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Departments of Pediatrics and of Neurology and Neurosurgery (G.S.T.), McGill University, and the Neuropeptide Physiology Laboratory, McGill University-Montréal Children’s Hospital Research Institute, Montreal, Québec, Canada H3H 1P3; Institut National de la Santé et de la Recherche Médicale, Unité 549 (J.E.), 75014 Paris, France; and Department of Medicine, Division of Endocrinology, Tulane University Medical Center (C.Y.B.), New Orleans, Louisiana 70112-2699
Address all correspondence and requests for reprints to: Dr. Gloria S. Tannenbaum, Neuropeptide Physiology Laboratory, McGill University-Montréal Children’s Hospital Research Institute, 2300 Tupper Street, Montréal, Québec, Canada H3H 1P3. E-mail: gloria.tannenbaum{at}mcgill.ca.
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Introduction |
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Regulation of the secretion of GH from the anterior pituitary gland is under the reciprocal control of two hypothalamic hormones, a stimulatory GHRH found in the arcuate nucleus, and an inhibitory hormone, somatostatin (SRIF), synthesized in the periventricular nucleus. Several lines of evidence suggest that in addition to the intricate patterned release of GHRH and SRIF regulating GH directly at the pituitary level, SRIF modulates GH secretion indirectly through central regulation of GHRH-containing neurons. The net result of these interactions is a striking pulsatile pattern of GH release as observed in the peripheral blood of both human and experimental animals (see Ref. 2 for review).
In recent years there has been intense interest in a novel class of peptide and nonpeptidyl synthetic compounds, termed GH secretagogues (GHSs), developed from the prototype hexapeptide GH-releasing peptide-6 (3) and shown to exert potent GH-releasing activity in multiple species, including the human (4, 5). A unique receptor for GHS (GHS-R) was cloned from the pituitary of humans (6) and rats (7), and numerous sites of expression of GHS-R were identified in brain (8). Double-labeling studies demonstrated the expression of GHS-R by GHRH mRNA-containing neurons in the hypothalamus (9, 10), suggesting that GHSs may directly modulate GHRH neurons. Moreover, GHS-R mRNA, in both brain (11) and pituitary (12, 13), was shown to be sensitive to changes in GH status. All of these findings supported the idea that a third neuroendocrine pathway may exist to regulate pulsatile GH secretion.
The breakthrough discovery, in late 1999, of the natural endogenous ligand for the GHS-R, termed ghrelin (14), has provided a new and intriguing dimension for GH research. The chemistry and the major anatomical site of origin of ghrelin are novel; it is a Ser3-octanoylated, 28-amino-acid peptide that surprisingly originates primarily from the stomach rather than the hypothalamus, although ghrelin-immunoreactive neurons have been detected in small amounts in the hypothalamic arcuate nucleus in rat (14, 15) and the infundibular nucleus in human (16). Ghrelin directly releases GH in vitro from primary rat pituitary cells (14) and is a potent GH secretagogue in vivo when administered systemically to rats and humans (14, 17, 18, 19, 20). Ghrelin has also been shown to stimulate GH release when administered into the central nervous system by some (21, 22), but not all (23), investigators, suggesting a central site of action. However, the neuroendocrine pathways through which ghrelin acts to release GH relative to the primary hypothalamic regulators of GH secretion (GHRH and SRIF) and its functional significance for GH regulation are largely unknown.
Intriguingly, ghrelin’s actions are not restricted to the GH axis. Ghrelin also functions as a powerful orexigenic hormone; it stimulates feeding and increases body weight when administered either peripherally or centrally (22, 24, 25), and these effects appear to be independent of changes in GH (24, 25). It is well known that the secretion of GH is exquisitely sensitive to perturbations in nutritional states (see Ref. 2 for review); thus, ghrelin may be a critical hormonal signal of nutritional status to the GH neuroendocrine axis.
