Growth Hormone Secretagogue Receptor mRNA Expression Research Articles

Intestinal expression profiles and hepatic expression of LEAP2, ghrelin and their common receptor, GHSR, in humans

Liver-expressed antimicrobial peptide 2 (LEAP2) and ghrelin have reciprocal effects on their common receptor, the growth hormone secretagogue receptor (GHSR). Ghrelin is considered a gastric hormone and LEAP2 a liver-derived hormone and both have been proposed to be involved in the pathophysiology of obesity and type 2 diabetes (T2D). We investigated the mRNA expression of LEAP2, ghrelin and GHSR along the intestinal tract of individuals with and without TD2, and in the liver of men with and without obesity. Mucosal biopsies retrieved with 30-cm intervals throughout the small intestine and from 7 well-defined locations along the large intestine from 12 individuals with T2D and 12 healthy controls together with liver biopsies from 15 men with obesity and 15 lean men were subjected to bulk transcriptomics analysis. Both in individuals with and without T2D, mRNA expression of LEAP2 increased through the small intestine until dropping at the ileocecal valve, with little LEAP2 mRNA expression in the large intestine. Pronounced LEAP2 expression was observed in the liver of men with and without obesity. Robust ghrelin mRNA expression was observed in the duodenum of individuals with and without T2D, gradually decreasing along the small intestine with little expression in the large intestine. Ghrelin mRNA expression was not detected in the liver biopsies, and GHSR mRNA expression was not. In conclusion, we provide unique mRNA expression profiles of LEAP2, ghrelin and GHSR along the human intestinal tract showing no T2D-associated changes, and in the liver showing no differences between men with and without obesity.

Energetic demands of lactation produce an increase in the expression of growth hormone secretagogue receptor in the hypothalamus and ventral tegmental area of the rat despite a reduction in circulating ghrelin.

Lactating rats show changes in the secretion of hormones and brain signals that promote hyperphagia and facilitate the production of milk. Little is known, however, about the role of ghrelin in the mechanisms sustaining lactational hyperphagia. Here, we used Wistar female rats that underwent surgery to sever the galactophores to prevent milk delivery (GC rats) and decrease the energetic drain of milk delivery. We compared plasma acyl-ghrelin concentrations and growth hormone secretagogue receptor (GHSR) mRNA expression in different brain regions of GC rats with those of sham operated lactating and nonlactating rats. Additional lactating and nonlactating rats were implanted with cannulae aimed at the lateral ventricles and were used to compare feeding responses to central ghrelin or GHSR antagonist infusions to those of nonlactating rats receiving similar infusions on day 14-16 postpartum (pp). Results show lower plasma acyl-ghrelin concentrations on day 15 pp sham operated lactating rats compared to GC or nonlactating rats. These changes occur in association with increased GHSR mRNA expression in the hypothalamic arcuate nucleus (ARC) and ventral tegmental area (VTA) of sham operated lactating rats. Despite lactational hyperphagia, infusions of ghrelin (0.25 or 1μg) resulted in similar increases in food intake in lactating and nonlactating rats. In addition, infusions of the GHSR antagonist JMV3002 (4μg in 1μl of vehicle) produced greater suppression of food intake in lactating rats than in nonlactating rats. These data suggest that, despite lower plasma ghrelin, the energetic drain of lactation increases sensitivity to the orexigenic effects of ghrelin in brain regions important for food intake and energy balance, and these events are associated with lactational hyperphagia.

Metabolic stress leads to divergent changes in the ghrelinergic system in goldfish (Carassius auratus) gonads

Various endocrine factors that regulate energy homeostasis are also implicated in the reproductive physiology of mammals. However, the hormonal link between metabolism and reproduction in fish is poorly understood. Ghrelin is a multifunctional hormone with both metabolic and reproductive roles in vertebrates. Post-translational acylation by ghrelin-O-acyltransferase (GOAT) is critical for its biological actions. The expression of ghrelin, ghrelin or growth hormone secretagogue receptor (GHSR), and GOAT (which forms the ghrelinergic system) in fish under metabolic stress remains unclear. In this research, we used RT-qPCR and Western blot analysis to determine the expression of the ghrelinergic system in goldfish (during the reproductively active phase) hypothalamus and gonads under 7 and 28 days of fasting. We found a significant increase in preproghrelin mRNA expresson in the ovary, and GOAT mRNA expression in the testis of goldfish deprived of food for 7 days. In fish deprived of food for 28 days, preproghrelin, GHSR and GOAT mRNA expression was significantly increased in the hypothalamus of male goldfish. Such differences were not observed in the hypothalamus of female fish, and in the testis of 28 days fasted fish. Meanwhile, preproghrelin, GHSR, and GOAT expression (both mRNA and protein) was significantly increased in the ovary of female fish fasted for 28 days. Ghrelin has been shown to suppress oocyte maturation in fish. The upregulation of a system that has ovarian inbititory roles suggests a role for ghrelin in maintaining reduced reproductive capability during metabolically challenging periods.

