower in corticotrope tumors compared with normal pituitary and other pituitary tumor types, the authors pointed out that corticotrope adenomas showed a high ratio of receptor to ghrelin expression (24), an observation that may be functionally relevant. It is important to emphasize that it is currently unknown why normal corticotropes do not show a significant response to ghrelin, whereas tumor corticotropes do. It seems conceivable that hypercortisolism might regulate GHS-R expression in corticotropes, because glucocorticoids up-regulate this receptor in rat hypothalamus (28, 29). Unfortunately, limited availability of corticotrope tumors and, especially, of normal corticotropes has precluded hitherto a more detailed analysis of this issue, and thus, our future studies should aim at further investigating the causes and underlying molecular mechanism for these changes. Likewise, the pathophysiological significance and regulation of locally produced ghrelin in corticotrope adenoma cells deserves additional investigation. We hope that our present findings provide new insights to understand ACTH hypersecretion in Cushing’s disease and may prompt new strategies to investigate medical treatment for this disease, for example, through the recently developed specific antagonists for human GHS-R (30).

	Acknowledgments

 
The contribution of Dr. Frederic Bartumeus, Dr. María-José Barahona, and Dr. Nuria Sucunza of the Hospital Sant Pau, Barcelona, and of Dr. Jose Manuel Montero Elena and Dr. Miguel Angel Japón of the Virgen del Rocio University Hospital, Sevilla, in obtaining pituitary samples is gratefully acknowledged.

	Footnotes

 
This work was supported by Junta de Andalucia (CVI-0139), Ministerio de Educación y Ciencia (BFI-2001-2007, BFU2004-03883), and Instituto de Salud Carlos III–Ministerio de Sanidad y Consumo (PI042082), Spain.

The authors have nothing to declare.

First Published Online March 21, 2006

¹ A.J.M.-F. and J.M.-F. contributed equally to this work and should be considered as first coauthors.

Abbreviations: [Ca²⁺]_i, Free cytosolic calcium; DDAVP, deamino--D-arginine vasopressin; GHRP, GH-releasing peptide; GHS-R, receptor for GH secretagogues; MRI, magnetic resonance imaging; POMC, proopiomelanocortin; UFC, urinary free cortisol.

Received February 1, 2006.

Accepted March 14, 2006.

	References

Top Abstract Introduction Patients and Methods Results Discussion References

 