In this paper we report on our physiological studies designed to further understand how ghrelin interacts with the well established, seemingly self-sufficient GHRH/SRIF neuronal system in the regulation of pulsatile GH secretion. To further assess the hypothalamic cell types through which ghrelin influences GH release and food intake, we performed dual chromogenic and autoradiographic in situ hybridization and compared the coexpression of the GHS-R with SRIF and GHRH, and also with neuropeptide Y (NPY; a potent orexigenic peptide), in three different hypothalamic nuclei.
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Materials and Methods |
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For the physiological studies, chronic intracerebroventricular (icv) and intracardiac venous cannulas were implanted under sodium pentobarbital (50 mg/kg, ip) anesthesia using previously described techniques (26, 27). The placement of the icv cannula was verified by both a positive drinking response to icv carbachol (100 ng/10 µl) injection on the day after surgery and methylene blue dye at the time of death. After surgery, the rats were placed directly in isolation test chambers with food and H2O freely available until body weight returned to preoperative levels (usually within 5–7 d). During this time the rats were handled daily to minimize any stress associated with handling on the day of the experiment. On the test day, food was removed 1.5 h before the start of sampling and was returned at the end.
In the first series of experiments we examined the temporal pattern and magnitude of the GH response to ghrelin, administered either systemically or centrally. Free moving rats were iv injected with either ghrelin (5 or 10 µg) or normal saline at two different time points during a 6-h sampling period. The times of 1100 and 1300 h were chosen because they reflect typical peak and trough periods of GH secretion, as previously documented (26, 28). The human ghrelin peptide (provided by Dr. K. Chang, Phoenix Pharmaceuticals, Inc., Belmont, CA) was diluted in normal saline just before use. To assess the central actions of ghrelin on pulsatile GH release, a 10-fold lower dose of ghrelin (500 ng) or normal saline was administered icv at the same time points. Blood samples (0.35 ml) were withdrawn every 15 min over the 6-h sampling period (1000–1600 h) from all animals. To document the rapidity of the GH response to ghrelin, an additional blood sample was obtained 5 min after each injection of the peptide. All blood samples were immediately centrifuged, and plasma was separated and stored at -20 C for subsequent assay of GH. To avoid hemodynamic disturbance, the red blood cells were resuspended in normal saline and returned to the animal after removal of the next blood sample.
In the second series of experiments, designed to assess the roles of endogenous GHRH and SRIF in mediating the GH responses to ghrelin, two groups of rats were administered 1–2 ml of specific GHRH or SRIF antisera iv at 1015 h after removal of the first of two blood samples. The ghrelin peptide (5 µg) was subsequently iv injected at 1100 and 1300 h. A third group of rats served as controls and received 1–2 ml of normal sheep serum and 5 µg ghrelin, iv, at the same time points. Blood samples were withdrawn from 1000–1600 h, as described above. The SRIF and GHRH antisera were the same as those described in our previous passive immunization studies (28, 29).
For the histochemical studies, adult male Sprague Dawley rats were killed by decapitation between 1100–1115 h. The brains were snap-frozen in isopentane at -40 C for 1 min and stored at -80 C. They were coronally sectioned with a cryostat (CM 3050 S, Leica Corp., Deerfield, IL) at 20-µm thickness, beginning at the joining of the anterior commissure and continuing caudally to the mammillary bodies. Sections were collected on poly-L-lysine (50 µg/ml)-coated slides, dried for 2 min at 37 C, and stored at -70 C until in situ hybridization was performed.
All animal-based procedures were approved by the McGill University animal care committee.
GH assay
Plasma GH concentrations were measured in duplicate by double antibody RIA using materials supplied by the NIDDK Hormone Distribution Program (Bethesda, MD). The averaged plasma GH values are reported in terms of the rat GH reference preparation (rGH RP-2). The standard curve was linear between 0.62 and 320 ng/ml; the least detectable concentration of plasma GH under the conditions used was 1.2 ng/ml. All samples with values above 320 ng/ml were reassayed at dilutions ranging from 1:2 to 1:10. The intra- and interassay coefficients of variation were 7.7% and 10.7%, respectively, for duplicate samples of pooled plasma containing a mean GH concentration of 60.7 ng/ml.
