Phosphatidylinositol-3-kinase/Akt/glycogen synthase kinase-3 beta and ERK1/2 pathways mediate protective effects of acylated and unacylated ghrelin against oxygen-glucose deprivation-induced apoptosis in primary rat cortical neuronal cells

Only acylated ghrelin (AG) binds GH secretagog receptor 1a (GHS-R1a) and has central endocrine activities. An anti-apoptotic effect of AG in neuronal cells has recently been reported. However, whether there is a neuroprotective effect of unacylated ghrelin (UAG), the most abundant form of ghrelin in plasma, is still unknown. Therefore, we investigated whether UAG was neuroprotective against ischemic neuronal injury using primary cultured rat cortical neurons exposed to oxygen and glucose deprivation (OGD). Both AG and UAG inhibited OGD-induced apoptosis. Exposure of cells to the receptor-specific antagonist D-Lys-3-GHRH-6 abolished the protective effects of AG against OGD, whereas those of UAG were preserved, suggesting the involvement of a receptor that is distinct from GHS-R1a. Chemical inhibition of MAPK and phosphatidylinositol-3-kinase (PI3K) blocked the anti-apoptotic effects of AG and UAG. Ghrelin siRNA enhanced apoptosis either during OGD or even in normoxic conditions. The protective effects of AG and UAG were accompanied by an increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2, Akt, and glycogen synthase kinase-3beta (GSK-3beta). Furthermore, treatment of cells with AG or UAG resulted in nuclear translocation of beta-catenin. In addition, both AG and UAG increased the Bcl-2/Bax ratio, prevented cytochrome c release, and inhibited caspase-3 activation. The data indicate that, independent of acylation, ghrelin can function as a neuroprotective agent that inhibits apoptotic pathways. These effects may be mediated via activation of the MAPK and PI3K/Akt pathways. Our data also suggest that PI3K/Akt-mediated inactivation of GSK-3beta and stabilization of beta-catenin contribute to the anti-apoptotic effects of ghrelin.     

Ghrelin induces cardiac lineage differentiation of human embryonic stem cells through ERK1/2 pathway

Background

Ghrelin, an endogenous ligand for growth hormone secretagogue receptor (GHS-R), shows cardioprotective activity and regulates the differentiation of several mesoderm-derived cells, including myocytes, adipocytes and osteoblasts. The effect of ghrelin on cardiogenesis and its underlying mechanism, however, have not been studied in detail.

Methods

The effects of ghrelin on cardiomyocyte differentiation were tested both in human embryonic stem cells (hESCs) cultured in embryoid body (EB)-based differentiation protocol, and in hESCs transplanted into rat hearts. The signaling mechanisms of ghrelin were further investigated under the EB-based culture condition.

Results

The generation of beating EBs and the expression of cardiac-specific markers including cardiac troponin I (cTnI) and α-myosin heavy chain (α-MHC) were 2 to 3-fold upregulated by ghrelin. Although GHS-R1α protein was expressed in differentiated EBs, the effects of exogenous ghrelin were unchanged by D-[lys³]-GHRP-6, a specific GHS-R1α antagonist. Moreover, des-acyl ghrelin, which does not bind to GHS-R1α, displayed similar effects with ghrelin. Importantly, activation of ERK1/2, but not Akt, was induced by ghrelin in the newly-formed EBs, and the ghrelin-induced effects of cardiomyocyte differentiation were abolished by adding specific ERK1/2 inhibitor PD98059, but not specific PI3K inhibitor Wortmannin. In addition, ghrelin promoted the differentiation of grafted hESCs into Sox9- and Flk1-positive mesodermal/cardiac progenitor cells in rat hearts.

Conclusions

These results suggest that ghrelin induces cardiomyocyte differentiation from hESCs via the activation of the ERK1/2 signaling pathway. Our study, therefore, indicates that using ghrelin may be an effective strategy to promote the differentiation of hESCs into cardiomyocytes.     