1. Nieman LK, Ilias I 2005 Evaluation and treatment of Cushing’s syndrome. Am J Med 118:1340–1346[CrossRef][Medline]
2. Bigos ST Somma M, Rasio E, Eastman RC, Lanthier A, Johnston HH, Hardy J 1980 Cushing’s disease: management by transsphenoidal pituitary microsurgery. J Clin Endocrinol Metab 50:348–354[Abstract]
3. Sonino N, Boscaro M, Merola G., Mantero F 1985 Prolonged treatment of Cushing’s disease by ketoconazole. J Clin Endocrinol Metab 61:718–722[Abstract]
4. Casanueva FF, Diéguez C 1998 Interaction between body composition, leptin and growth hormone status. Baillieres Clin Endocrinol Metab 12:297–314[Medline]
5. Leal-Cerro A, Pereira JL, Garcia-Luna PP, Astorga R, Cordido F, Casanueva FF, Diéguez C 1990 Effect of enhancement of endogenous cholinergic tone with pyridostigmine on growth hormone (GH) responses to GH-releasing hormone in patients with Cushing’s syndrome. Clin Endocrinol (Oxf) 33:291–295[Medline]
6. Leal-Cerro A, Pumar A, Villamil F, Astorga R, Diéguez C, Casanueva FF 1993 Growth hormone releasing hormone priming increases growth hormone secretion in patients with Cushing’s syndrome. Clin Endocrinol (Oxf) 38:399–403[Medline]
7. Leal-Cerro A, Pumar A, Garcia-Garcia E, Diéguez C, Casanueva FF 1994 Inhibition of growth hormone release after the combined administration of GHRH and GHRP-6 in patients with Cushing’s syndrome. Clin Endocrinol (Oxf) 41:649–654[Medline]
8. De Boer H, Blok GJ, Van der Veen EA 1995 Clinical aspects of growth hormone deficiency in adults. Endocr Rev 16:63–86[CrossRef][Medline]
9. Ghigo E, Arvat E, Ramunni J, Colao A, Gianotti L, Deghenghi R, Lombardi G, Camanni F 1997 Adrenocorticotropin- and cortisol-releasing effect of hexarelin, a synthetic growth hormone-releasing peptide, in normal subjects and patients with Cushing’s syndrome. J Clin Endocrinol Metab 82:2439–2444[Abstract/Free Full Text]
10. Arvat E, Maccario M, Di Vito L, Broglio F, Benso A, Gottero C, Papotti M, Muccioli G, Dieguez C, Casanueva FF, Deghenghi R, Camanni F, Ghigo E 2001 Endocrine activities of ghrelin, a natural growth hormone secretagogue (GHS), in humans: comparison and interactions with hexarelin, a nonnatural peptidyl GHS, and GH-releasing hormone. J Clin Endocrinol Metab 86:1169–1174[Abstract/Free Full Text]
11. van der Lely AJ, Tschop M, Heiman ML, Ghigo E 2004 Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin. Endocr Rev 25:426–457[Abstract/Free Full Text]
12. Leal-Cerro A, Torres E, Soto A, Dios E, Deghenghi R, Arvat E, Ghigo E, Diéguez C, Casanueva FF 2002 Ghrelin is no longer able to stimulate growth hormone secretion in patients with Cushing’s syndrome but instead induces exaggerated corticotropin and cortisol responses. Neuroendocrinology 76:390–396[CrossRef][Medline]
13. Giordano R, Picu A, Broglio F, Bonelli L, Baldi M, Berardelli R, Ghigo E, Arvat E 2004 Ghrelin, hypothalamus-pituitary-adrenal (HPA) axis and Cushing’s syndrome. Pituitary 7:243–248[CrossRef][Medline]
14. Giordano R, Picu A, Pagotto U, De Iasio R, Bonelli L, Prodam F, Broglio F, Marafetti L, Pasquali R, Maccario M, Ghigo E, Arvat E 2005 The negative association between total ghrelin levels, body mass and insulin secretion is lost in hypercortisolemic patients with Cushing’s disease. Eur J Endocrinol 153:535–543[Abstract/Free Full Text]
15. Otto B, Tschop M, Heldwein W, Pfeiffer AF, Diederich S 2004 Endogenous and exogenous glucocorticoids decrease plasma ghrelin in humans. Eur J Endocrinol 151:113–117[Abstract]
16. Kim K, Arai K, Sanno N, Osamura RY, Teramoto A, Shibasaki T 2001 Ghrelin and growth hormone (GH) secretagogue receptor (GHSR) mRNA expression in human pituitary adenomas. Clin Endocrinol (Oxf) 54:759–768[CrossRef][Medline]