Probe preparation
GHS-R.
Single-stranded sense and antisense RNA probes were generated from constructed full-length rat GHS-R type 1a cDNA (a gift from Dr. Andrew Howard, Merck & Co., Inc., Rahway, NJ) inserted into the mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, CA). To obtain GHS-R antisense probes, the cDNA templates were produced by linearization of the vector with EcoRI and transcription with the Gemini II system (Promega Corp., Madison, WI) using SP6 RNA polymerase and [35S]uridine 5'-[
-thio]triphosphate (NEN Life Science Products, Boston, MA). Sense probes were prepared from NotI-linearized plasmid DNA in the presence of T7 RNA polymerase. Aliquots were stored at -70 C. Before use, the identity and integrity of the probes were verified by PAGE against known standards. The final probe specific activity was approximately 1.6 x 109 dpm/µg.
GHRH.
The rat prghrf-2 plasmid was obtained from Dr. Kelly Mayo, (Northwestern University, Evanston, IL). A 217-bp fragment including the entire GHRH-43 coding sequence was subcloned into the transcription vector pGEM-4 (Promega Corp.), and a digoxigenin (DIG)-labeled antisense cRNA probe was made in vitro using DIG RNA Labeling Mix (Roche Molecular Biochemicals, Laval, Canada) containing 3.5 mM DIG-II-UTP; 6.5 mM unlabeled uridine triphosphate; 10 mM GTP, ATP, and CTP; and T7 RNA polymerase.
SRIF and NPY.
The rat pSRIF-28 and rat pBLNPY-1 plasmids were obtained from Dr. Robert Steiner (University of Washington School of Medicine, Seattle, WA). DIG-labeled antisense cRNA probes were made in vitro using DIG RNA Labeling Mix described above and SP6 RNA polymerase (SRIF probe) or T3 RNA polymerase (NPY probe).
Double label in situ hybridization
We performed dual chromogenic and autoradiographic in situ hybridization using a protocol described previously (9, 30). Separate experiments were carried out for each of the DIG-labeled probes. Briefly, processed sections were hybridized with 35S-labeled antisense GHS-R probe (3–6 x 106 cpm/ml) and either 3.75 µl/ml (GHRH), 2.5 µl/ml (SRIF), or 5 µl/ml (NPY) DIG-labeled antisense probes in hybridization buffer. Overnight hybridization at 60 C was followed by ribonuclease treatment, a series of stringent washes in standard saline citrate, and a wash at 60 C. The slides were then blocked with 2% normal sheep serum and incubated overnight at room temperature with 150 µl anti-DIG antibody conjugated to alkaline phosphatase (Roche Molecular Biochemicals; 1:1000). Slides were rinsed in buffer before applying 150 µl chromogen and incubated at 37 C for 7 h until color development. They were then washed in 1 M Tris-HCl (pH 8) and 0.5 M EDTA, dehydrated in 70% ethanol, air-dried, dipped in 3% parlodion, and dried overnight. All slides were coated with NTB2 photographic emulsion (Eastman Kodak Co., Rochester, NY) diluted 1:1 with distilled H2O and exposed for 5–6 wk at 4 C.
Image analysis
Light microscopic autoradiograms of hybridized brain sections were analyzed under epifluorescence illumination using a computer-assisted image analysis system (Biocom, Les Ulis, France) coupled to a Diaplan microscope (Leitz, Rockleigh, NJ). Twenty tissue sections per rat for each probe were analyzed. First, the numbers of GHS-R-, GHRH-, SRIF-, and NPY-labeled cells were quantified using Histo and Rag programs; purple-stained DIG-labeled GHRH, SRIF, or NPY cells were outlined and counted under bright-field illumination. Second, the number of silver grains overlying individual DIG-labeled cells was counted. Cells were considered double-labeled if the density of silver grains counted over them was at least three times higher than background (determined in another area of the hypothalamus). Results were expressed either as the percentage (mean ± SE) of GHRH, SRIF, or NPY mRNA-positive cells dually stained for GHS-R mRNA or as the percentage of GHS-R mRNA-positive cells expressing GHRH, SRIF, or NPY in the arcuate (ARC), ventromedial (VMN), and periventricular (PeV) nuclei of the hypothalamus.