Unacylated ghrelin is not a functional antagonist but a full agonist of the type 1a growth hormone secretagogue receptor (GHS-R)

Recent findings demonstrate that the effects of ghrelin can be abrogated by co-administered unacylated ghrelin (UAG). Since the general consensus is that UAG does not interact with the type 1a growth hormone secretagogue receptor (GHS-R), a possible mechanism of action for this antagonistic effect is via another receptor. However, functional antagonism of the GHS-R by UAG has not been explored extensively. In this study we used human GHS-R and aequorin expressing CHO-K1 cells to measure [Ca(2+)](i) following treatment with UAG. UAG at up to 10(-5)M did not antagonize ghrelin induced [Ca(2+)](i). However, UAG was found to be a full agonist of the GHS-R with an EC(50) of between 1.6 and 2 microM using this in vitro system. Correspondingly, UAG displaced radio-labeled ghrelin from the GHS-R with an IC(50) of 13 microM. In addition, GHS-R antagonists were found to block UAG induced [Ca(2+)](i) with approximately similar potency to their effect on ghrelin activation of the GHS-R, suggesting a similar mode of action. These findings demonstrate in a defined system that UAG does not antagonize activation of the GHS-R by ghrelin. But our findings also emphasize the importance of assessing the concentration of UAG used in both in vitro and in vivo experimental systems that are aimed at examining GHS-R independent effects. Where local concentrations of UAG may reach the high nanomolar to micromolar range, assignment of GHS-R independent effects should be made with caution.     

Ghrelin stimulates, whereas des-octanoyl ghrelin inhibits, glucose output by primary hepatocytes

Ghrelin exerts various metabolic activities, including regulation of glucose levels in humans. To verify whether the glucose response to ghrelin reflects a modulation of an insulin-independent hepatic phenomenon, we studied glucose output by primary porcine hepatocytes in suspension culture, after incubation with acylated ghrelin (AG), unacylated ghrelin (UAG), and hexarelin (HEX). AG induced glucose output dose dependently after 20 min of incubation (P < 0.001), whereas HEX, a GH secretagogue receptor type 1a (GHS-R1a) agonist, had no effect. UAG inhibited glucose release also dose dependently and after 20 min (P < 0.001). Moreover, UAG completely reversed AG-induced glucose output (P < 0.01). Using real-time PCR, GHS-R1a gene expression was undetectable in all the hepatocyte preparations studied. The lack of efficacy of HEX, the efficacy of UAG, and the absence of GHS-R1a expression indicate the involvement of a yet uncharacterized ghrelin receptor type. In conclusion, glucose output by primary hepatocytes is time- and dose-dependently stimulated by AG and inhibited by UAG. Moreover, UAG counteracts the stimulatory effect of AG on glucose release. These actions might be mediated by a different receptor than GHS-R1a, and apparently, we must consider AG and UAG as separate hormones that can modify each other's actions on glucose handling, at least in the liver.     

Unacylated ghrelin is active on the INS-1E rat insulinoma cell line independently of the growth hormone secretagogue receptor type 1a and the corticotropin releasing factor 2 receptor

Both unacylated ghrelin (UAG) and acylated ghrelin (AG) exert metabolic effects. To investigate the interactions between AG and UAG on ghrelin receptors we evaluated the effects of AG and UAG on INS-1E rat insulinoma cells, using insulin secretion after 30min static incubation as a read-out. A possible involvement of the growth hormone secretagogue receptor type 1a (GHS-R1a) or the corticotropin-releasing factor 2 (CRF2) receptor (CRF2R), as a putative receptor for UAG, was also studied determining their mRNA expression and the functional effects of receptor antagonists on insulin release. Both UAG and AG stimulated insulin release dose-dependently in the nanomolar range. The AG-induced insulin output was antagonized by two GHS-R1a antagonists ([d-Lys(3)]GHRP-6 and BIM28163), which did not block UAG actions. These effects occurred in the presence of low levels of GHS-R1a mRNA. Neither CRF2R expression nor effects of the CRF2R antagonist (astressin(2)B) on insulin output were observed. In conclusion, we provide a sensitive and reproducible assay for specific effects of UAG, which in this study is responsible for insulin release by INS-1E cells. Our data support the existence of a specific receptor for UAG, other than the CRF2R and GHS-R1a. The stimulatory effect on insulin secretion by AG in this cell line is mediated by the GHS-R1a.     