Effects of IBDV infection on expression of ghrelin and ghrelin-related genes in chicken

Ghrelin is a peptide hormone that plays a modulatory role in the immune system. Studies have demonstrated that mammal ghrelin level is influenced by pathological status. However, it has not been reported whether chicken ghrelin level changes during pathogen infection. This study was designed to investigate changes of ghrelin levels in chickens infected with infectious bursal disease virus (IBDV) and to explore the relationship between ghrelin changes and bursal damage, and inflammatory cells infiltration induced by IBDV. The results showed that (1) plasma ghrelin concentration increased after IBDV infection. It reached a peak at 10443.6 ± 2612.9 pg/mL on 2 dpi, which was about 100-fold as high as that of the control. Then it decreased sharply on 3 dpi, which was only 31.7% as that of 2 dpi, and remained stable until 5 dpi. Meanwhile, ghrelin and ghrelin-related gene, ghrelin-o-acyltransferase (GOAT), and growth hormone secretagogue receptor (GHSR) mRNA expression levels in bursa were also increased after IBDV infection, and reached the peak on 2 dpi at 149, 28.8, and 117.2-fold higher than that of the control, respectively. Then they decreased and remained at a higher status. Correlation analysis showed that plasma ghrelin concentration and ghrelin, GOAT, and GHSR mRNA expressions in bursa were strongly associated with IBDV VP2 mRNA expression in bursa. (2) The damage of bursa was the most severe on 5 dpi with a histopathological score of 12. It had no direct correlation with plasma ghrelin level and ghrelin, GOAT, and GHSR mRNA expressions in bursa. However, the number of inflammatory cells infiltrating into bursa, which was the highest on 2 and 3 dpi, showed significant a positive correlation with the ghrelin and GHSR mRNA expression. Presumably chicken ghrelin may function as an anti-inflammatory factor. In conclusion, IBDV infection upregulates the expression of ghrelin and ghrelin-related gene in chickens, and chicken ghrelin may play an important regulatory role during pathogen infection.

Development and initial characterization of a novel ghrelin receptor CRISPR/Cas9 knockout wistar rat model.

Background/objectivesGhrelin, a stomach-derived hormone implicated in numerous behaviors including feeding, reward, stress, and addictive behaviors, acts through binding to the growth hormone secretagogue receptor (GHSR). Here, we present the development, verification and initial characterization of a novel GHSR knockout (KO) Wistar rat model created with CRISPR genome editing.MethodsUsing CRISPR/Cas9, we developed a GHSR knockout (KO) in a Wistar background. Loss of GHSR mRNA expression was histologically verified using RNAscope in wild-type WT (n = 2) and KO (n = 2) rats. We tested the effects of intraperitoneal acyl-ghrelin administration on food consumption and plasma growth hormone (GH) concentrations in WT (n = 8) and KO (n = 8) rats. We also analyzed locomotion, food consumption, and body fat composition in these animals. Body weight was monitored from early development to adulthood.ResultsThe RNAscope analysis revealed an abundance of GHSR mRNA expression in the hypothalamus, midbrain, and hippocampus in WTs, and no observed probe binding in KOs. Ghrelin administration increased plasma GH levels (p = 0.0067) and food consumption (p = 0.0448) in WT rats but not KOs. KO rats consumed less food overall at basal conditions and weighed significantly less compared with WTs throughout development (p = 0.0001). Compared with WTs, KOs presented higher concentrations of brown adipose tissue (BAT) (p = 0.0322).ConclusionsWe have verified GHSR deletion in our KO model using histological, physiological, neuroendocrinological and behavioral measures. Our findings indicate that GHSR deletion in rats is not only associated with a lack of response to ghrelin, but also associated with decreases in daily food consumption and body growth, and increases in BAT. This GHSR KO Wistar rat model provides a novel tool for studying the role of the ghrelin system in obesity and in a wide range of medical and neuropsychiatric disorders.