Statistical analyses
ANOVA and t tests for unpaired and paired data, as appropriate, were used for statistical comparisons between and within experimental groups. The integrated area under the GH response curve (AUC) was calculated by the linear trapezoidal method. The results are expressed as the mean ± SE. P < 0.05 was considered significant.
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Results |
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illustrates the mean plasma GH responses evoked by ghrelin administered iv during peak and trough periods of GH secretion compared with normal saline (NS) iv-injected controls. Injection of 5 µg ghrelin during a time of a spontaneous GH secretory episode (1100 h) caused a rapid 5- to 8-fold increase in plasma GH levels within 5 min after injection; plasma GH levels remained significantly elevated for approximately 30 min compared with NS-treated controls (30-min GH AUC, 221 ± 44 vs. 70 ± 10 ng/ml·h; P < 0.001). In contrast, administration of ghrelin during a trough period (1300 h), when endogenous SRIF release is known to be high (28), resulted in a markedly attenuated GH response at 5 min after injection compared with that observed at 1100 h (35 ± 9 vs. 475 ± 80 ng/ml; P < 0.008; Figs. 1B and 2). However, by 15 min, the amount of GH released was similar to that observed at peak times (Figs. 1B and 2). This time-dependent GH-releasing activity of ghrelin was also observed with the 10-µg dose; doubling the dose of ghrelin did not significantly alter either the amplitude or the temporal pattern of the GH response compared with the 5-µg dose (Fig. 2).
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illustrates individual, representative plasma GH responses to 500 ng ghrelin injected icv at 1100 and 1300 h compared with those in NS icv-injected controls. As shown in Fig. 3B, administration of ghrelin during a time of a spontaneous GH peak strongly stimulated plasma GH levels within 5–15 min after icv injection compared with NS icv-injected controls. Plasma GH levels in these animals reached values as high as those observed after iv administration (see Fig. 1B
) despite the 10-fold lower dose of ghrelin and remained significantly elevated for approximately 30 min (30-min GH AUC, 217 ± 50 vs. 65 ± 20 ng/ml·h in NS controls; P < 0.03). However, in contrast to the temporal pattern observed in rats given ghrelin systemically, icv injection of ghrelin at trough times failed to significantly increase plasma GH levels compared with NS icv-injected controls (30-min GH AUC, 28 ± 12 vs. 25 ± 19 ng/ml·h; Fig. 3B). The GH response evoked by ghrelin at 1300 h was approximately 8-fold lower than that observed at 1100 h (P < 0.001).
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), similar to those observed in untreated control rats described above. Administration of SRIF antiserum reversed the blunted 5-min GH response to ghrelin during trough periods to levels as high as those observed during peak periods. Immunoneutralization of endogenous SRIF did not significantly alter the GH responses to ghrelin, compared with NSS-treated controls, at all other time points examined (Fig. 4). The 30-min GH AUC response to ghrelin at peak times in SRIF antiserum-treated rats (236 ± 24 ng/ml·h) was similar to that observed in NSS-treated controls (197 ± 38 ng/ml·h).
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). The 30-min GH AUC response to ghrelin at 1100 h in animals pretreated with GHRH antiserum (23 ± 9 ng/ml·h) was approximately 9-fold lower than that observed in NSS-treated controls (197 ± 38 ng/ml·h) and was 13-fold lower at trough times (9 ± 2 ng/ml·h).
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); more sparsely distributed and/or less intensely labeled neurons were evident in the PeV. SRIF-expressing neurons were also found in these three nuclei, but in greater amounts in the PeV. GHRH neurons were only located in the ARC and outer lateral rim of the VMN. NPY expression was restricted to the boundaries of the ARC, in close opposition with the third ventricle.