Acylated and unacylated ghrelin promote proliferation and inhibit apoptosis of pancreatic beta-cells and human islets: involvement of 3',5'-cyclic adenosine monophosphate/protein kinase A, extracellular signal-regulated kinase 1/2, and phosphatidyl inositol 3-Kinase/Akt signaling

Among its pleiotropic actions, ghrelin modulates insulin secretion and glucose metabolism. Herein we investigated the role of ghrelin in pancreatic beta-cell proliferation and apoptosis induced by serum starvation or interferon (IFN)-gamma/TNF-alpha, whose synergism is a major cause for beta-cell destruction in type I diabetes. HIT-T15 beta-cells expressed ghrelin but not ghrelin receptor (GRLN-R), which binds acylated ghrelin (AG) only. However, both unacylated ghrelin (UAG) and AG recognized common high-affinity binding sites on these cells. Either AG or UAG stimulated cell proliferation through Galpha(s) protein and prevented serum starvation- and IFN-gamma/TNF-alpha-induced apoptosis. Antighrelin antibody enhanced apoptosis in either the presence or absence of serum but not cytokines. AG and UAG even up-regulated intracellular cAMP. Blockade of adenylyl cyclase/cAMP/protein kinase A signaling prevented the ghrelin cytoprotective effect. AG and UAG also activated phosphatidyl inositol 3-kinase (PI3K)/Akt and ERK1/2, whereas PI3K and MAPK inhibitors counteracted the ghrelin antiapoptotic effect. Furthermore, AG and UAG stimulated insulin secretion from HIT-T15 cells. In INS-1E beta-cells, which express GRLN-R, AG and UAG caused proliferation and protection against apoptosis through identical signaling pathways. Noteworthy, both peptides inhibited cytokine-induced NO increase in either HIT-T15 or INS-1E cells. Finally, they induced cell survival and protection against apoptosis in human islets of Langerhans. These expressed GRLN-R but showed also UAG and AG binding sites. Our data demonstrate that AG and UAG promote survival of both beta-cells and human islets. These effects are independent of GRLN-R, are likely mediated by AG/UAG binding sites, and involve cAMP/PKA, ERK1/2, and PI3K/Akt.     

Expression of ghrelin and biological activity of specific receptors for ghrelin and des-acyl ghrelin in human prostate neoplasms and related cell lines

BACKGROUND: Ghrelin, a natural growth hormone secretagogue (GHS), has been identified in prostate carcinoma cell lines. OBJECTIVES: To investigate the presence of ghrelin and its receptors in human prostate tumours and in DU-145, PC-3 and LNCaP prostate carcinoma cell lines, and to assess the effects of ghrelin and its more abundant circulating form, des-octanoyl ghrelin, on cell proliferation. METHODS: Ghrelin and types 1a and 1b GHS receptor (GHS-R) were determined at the mRNA and protein levels by RT-PCR, in situ hybridization, immunohistochemistry and enzyme immunoassay in tissues, cell lines and culture medium. Ghrelin binding was determined by radioreceptor assay. The effects on cell proliferation were evaluated by growth curves. RESULTS: Ghrelin mRNA was found in prostatic carcinomas and benign hyperplasias, but immunohistochemistry was negative. GHS-R1a and 1b mRNAs were absent from carcinomas, but GHS-R1b mRNA was present in 50% of hyperplasias. Ghrelin peptide and mRNA were present in PC-3 cells exclusively, whereas GHS-R1a and 1b mRNAs were expressed in DU-145 cells only. Specific [125I]Tyr4-ghrelin binding was detected in prostate tumour, DU-145 and PC-3 cell membranes and the binding was displaced by ghrelin, synthetic GHS and des-octanoyl ghrelin, which is devoid of GHS-R1a binding affinity and GH-releasing activity. Ghrelin and des-acyl ghrelin inhibited DU-145 cell proliferation, displayed a biphasic effect in PC-3 cells and were ineffective in LNCaP cells. CONCLUSIONS: Specific GHS binding sites, other than GHS-R1a and 1b, are present in human prostatic neoplasms. Ghrelin, in addition to des-acyl ghrelin, exerts different effects on cell proliferation in prostate carcinoma cell lines.     