Correlation of ghrelin and growth hormone secretagogue receptor expression with clinical features in human pituitary adenomas.

Ghrelin, as a brain-gut peptide, has growth hormone (GH)-releasing and appetite-inducing activities and a widespread tissue distribution. Furthermore, ghrelin is an endogenous ligand of the GH secretagogue receptor (GHSR), and both ghrelin and GHSR are expressed in the pituitary; however, the data regarding the expression of ghrelin and GHSR in pituitary adenomas are divergent and conflicting. In the present study, therefore, the expression of ghrelin and GHSR was examined in the full spectrum of human pituitary adenoma subtypes (n=34) and in normal pituitary tissue (n=3). The mRNA and protein expression levels were quantified using a competitive reverse transcription-polymerase chain reaction and western blotting and the correlation of the results with the clinical parameters was assessed. mRNA and protein expression of ghrelin and GHSR was detected in all samples with the highest mean level in GH adenomas, a moderate level in clinically non-functioning adenomas and the lowest level in adrenocorticotropin adenomas. A significant correlation between the ghrelin and GHSR mRNA expression levels was observed in the GH adenomas (n=12) (r=0.8435, P=0.0006). The ghrelin mRNA expression level in the GH adenomas correlated positively with the basic serum GH level (n=12) (r=0.6488, P=0.0225). Furthermore, the mean level of ghrelin mRNA expression was significantly higher in invasive adenomas than in noninvasive adenomas (P<0.01). Collectively, the results of the study provided evidence that ghrelin and GHSR are expressed in the various subtypes of pituitary adenoma, with specific overexpression in GH adenomas. The study suggests that the binding of ghrelin to GHSR promotes the secretion of GH and plays an important role in the development of GH adenomas via autocrine and/or paracrine effects.

Neuroanatomical characterization of a growth hormone secretagogue receptor-green fluorescent protein reporter mouse.

Growth hormone secretagogue receptor (GHSR) 1a is the only molecularly identified receptor for ghrelin, mediating ghrelin-related effects on eating, body weight, and blood glucose control, among others. The expression pattern of GHSR within the brain has been assessed previously by several neuroanatomical techniques. However, inherent limitations to these techniques and the lack of reliable anti-GHSR antibodies and reporter rodent models that identify GHSR-containing neurons have prevented a more comprehensive functional characterization of ghrelin-responsive neurons. Here we have systematically characterized the brain expression of an enhanced green fluorescence protein (eGFP) transgene controlled by the Ghsr promoter in a recently reported GHSR reporter mouse. Expression of eGFP in coronal brain sections was compared with GHSR mRNA expression detected in the same sections by in situ hybridization histochemistry. eGFP immunoreactivity was detected in several areas, including the prefrontal cortex, insular cortex, olfactory bulb, amygdala, and hippocampus, which showed no or low GHSR mRNA expression. In contrast, eGFP expression was low in several midbrain regions and in several hypothalamic nuclei, particularly the arcuate nucleus, where robust GHSR mRNA expression has been well-characterized. eGFP expression in several brainstem nuclei showed high to moderate degrees of colocalization with GHSR mRNA labeling. Further quantitative PCR and electrophysiological analyses of eGFP-labeled hippocampal cells confirmed faithful expression of eGFP within GHSR-containing, ghrelin-responsive neurons. In summary, the GHSR-eGFP reporter mouse model may be a useful tool for studying GHSR function, particularly within the brainstem and hippocampus; however, it underrepresents GHSR expression in nuclei within the hypothalamus and midbrain.

Ghrelin Decreases Firing Activity of Gonadotropin-Releasing Hormone (GnRH) Neurons in an Estrous Cycle and Endocannabinoid Signaling Dependent Manner