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, C and D, and Table 1). However, when expressed as a percentage of peptide colocalization, the proportions of ARC and VMN GHRH mRNA-containing neurons coexpressing GHS-R rose to 27% and 22%, respectively (Table 1). Only a weak hybridization signal for GHS-R was detected in SRIF mRNA-containing neurons in the ARC, VMN, and PeV nuclei (Fig. 6, B and E); the extent of colocalization was lowest for hypothalamic SRIF neurons compared with NPY and GHRH (Table 1).
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Discussion |
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Of importance to note, however, is that full recovery of the GH response to iv ghrelin during the trough period was evident by 15 min after injection in all groups. This temporal pattern of GH responsiveness to ghrelin differs from that previously found with iv-administered GHRH, wherein GHRH-induced GH release remained blunted throughout the GH trough period (28). The present in vivo results, therefore, suggest that ghrelin is a functional antagonist of SRIF and are in conformity with earlier in vitro studies of the GHSs demonstrating that GHSs behave as functional antagonists of SRIF activity at the level of the pituitary gland (31). Interestingly, the GH response to ghrelin in humans was recently shown to be partially refractory to the inhibitory effect of exogenous SRIF (32).
The effectiveness of low-dose, icv-administered ghrelin to potently stimulate spontaneous pulsatile GH release supports the idea that the central nervous system is an important target for ghrelin’s actions on GH. However, a different pattern of GH responsiveness to ghrelin emerged after central injection; ghrelin failed to significantly increase plasma GH during the trough period at all time points examined. Mathematical modeling of the GH neuroendocrine axis led us to propose that SRIF is released episodically in brain at GH trough times to affect hypothalamic arcuate GHRH neurons (33) via specific SRIF receptor subtypes known to be located on GHRH cells (30). Thus, SRIF may behave as a functional antagonist of ghrelin acting centrally as well as at the level of the pituitary gland. Indeed, central administration of a SRIF analog completely blocked the GH response to GHS given icv (34). On the other hand, we cannot discount the possibility that the weak GH response evoked by icvadministered ghrelin at the second injection was due to down-regulation of GHS/ghrelin receptors. Whether such a mechanism is at play here remains to be established.
In striking contrast to the effects of anti-SRIF serum, immunoneutralization of endogenous GHRH virtually obliterated the GH responses to ghrelin regardless of the time of administration, strongly indicating that the GH response to ghrelin in vivo requires an intact GHRH system. This finding is congruent with previous reports indicating that GHS-induced GH release is attenuated by GHRH antiserum (35, 36, 37). Indeed, there is convincing evidence that GHSs/ghrelin stimulate GH release via GHRH-dependent pathways. Both GHSs (38) and ghrelin (39) have been shown to activate a subpopulation of hypothalamic arcuate neurons, and GHS-induced c-fos expression was observed in GHRH mRNA-containing cells in the ARC (40). Moreover, GHS administration to conscious sheep provokes the release of GHRH into hypophyseal portal blood, but does not influence SRIF release (41). In humans, GHS-induced GH release was shown to be dependent on a functional endogenous GHRH system (42, 43). Together, these results implicate GHRH neurons as targets for ghrelin. We interpret all these findings to indicate that ghrelin does not act by altering hypothalamic SRIF release, but, rather, stimulates GH release via GHRH-dependent pathways.