Growth hormone releasing peptide (ghrelin) is synthesized and secreted by cardiomyocytes

OBJECTIVE: Ghrelin, the endogenous ligand of growth hormone secretagogue receptor (GHS-R), acts on the pituitary and the hypothalamus to stimulate the release of growth hormone (GH) and promotes appetite and adiposity. It has also been reported to increase myocardial contractility, induce vasodilation, and protect against myocardial-infarction-induced heart failure. Though principally gastric in origin, it is also produced by other tissues. This work investigated whether cardiomyocytes synthesize and secrete ghrelin, and how its production in these cells responds to stress and exogenous apoptotic agents. METHODS: Ghrelin and its receptor expression was studied by RT-PCR, immunohistochemistry, and competitive binding studies in mouse adult cardiomyocyte cell line HL-1, and primary cultured human cardiomyocytes. Ghrelin accumulation in cardiomyocyte culture medium was measured by radioimmunoassay. Viability and apoptosis assays were carried on by MTT and Hoechst dye vital staining, respectively. RESULTS: RT-PCR showed that HL-1 cells produce mRNAs for both ghrelin and GHS-R, and that GHS-R1a is expressed in human cardiomyocytes; and competitive binding studies using (125)I-labelled ghrelin showed efficient constitutive expression of GHS-R at the surface of HL-1 cells. Immunohistochemistry confirmed the presence of ghrelin in the cytoplasm of HL-1 cells and of isolated human cardiomyocytes in primary culture. Radioimmunoassay showed that ghrelin was secreted by HL-1 cells and human cardiomyocytes into the culture medium. Ghrelin did not modify the viability of HL-1 cells subjected to 12-h starvation, but did protect against the apoptosis inducer cytosine arabinoside (AraC). Finally, production of ghrelin mRNA in HL-1 cardiomyocytes was reduced by AraC but increased if exposure to AraC was preceded by GH treatment. CONCLUSIONS: Ghrelin is synthesized and secreted by isolated murine and human cardiomyocytes, probably with paracrine/autocrine effects, and may be involved in protecting these cells from apoptosis.     

Ghrelin exerts a proliferative effect on a rat pituitary somatotroph cell line via the mitogen-activated protein kinase pathway