The orexigenic peptide, ghrelin is known to influence function of GnRH neurons, however, the direct effects of the hormone upon these neurons have not been explored, yet. The present study was undertaken to reveal expression of growth hormone secretagogue receptor (GHS-R) in GnRH neurons and elucidate the mechanisms of ghrelin actions upon them. Ca2+-imaging revealed a ghrelin-triggered increase of the Ca2+-content in GT1-7 neurons kept in a steroid-free medium, which was abolished by GHS-R-antagonist JMV2959 (10µM) suggesting direct action of ghrelin. Estradiol (1nM) eliminated the ghrelin-evoked rise of Ca2+-content, indicating the estradiol dependency of the process. Expression of GHS-R mRNA was then confirmed in GnRH-GFP neurons of transgenic mice by single cell RT-PCR. Firing rate and burst frequency of GnRH-GFP neurons were lower in metestrous than proestrous mice. Ghrelin (40nM-4μM) administration resulted in a decreased firing rate and burst frequency of GnRH neurons in metestrous, but not in proestrous mice. Ghrelin also decreased the firing rate of GnRH neurons in males. The ghrelin-evoked alterations of the firing parameters were prevented by JMV2959, supporting the receptor-specific actions of ghrelin on GnRH neurons. In metestrous mice, ghrelin decreased the frequency of GABAergic mPSCs in GnRH neurons. Effects of ghrelin were abolished by the cannabinoid receptor type-1 (CB1) antagonist AM251 (1µM) and the intracellularly applied DAG-lipase inhibitor THL (10µM), indicating the involvement of retrograde endocannabinoid signaling. These findings demonstrate that ghrelin exerts direct regulatory effects on GnRH neurons via GHS-R, and modulates the firing of GnRH neurons in an ovarian-cycle and endocannabinoid dependent manner.

A relationship between motilin and growth hormone secretagogue receptors

The motilin receptor (MR) belongs to a family of Class I G protein-coupled receptors that also includes growth hormone secretagogue receptor (GHSR). Their potentially unique structure and the molecular basis of their binding and activation are not yet clear. We previously reported that the perimembranous residues in the predicted extracellular loops and amino-terminal tail of the MR were important for responses to the natural peptide ligand, motilin, and the transmembrane domains of the MR were important for a non-peptidyl ligand, erythromycin. We also reported that the perimembranous residues in the second extracellular loop of the GHSR were critical for natural ligand ghrelin binding and activity. The MR is 52% identical to GHSR, with 86% sequence identity in the transmembrane domains. In the current work, to gain insight into a relationship between MR and GHSR, we studied functional responses to motilin, erythromycin and ghrelin of expression cells of chimeric constructs of MR and GHSR and co-expression cells of both MR and GHSR. We also generated human MR transgenic mice, and clarified a relationship between motilin and ghrelin. MR(1–62)/GHSR(68–366) construct responded only to ghrelin, MR(1–102)/GHSR(108–366) responded to ghrelin and erythromycin, and MR(1–129)/GHSR(135–366) and MR(1–178)/GHSR(184–366) responded to erythromycin, while GHSR(1–183)/MR(179–412) responded to neither motilin, erythromycin nor ghrelin. MR and GHSR co-expression cells have no additional responses to these ligands. Motilin or erythromycin administration to human MR transgenic mice resulted in a decrease of serum acyl-ghrelin levels, while MR and GHSR mRNA expression in the gastrointestinal tracts were not changed. These data suggested that in species expressing both motilin–MR and ghrelin–GHSR, there is a compensatory relationship in vivo.

Phenotypical and functional study of ghrelin and its receptor in the pathogenesis of Crohnʼs disease

Ghrelin, a novel endogenous ligand for the growth hormone secretagogue receptor (GHSR), has been demonstrated to possess multiple functions including antiinflammatory effects. The aim of this study was to investigate the expression of ghrelin and GHSR and the function of ghrelin in inflammatory bowel disease (IBD). The expression of ghrelin and GHSR mRNA was quantified in mucosal biopsy specimens from 9 controls, 15 patients with Crohn's disease (CD), and 15 patients with ulcerative colitis (UC) using quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). The locations of ghrelin and GHSR were investigated immunohistochemically in surgically resected specimens. We also evaluated the percentage of GHSR-positive peripheral blood mononuclear cells (PBMCs) in healthy controls and patients with CD by flow cytometry. In addition, we investigated the immunoregulatory function of ghrelin in peripheral blood T cells. Ghrelin mRNA levels in colonic mucosa of IBD were higher than control level. The GHSR-1a mRNA level in active CD was also significantly higher than the control level. Ghrelin and GHSR-1a were expressed on CD3- and CD68-positive cells. The percentage of GHSR-1a-positive peripheral blood T cells in patients with CD was significantly higher than the control level. Stimulation of human T cells with ghrelin increased levels of IL-4 and IL-13 proteins and decreased levels of IFN-gamma protein. Reactivity to ghrelin was low in CD compared with the control level. Our findings demonstrate that ghrelin may play an important role in the immune system in CD. The dysregulation of reactivity of T cells induced by ghrelin suggests that ghrelin might participate in the pathogenesis of CD.