17. Barlier A, Zamora AJ, Grino M, Gunz G, Pellegrini-Bouiller I, Morange-Ramos I, Figarella-Branger D, Dufour H, Jaquet P, Enjalbert A 1999 Expression of functional growth hormone secretagogue receptors in human pituitary adenomas: polymerase chain reaction, triple in-situ hybridization and cell culture studies. J Neuroendocrinol 11:491–502[CrossRef][Medline]
18. Skinner MM, Nass R, Lopes B, Laws ER, Thorner MO 1998 Growth hormone secretagogue receptor expression in human pituitary tumors. J Clin Endocrinol Metab 83:4314–4320[Abstract/Free Full Text]
19. Lania A, Ballare E, Corbetta S, Filopanti M, Persani L, Spada A 1998 Growth hormone-releasing hexapeptide (GHRP-6) increases intracellular calcium concentrations in cultured cells from human pituitary adenomas of different types. Eur J Endocrinol 139:343–348[Abstract]
20. Malerbi DA, Mendonca BB, Liberman B, Toledo SP, Corradini MC, Cunha-Neto MB, Fragoso MC, Wajchenberg BL 1993 The desmopressin stimulation test in the differential diagnosis of Cushing’s syndrome. Clin Endocrinol (Oxf) 38:463–472[Medline]
21. Malagón MM, Luque RM, Ruiz-Guerrero E, Rodriguez-Pacheco F, Garcia-Navarro S, Casanueva FF, Gracia-Navarro F, Castaño JP 2003 Intracellular signaling mechanisms mediating ghrelin-stimulated growth hormone release in somatotropes. Endocrinology 144:5372–5380[Abstract/Free Full Text]
22. Castaño JP, Torronteras R, Ramirez JL, Gribouval A, Sanchez-Hormigo A, Ruiz-Navarro A, Gracia-Navarro F 1996 Somatostatin increases growth hormone (GH) secretion in a subpopulation of porcine somatotropes: evidence for functional and morphological heterogeneity among porcine GH-producing cells. Endocrinology 137:129–136[Abstract]
23. Arvat E, Ramunni J, Giordano R, Maccagno B, Broglio F, Benso A, Deghenghi R, Ghigo E 1999 Effects of the combined administration of hexarelin, a synthetic peptidyl GH secretagogue, and hCRH on ACTH, cortisol and GH secretion in patients with Cushing’s disease. J Endocrinol Invest 22:23–28[Medline]
24. Korbonits M, Bustin SA, Kojima M, Jordan S, Adams EF, Lowe DG, Kangawa K, Grossman AB 2001 The expression of the growth hormone secretagogue receptor ligand ghrelin in normal and abnormal human pituitary and other neuroendocrine tumors. J Clin Endocrinol Metab 86:881–887[Abstract/Free Full Text]
25. Nanzer AM, Khalaf S, Mozid AM, Fowkes RC, Patel MV, Burrin JM, Grossman AB, Korbonits M 2004 Ghrelin exerts a proliferative effect on a rat pituitary somatotroph cell line via the mitogen-activated protein kinase pathway. Eur J Endocrinol 151:233–240[Abstract]
26. Libe R, Morpurgo PS, Cappiello V, Maffini A, Bondioni S, Locatelli M, Zavanone M, Beck-Peccoz P, Spada A 2005 Ghrelin and adiponectin in patients with Cushing’s disease before and after successful transsphenoidal surgery. Clin Endocrinol (Oxf) 62:30–36[CrossRef][Medline]
27. Tschop M, Weyer C, Tataranni PA, Devanarayan V, Ravussin E, Heiman ML 2001 Circulating ghrelin levels are decreased in human obesity. Diabetes 50:707–709[Abstract/Free Full Text]
28. Thomas GB, Bennett PA, Carmignac DF, Robinson ICAF 2000 Glucocorticoid regulation of growth hormone (GH) secretagogue-induced growth responses and GH secretagogue receptor expression in the rat. Growth Horm IGF Res 10:45–52[CrossRef][Medline]
29. Tamura H, Kamegai J, Sugihara H, Kineman RD, Frohman LA, Wakabayashi I 2000 Glucocorticoids regulate pituitary growth hormone secretagogue receptor gene expression. J Neuroendocrinol 12:481–485[CrossRef][Medline]
30. Halem HA, Taylor JE, Dong JZ, Shen Y, Datta R, Abizaid A, Diano S, Horvath TL, Culler MD 2005 A novel growth hormone secretagogue-1a receptor antagonist that blocks ghrelin-induced growth hormone secretion but induces increased body weight gain. Neuroendocrinology 81:339–349[Medline]

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