OBJECTIVES: Ghrelin is a brain-gut peptide with GH-releasing and appetite-inducing activities and a widespread tissue distribution. Ghrelin is the endogenous ligand of the GH secretagogue receptor type 1a (GHS-R1a), and both ghrelin and the GHS-R1a are expressed in the pituitary. There are conflicting data regarding the effects of ghrelin on cell proliferation. A positive effect on proliferation and activation of the mitogen-activated protein kinase (MAPK) pathway has been found in hepatoma, adipose, cardiomyocyte and prostate cell lines. However, ghrelin has also been shown to have anti-proliferative effects on breast, lung and thyroid cell lines. We therefore examined the effect of ghrelin on the rat pituitary cell line GH3. METHODS: RT-PCR was used for the detection of GHS-R1a and pre-proghrelin mRNA expression in GH3 cells. The effect of ghrelin on cell proliferation was studied using [(3)H]thymidine incorporation; cell counting and the activation of the MAPK pathway were studied using immunoblotting and inhibitors of the extracellular signal-regulated kinase 1 and 2 (ERK 1/2), protein kinase C (PKC) and tyrosine phosphatase pathways. RESULTS: GHS-R1a and ghrelin mRNA expression were detected in GH3 cells. Ghrelin, at 10(-10) to 10(-6) M concentrations, significantly increased [(3)H]thymidine incorporation (at 10(-9) M, 183+/-13% (means+/-s.e.m.) compared with untreated controls), while 12-phorbol 13-myristate acetate (PMA) at 10(-7) M (used as a positive control) caused a 212+/-14% increase. A reproducible stimulatory effect of desoctanoyl ghrelin was also observed on [(3)H]thymidine incorporation (135+/-5%; P<0.01 at 10(-9) M compared with control), as well as on the cell count (control 6.8 x 10(4)+/-8.7 x 10(3) cells/ml vs desoctanoyl ghrelin (10(-9) M) 1.04 x 10(5)+/-7.5 x 10(3) cells/ml; P<0.01). Ghrelin caused a significant increase in phosphorylated ERK 1/2 in immunoblotting, while desoctanoyl ghrelin showed a smaller but also significant stimulatory effect. The positive effect of ghrelin and desoctanoyl ghrelin on [(3)H]thymidine incorporation was abolished by the MAPK kinase inhibitor U0126, the PKC inhibitor GF109203X and the tyrosine kinase inhibitor tyrphostin 23, suggesting that the ghrelin-induced cell proliferation of GH3 cells is mediated both via a PKC-MAPK-dependent pathway and via a tyrosine kinase-dependent pathway. This could also be clearly demonstrated by Western blot analysis, where a transient increase in ERK 1/2 phosphorylation by ghrelin was attenuated by all three inhibitors. CONCLUSION: We have shown a novel role for ghrelin in stimulating the proliferation of a somatotroph pituitary tumour cell line, suggesting that ERK activation is involved in mediating the effects of ghrelin on cell proliferation. Desoctanoyl ghrelin showed a similar effect. As ghrelin has been shown to be expressed in both normal and adenomatous pituitary tissue, locally produced ghrelin may play a role in pituitary tumorigenesis via an autocrine/paracrine pathway.     

Ghrelin以非经典受体依赖性方式促进人胚胎干细胞定向分化为心肌细胞的研究

目的 探讨Ghrelin对人胚胎干（hES）细胞定向分化为心肌细胞的影响. 方法 以不同浓度Ghrelin诱导hES细胞定向分化为心肌细胞,显微镜下计数搏动细胞团的比例,应用实时PCR检测心肌特异性标志物的表达.应用RT-PCR、Western blot及免疫荧光染色检测分化细胞中生长激素促分泌物受体1α（GHSR1α）的表达,添加GHS-R1α拮抗剂[D-lys3]-GHRP-6,观察其对Ghrelin促分化作用的影响. 结果 与对照组比较,10-10、10-9及10-8 mol/L Ghrelin均可增加搏动细胞团的比例[搏动比例分别为（12.9±1.4）％％,（19.5±2.2）％及（13.5±3.1）％vs（10.3±2.2）％,P＜0.05].RT-PCR分析显示,Ghrelin可上调α-肌球蛋白重链（α-MHC）和心肌肌钙蛋白I（cTnI）的表达[α-MHC：100％ vs （40.1±13.2）％,P＜0.001;cTnI：100％ vs （52.6±9.8）％,P＜0.001].RT-PCR、Western blot及免疫荧光染色均显示GHS-R1α在不同阶段的分化细胞中表达,但酰基化和非酰基化Ghrelin的促分化作用相似,且添加GHS-R1α拮抗剂不能阻断Ghrelin的促分化作用. 结论 Ghrelin可促进hES细胞定向分化为心肌细胞,该作用可能由GHS-R1α以外的信号途径所介导.     