S1663 Enhanced Ghrelin Production and Secretion and Reduced Expression of Ghrelin Receptor in Rat Stomach with Delayed Gastric Emptying

Background. Ghrelin is a gastrointestinal peptide that stimulates growth hormone secretion, food intake, body weight gain and upper gastrointestinal motility. Ghrelin has distinct effects on gastrointestinal motility, which may be mediated through the growth hormone secretagogue receptor (GHS-R) on the vagus nerve and enteric nerve endings. We previously reported that plasma ghrelin levels were significantly higher in patients with dysmotilitylike functional dyspepsia (Aliment Pharmacol Ther. S4:104-110, 2006). Plasma ghrelin was also increased in obese patients after gastric banding operation (Obese Surg. 15, 1024-9, 2005). The aim of the present study was to investigate the influence of delayed gastric emptying caused by surgical manipulation on the dynamics of ghrelin and GHS expression in rats.Methods. 7-week-old male Sprague-Dawley rats with or without duodenal strictures were used as models of delayed gastric emptying. After 24 hours' fasting, the rats were divided into two groups, as follows; Control group (n=14; sham operation with abdominal incision alone), impaired gastric emptying group (n=14; proximal duodenal stricture with an 18 Fr.-Nelaton catheter) As weight loss is known to increase the circulating levels of ghrelin, a pair-feeding method was applied to equalize the body weight. Rats were sacrificed 2 weeks after the operation after the 24 hours' food deprivation. Gastric emptying rate was evaluated by the phenol red method. Plasma and gastric levels of ghrelin levels were measured by radioimmunoassay. Gastric expressions of preproghrelin mRNA and GHS-R mRNA were evaluated by quantitative RT-PCR, and immunohistochemistry was performed to determine gastric ghrelin localization. Results. No significant differences in the body weight were observed among two groups. The gastric emptying rate in groups with duodenal stricture was significantly lower compared with control group (9.8%, 61.0%, p<0.05). Rats with duodenal strictures showed significant increase in the preproghrelin mRNA expression (p<0.05) and density of ghrelin positive cells in the stomach (p<0.01), significant decrease of gastric total ghrelin levels (p<0.05), and increase of the plasma total ghrelin levels (p<0.01), while they showed a significant decrease in the levels of GHS-R mRNA expression (p<0.05). Conclusion. The present study has provided the first evidence that production and secretion of gastric ghrelin is enhanced in rats with delayed gastric emptying. It suggests that deregulation of gastric accommodation and emptying would induce the enhancement of ghrelin dynamics and the down-regulation of GHS-R.

GH-releasing peptides improve cardiac dysfunction and cachexia and suppress stress-related hormones and cardiomyocyte apoptosis in rats with heart failure

Growth hormone (GH)-releasing peptides (GHRP), a class of synthetic peptidyl GH secretagogues, have been reported to exert a cardioprotective effect on cardiac ischemia. However, whether GHRP have a beneficial effect on chronic heart failure (CHF) is unclear, and the present work aims to clarify this issue. At 9 wk after pressure-overload CHF was created by abdominal aortic banding in rats, one of four variants of GHRP (GHRP-1, -2, and -6 and hexarelin, 100 mug/kg) or saline was injected subcutaneously twice a day for 3 wk. Echocardiography and cardiac catheterization were performed to monitor cardiac function and obtain blood samples for hormone assay. GHRP treatment significantly improved left ventricular (LV) function and remodeling in CHF rats, as indicated by increased LV ejection fraction, LV end-systolic pressure, and diastolic posterior wall thickness and decreased LV end-diastolic pressure and LV end-diastolic dimension. GHRP also significantly alleviated development of cardiac cachexia, as shown by increases in body weight and tibial length in CHF rats. Plasma CA, renin, ANG II, aldosterone, endothelin-1, and atrial natriuretic peptide were significantly elevated in CHF rats but were significantly decreased in GHRP-treated CHF rats. GHRP suppressed cardiomyocyte apoptosis and increased cardiac GH secretagogue receptor mRNA expression in CHF rats. GHRP also decreased myocardial creatine kinase release in hypophysectomized rats subjected to acute myocardial ischemia. We conclude that chronic administration of GHRP alleviates LV dysfunction, pathological remodeling, and cardiac cachexia in CHF rats, at least in part by suppressing stress-induced neurohormonal activations and cardiomyocyte apoptosis.