Our dual chromogenic and autoradiographic in situ hybridization experiments provide anatomical evidence to support this idea. Quantitative analysis of double-labeled cells revealed that GHRH mRNA-containing neurons in ARC and VMN of the hypothalamus expressed the GHS-R, implying that ghrelin may directly modulate GHRH release into hypophyseal portal blood, and thereby influence GH secretion, through interaction with the GHS-R on GHRH-containing neurons. A very weak hybridization signal was detected in SRIF mRNA-containing neurons in the ARC and VMN as well as in the PeV, the primary source of hypophysiotrophic SRIF neurons projecting to the median eminence (44), suggesting that SRIF cells are only minor direct targets for ghrelin’s actions on GH. Nevertheless, it is possible that putative ghrelin effects on SRIF neurons could be mediated by a different GHS-R, as there are studies indicating the possible existence of different subtypes of ghrelin receptors (see Ref. 45 for review). The largest proportion (30%) of GHS-R-expressing cells was colocalized in hypothalamic ARC neurons containing NPY, one of the most potent orexigenic peptides (46, 47). The present findings are partly consistent with an earlier study restricted to the ARC nucleus in adult female Wistar rats (10) in which the percentage of GHRH neurons coexpressing GHS-R mRNA (20–25%) was similar. However, the percentages of NPY and SRIF neurons coexpressing GHS-R mRNA (94% and 30%, respectively) were far greater than those found in the present work. These quantitative differences may be due to strain or gender differences. Moreover, in this previous study only a small number of tissue sections (one or two per rat) were analyzed for NPY and SRIF mRNA-containing neurons coexpressing GHS-R mRNA. In both studies, however, NPY ARC neurons were those that expressed GHS-R in the greatest proportion, when quantified in terms of peptide-expressing neurons or, as in our study, GHS-R-expressing neurons. These anatomical findings provide compelling evidence that ghrelin’s orexigenic effect is mediated at least in part via stimulation of NPY-expressing ARC neurons. Indeed, centrally administered ghrelin increases hypothalamic NPY mRNA expression (48, 49) and pretreatment with either antiserum to NPY (25) or specific NPY receptor antagonists (49) significantly interfered with ghrelin’s appetite-stimulating effect. However, it should be noted that the mechanism by which ghrelin promotes appetite is not entirely clear, as ghrelin induced a small, but significant, increase in food intake in NPY-deficient mice, suggesting that the presence of NPY is not obligatory for the stimulation of food intake (24). As the appetite-stimulating neuropeptide, agouti-related protein, is colocalized with NPY in ARC neurons (50, 51), and its mRNA levels are increased after central administration of ghrelin (23, 48), ghrelin’s effects on energy balance may be mediated through activation of hypothalamic NPY/agouti-related protein pathways. The finding that systemically administered ghrelin induced c-fos expression in a subpopulation of arcuate neurons where NPY cells are located (39) suggests that stomach-derived ghrelin may reach hypothalamic sites by penetrating the blood-brain barrier (perhaps due to its octanoyl moiety adding hydrophobicity to the molecule).
In summary, the results of the present study demonstrate that 1) ghrelin, administered either centrally or peripherally, exerts potent, time-dependent stimulation of spontaneous pulsatile GH secretion under physiological conditions; 2) ghrelin is a functional antagonist of SRIF at the pituitary level, but at this level its GH-releasing activity is not dependent on inhibiting endogenous SRIF release; 3) SRIF antagonizes the actions of ghrelin acting at the level of the pituitary gland; 4) the GH response to ghrelin requires an intact endogenous GHRH system; and 5) hypothalamic ARC GHRH- and NPY-containing neurons, but not PeV SRIF-expressing cells, are major direct targets for ghrelin. The dual actions of ghrelin on GH secretion and food intake in conjunction with the finding that the stomach, rather than the hypothalamus, is the major anatomical origin of ghrelin support the idea that ghrelin may be a critical hormonal signal of nutritional status to the GH neuroendocrine axis serving to integrate energy balance and the growth process. The challenge ahead is to determine whether hypothalamic ghrelin vs. stomach ghrelin, or both, play an important role in genesis of GH pulsatility at the level of either the hypothalamus or pituitary under normal physiological conditions, or whether ghrelin’s role in GH regulation only becomes more active and more prominent during states of negative energy balance.
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Acknowledgments |
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Footnotes |
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Abbreviations: ARC, Arcuate nucleus; AUC, area under the curve; DIG, digoxigenin; GHS, GH secretagogue; GHS-R, GHS receptor; icv, intracerebroventricular; NPY, neuropeptide Y; NS, normal saline; NSS, normal sheep serum; PeV, periventricular nucleus; SRIF, somatostatin; SRIF AS, SRIF antiserum; VMN, ventromedial nucleus.
Received August 14, 2002.
Accepted for publication November 12, 2002.
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