The truncated ghrelin receptor polypeptide (GHS-R1b) is localized in the endoplasmic reticulum where it forms heterodimers with ghrelin receptors (GHS-R1a) to attenuate their cell surface expression

The ghrelin receptor (GHS-R1a) is remarkable amongst G-protein-coupled receptors for its high degree of constitutive activity, and this agonist-independent activity may be important for its physiological function in the control of food intake and body weight. Ghrelin receptors form heterodimers with the truncated ghrelin receptor polypeptide (GHS-R1b), which has a dominant-negative effect on ghrelin receptor function. Here we show that GHS-R1b has an intracellular localization distinct from ghrelin receptors, being primarily localized in the endoplasmic reticulum. Immunocytochemical studies suggest that GHS-R1b decreases the plasma membrane expression of ghrelin receptors, but the overall distribution profile of ghrelin receptors in isolated subcellular fractions is unaffected by GHS-R1b. Using bioluminescence resonance energy transfer methods, we have shown that while ghrelin receptor homodimers are evenly distributed in all subcellular fractions, GHS-R1a/GHS-R1b heterodimers are concentrated within the endoplasmic reticulum and these results suggest that GHS-R1b traps ghrelin receptors within the endoplasmic reticulum by the process of oligomerization. Furthermore, ghrelin receptors constitutively activated extracellular signal-regulated kinases 1/2 in the endoplasmic reticulum, but this small response was not affected by GHS-R1b and its physiological relevance is uncertain. Taken together, these results suggest that ghrelin receptors can be retained in the endoplasmic reticulum by heterodimerization with GHS-R1b, and constitutive activation of phospholipase C is attenuated due to decreased cell surface expression of ghrelin receptors. However, sufficient ghrelin receptor homodimers can still be expressed on the cell surface for maximal responses to agonist stimulation.     

The expression of the growth hormone secretagogue receptor ligand ghrelin in normal and abnormal human pituitary and other neuroendocrine tumors

Ghrelin is a recently identified endogenous ligand of the GH secretagogue (GHS) receptor. It was originally isolated from the stomach, but has also been shown to be present in the rat hypothalamus. It is a 28-amino acid peptide with an unusual octanoylated serine 3 at the N-terminal end of the molecule, which is crucial for its biological activity. Synthetic GHSs stimulate GH release via both the hypothalamus and the pituitary, and the GHS receptor (GHS-R) has been shown by us and others to be present in the pituitary. We investigated whether ghrelin messenger ribonucleic acid (mRNA) and peptide are present in the normal human hypothalamus and in normal and adenomatous human pituitary. RNA was extracted from pituitary tissue removed at autopsy and transsphenoidal surgery (n = 62), and ghrelin and GHS-R type 1a and 1b mRNA levels were investigated using real-time RT-PCR. Both ghrelin and GHS-R mRNA were detected in all samples. Corticotroph tumors showed significantly less expression of ghrelin mRNA, whereas GHS-R mRNA levels were similar to those in normal pituitary tissue. Gonadotroph tumors showed a particularly low level of expression of GHS-R mRNA. Immunohistochemistry, using a polyclonal antibody against the C-terminal end of the ghrelin molecule, revealed positive staining in the homolog of the arcuate nucleus in the human hypothalamus and in both normal and abnormal human pituitary. Pituitary tumor ghrelin peptide content was demonstrated using two separate RIA reactions for the N-terminal and C-terminal ends of the molecule. Both forms were present in normal and abnormal pituitaries, with 5 +/- 2.5% octanoylated (active) ghrelin (mean +/- SD) present as a percentage of the total. We suggest that the presence of ghrelin mRNA and peptide in the pituitary implies that the locally synthesized hormone may have an autocrine/paracrine modulatory effect on pituitary hormone release.     