Ghrelin is expressed in a novel endocrine cell type in developing rat islets and inhibits insulin secretion from INS-1 (832/13) cells.

Ghrelin is produced mainly by endocrine cells in the stomach and is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). It also influences feeding behavior, metabolic regulation, and energy balance. It affects islet hormone secretion, and expression of ghrelin and GHS-R in the pancreas has been reported. In human islets, ghrelin expression is highest pre- and neonatally. We examined ghrelin and GHS-R in rat islets during development with immunocytochemistry and in situ hybridization. We also studied the effect of ghrelin on insulin secretion from INS-1 (832/13) cells and the expression of GHS-R in these cells. We found ghrelin expression in rat islet endocrine cells from mid-gestation to 1 month postnatally. Islet expression of GHS-R mRNA was detected from late fetal stages to adult. The onset of islet ghrelin expression preceded that of gastric ghrelin. Islet ghrelin cells constitute a separate and novel islet cell population throughout development. However, during a short perinatal period a minor subpopulation of the ghrelin cells co-expressed glucagon or pancreatic polypeptide. Markers for cell lineage, proliferation, and duct cells revealed that the ghrelin cells proliferate, originate from duct cells, and share lineage with glucagon cells. Ghrelin dose-dependently inhibited glucose-stimulated insulin secretion from INS-1 (832/13) cells, and GHS-R was detected in the cells. We conclude that ghrelin is expressed in a novel developmentally regulated endocrine islet cell type in the rat pancreas and that ghrelin inhibits glucose-stimulated insulin secretion via a direct effect on the beta-cell.

Ghrelin mRNA and GH secretagogue receptor mRNA in human GH-producing pituitary adenomas is affected by mutations in the alpha subunit of G protein.

Ghrelin and its receptor, growth hormone secretagogue (GHS) receptor (GHSR), are expressed in the normal pituitary gland and various types of pituitary adenoma. Somatic mutations in the subunit of Gs alpha protein (gsp), which led to a constitutive activation of adenylyl cyclase, are reported in GH-producing pituitary adenomas. We analysed the relationship between ghrelin mRNA and GHSR mRNA expression levels in gsp mutation-positive and -negative GH-producing pituitary adenomas. Pituitary adenoma tissue was obtained at surgery from 20 patients with acromegaly. The expression levels of human ghrelin mRNA and GHSR mRNA were quantified using a competitive RT-PCR method. To detect the gsp mutations, amplified Gs alpha subunit cDNA fragments were sequenced directly using RT-PCR method. There was no significant difference in the expression of ghrelin mRNA between mutation-positive and -negative adenomas. The expression of GHSR mRNA was significantly lower in gsp mutation-positive than -negative adenomas. There was a significant negative correlation between the levels of ghrelin mRNA and GHSR mRNA expression in mutation-negative adenomas; no such correlation was found in mutation-positive adenomas. These results suggest that GHSR mRNA expression is downregulated by ghrelin in gsp mutation-negative GH-producing pituitary adenomas, and that changes in intracellular signalling pathways in gsp mutation-positive GH-producing pituitary adenomas affect the expression of G protein-coupled receptors such as GHSR. The absence of negative correlation between ghrelin and GHSR expression might be induced by lowered GHSR expression in gsp mutation-positive GH-producing adenomas.

Pattern-dependent suppression of growth hormone (GH) pulsatility by ghrelin and GH-releasing peptide-6 in moderately GH-deficient rats.

The peptide hormone ghrelin binds to the GH secretagogue receptor (GHS-R), stimulates GH secretion, and promotes adipogenesis. However, continuous GHS infusion does not stimulate skeletal growth and is associated with desensitization to further GH secretagogue treatment. In this study, 7-d intermittent (i.e. every 3 h) infusion of ghrelin, or the GH secretagogue, GH-releasing peptide-6, in the moderately GH- deficient transgenic growth-retarded rat, augmented GH secretion, leading to a sustained acceleration in skeletal growth. In contrast, continuous infusion of ghrelin, or GH-releasing peptide-6, suppressed the amplitude of spontaneous GH secretory episodes and produced only a transient increase in body weight gain. The reduction in GH secretion seen with continuous GHS-R activation was not associated with a desensitization of the pituitary to GH-releasing factor or to down-regulation of hypothalamic GHS-R mRNA expression. Continuous ghrelin treatment elicited an increase in somatostatin mRNA expression in the periventricular nuclei. Thus, exposure to continuously elevated circulating ghrelin may be responsible for the suppression of GH secretion reported in rats after prolonged starvation.

Growth hormone secretagogue receptor expression in the cells of the stomach-projected afferent nerve in the rat nodose ganglion

Growth hormone secretagogue receptor (GHS-R) is widely expressed in various regions of the body, such as the brain, pituitary gland, heart and gastrointestinal tract. Recently, ghrelin, an endogenous ligand for GHS-R, was found in the rat stomach, and several studies have suggested that ghrelin acts via the vagal afferent nerve. In this study, we studied the expression of GHS-R mRNA in the rat nodose ganglion by reverse transcriptase-polymerase chain reaction and in situ hybridization, the results of which clearly demonstrated the presence of GHS-R mRNA and GHS-R producing cells in the rat nodose ganglion. We also studied the retrograde tracing of nodose ganglion cells to the stomach and found that some GHS-R mRNA-expressing cells contain the retrograde rebelling. Our results provide direct morphological evidence that GHS-R is produced in afferent neurons of the nodose ganglion and suggest that ghrelin signals from the stomach are transmitted to the brain via vagal afferent nerves.

Presence of ghrelin in normal and adenomatous human pituitary.

Recently, an endogenous ligand has been described for the growth hormone secretagogue receptor (GHS-R), named ghrelin. It was originally isolated from the stomach, but it is also present in the hypothalamus, where the highest concentration of GHS-R has been detected. It is well established that synthetic GHSs exert their effects on the growth hormone (GH) axis principally via the hypothalamus, although they are also able to stimulate GH release directly from the pituitary. We have previously demonstrated the presence of GHS-R mRNA expression in normal and abnormal human pituitary. We have therefore now investigated the expression of the newly recognized endogenous ligand in rat as well as in human pituitary. We readily detected ghrelin mRNA message in normal rat pituitary using reverse transcriptase polymerase chain reaction with published primers. We then designed primers to the corresponding region on the human ghrelin sequence and successfully detected mRNA message in normal human pituitary, as well as in somatotroph, lactotroph, corticotroph, thyrotroph, and nonfunctioning adenomas. We confirmed the expected polymerase chain reaction product by direct sequencing. In conclusion, we suggest that in addition to the probable hypothalamic effects of ghrelin, the peptide is synthesized locally within the pituitary gland, where it may influence the release of GH in an autocrine or paracrine manner.

Developmentally and Regionally Regulated Expression of Growth Hormone Secretagogue Receptor mRNA in Rat Brain and Pituitary Gland

Distribution and development of growth hormone secretagogue receptor (GHS-R) mRNA expression in rat brain and pituitary gland were examined using ribonuclease protection assay. In adult male rats, GHS-R mRNA levels were highest in the pituitary gland, whereas those in the hypothalamus and hippocampus were 57 and 30% of those in the pituitary gland, respectively. Less abundant but detectable levels of GHS-R mRNA were found in the midbrain, pons, and medulla oblongata, but expression was barely detectable in the cerebellum and cerebral cortex. The expression of GHS-R mRNA was detected at late gestation (embryonic day 19) in the pituitary gland, hypothalamus, and brainstem. The mRNA levels increased with age in the pituitary gland, and decreased postnatally in the brainstem, while they remained constant in the hypothalamus during development. In contrast, GHS-R mRNA was not detectable in the hippocampus during the fetal period, but was first detected on postnatal day 7. Expression of GHS-R mRNA was also examined in the spontaneous dwarf rat (SDR), a model for isolated GH deficiency, to examine alterations in GHS-R mRNA expression in a GH-deficient state. GHS-R mRNA levels in the pituitary gland of SDRs were higher than those of control rats, suggesting negative regulation of GHS-R mRNA by GH in this region. GHS-R mRNA levels increased in the hypothalamus of female, but not in male SDRs. In contrast, GHS-R mRNA levels were not affected by GH in the brainstem and hippocampus. These results indicate that region-specific, developmentally regulated expression of GHS-R mRNA may reflect divergent physiological roles of GHS/GHS-R in distinct regions of the central nervous system and the pituitary gland.