Expression and action of the growth hormone releasing peptide ghrelin and its receptor in prostate cancer cell lines

This study has examined the expression of two new facets of the growth hormone axis, the growth hormone secretagogue receptor (GHS-R) and its recently identified putative natural ligand ghrelin, in prostate cancer cells. GHS-R 1a and 1b isoforms and ghrelin mRNA expression were detected by RT-PCR in the ALVA-41, LNCaP, DU145 and PC3 prostate cancer cell lines. A normal prostate cDNA library expressed GHS-R1a, but not the 1b isoform or ghrelin. Immunohistochemical staining for the GHS-R 1a isoform and ghrelin was positive in the four cell lines studied. PC3 cells showed increased cell proliferation in vitro in response to ghrelin to levels 33% above untreated controls, implying a potential tumour-promoting role for ghrelin in this tissue. This study is the first to demonstrate the co-expression of the GHS-R and ghrelin in prostate cancer cells. It is also the first study to provide evidence that a previously unrecognised autocrine/paracrine pathway involving ghrelin, that is capable of stimulating growth, exists in prostate cancer.     

Ghrelin inhibits steroid biosynthesis by cultured granulosa-lutein cells

CONTEXT: Growing evidence indicates that ghrelin may participate in the regulation of different aspects of reproductive function. The genes encoding for this peptide and its receptor are expressed in the human ovary, but their functional role is still unknown. OBJECTIVE: The aim of our study was to assess whether ghrelin has any effect on steroid synthesis by human granulosa-lutein cells and to identify the receptor isoform through which this potential effect is exerted. DESIGN, PATIENTS, AND METHODS: Thirty-five women with spontaneous ovulatory cycles undergoing in vitro fertilization for infertility due to uni- or bilateral tubal impatency or male factor were studied. Granulosa-lutein cells obtained from follicular fluid were incubated with increasing amounts of human acylated ghrelin (10(-11) to 10(-7) mol/liter) either alone or together with a 1:500 concentration of a specific anti-ghrelin receptor antibody [GH secretagogue receptor 1a (GHS-R1a)]. Culture media were tested for estradiol (E(2)) and progesterone (P(4)). The expression of GHS-R1a and GHS-R1b in human granulosa-lutein cells was also studied by real-time quantitative PCR. RESULTS: E(2) and P(4) concentrations in the culture media were significantly reduced by ghrelin in a dose-dependent fashion. The maximal decrease in E(2) (25%) and P(4) (20%) media concentrations was obtained with the 10(-7) and 10(-8) mol/liter ghrelin concentrations, respectively. The inhibitory effect of all ghrelin concentrations used was antagonized by the specific anti-ghrelin receptor-1a antibody added to the culture media and not by the specific anti-ghrelin receptor-1b antibody. Both 1a and 1b isoforms of the GHS-R were expressed in human granulosa-lutein cells, with the latter exceeding the former's expression (GHS-R1b/GHS-R1a ratio, 143.23 +/- 28.15). CONCLUSIONS: Ghrelin exerts an inhibitory effect on granulosa-lutein cells steroidogenesis by acting through its functional GHS-R1a. This suggests that ghrelin may serve an autocrine-paracrine role in the control of gonadal function and be part of a network of molecular signals responsible for the coordinated control of energy homeostasis and reproduction.     

The role of ghrelin and growth hormone secretagogues receptor on rat adipogenesis

Recent research progress indicates a close link between ghrelin, a natural ligand of GH secretagogues receptor (GHS-R), and both the metabolic balance and body composition. To clarify the involvement of ghrelin and GHS-R in the process of adipogenesis, we measured the expression of GHS-R and peroxisome proliferator-activated receptor gamma 2 (PPAR-gamma 2) mRNA in rat adipocytes using semiquantitative RT-PCR methods. The levels of GHS-R mRNA increased by up to 4-fold in adipose tissue from epididymal and parametrial regions as the rat aged from 4-20 wk and were significantly elevated during the differentiation of preadipocytes in vitro. Ghrelin (10(-8) M for 10 d) stimulated the activity of glycerol-3-phosphate dehydrogenase and the differentiation of rat preadipocytes in vitro. Ghrelin treatment also significantly increased the levels of PPAR-gamma 2 mRNA in primary cultured rat differentiated adipocytes. In addition, isoproterenol (10(-8) M, 40 min)-stimulated lipolysis was significantly reduced by simultaneous ghrelin treatment in a dose-dependent manner in vitro. In conclusion, the expression of GHS-R in rat adipocytes increases with the age and during adipogenesis. Ghrelin in vitro stimulates the differentiation of preadipocytes and antagonizes lipolysis. Ghrelin may therefore play an important role in the process of adipogenesis in rats.     

Expression of ghrelin and its functional receptor, the type 1a growth hormone secretagogue receptor, in normal human testis and testicular tumors

Ghrelin, the endogenous ligand for the GH secretagogue receptor (GHS-R), has been primarily linked to the central neuroendocrine regulation of GH secretion and food intake, although additional peripheral actions of ghrelin have also been reported. In this context, the expression of ghrelin and its cognate receptor has been recently demonstrated in rat testis, suggesting a role for this molecule in the direct control of male gonadal function. However, whether this signaling system is present in human testis remains largely unexplored. In this study we report the expression and cellular location of ghrelin and its functional receptor, the type 1a GHS-R, in adult human testis. In addition, evaluation of ghrelin and GHS-R1a immunoreactivity in testicular tumors and dysgenetic tissue is presented. The expression of the mRNAs encoding ghrelin and GHS-R1a was demonstrated in human testis specimens by RT-PCR, followed by direct sequencing. In normal testis, ghrelin immunostaining was demonstrated in interstitial Leydig cells and, at lower intensity, in Sertoli cells within the seminiferous tubules. In contrast, ghrelin was not detected in germ cells at any stage of spermatogenesis. The cognate ghrelin receptor showed a wider pattern of cellular distribution, with detectable GHS-R1a protein in germ cells, mainly in pachytene spermatocytes, as well as in somatic Sertoli and Leydig cells. Ghrelin immunoreactivity was absent in poorly differentiated Leydig cell tumor, which retained the expression of GHS-R1a peptide. In contrast, highly differentiated Leydig cell tumors expressed both the ligand and the receptor. The expression of ghrelin and GHS-R1a was also detected in dysgenetic Sertoli cell-only seminiferous tubules, whereas germ cell tumors (seminoma and embryonal carcinoma) were negative for ghrelin and were weakly positive for GHS-R1a. In conclusion, our results demonstrate that ghrelin and the type 1a GHS-R are expressed in adult human testis and testicular tumors. Overall, the expression of ghrelin and its functional receptor in human and rat testis, with roughly similar patterns of cellular distribution, is highly suggestive of a conserved role for this newly discovered molecule in the regulation of mammalian testicular function.     

Expression of ghrelin and of the GH secretagogue receptor by pancreatic islet cells and related endocrine tumors

Ghrelin is a novel gastrointestinal hormone produced by rat and human gastric X-like neuroendocrine cells, which strongly stimulates GH secretion and influences energy balance, gastric motility, and acid secretion. Ghrelin is expressed in pituitary and gastrointestinal endocrine tumors. It binds to the GH secretagogue receptor (GHS-R), which is present in a wide variety of central and peripheral human tissues. The aim of the present study was 2-fold: 1) to determine, by immunohistochemistry and mRNA analysis, whether pancreatic islet cells produce ghrelin and express GHS-R; and 2) to investigate ghrelin and GHS-R expression in pancreatic endocrine tumors. Seven cases of nonneoplastic pancreatic tissue and 28 endocrine tumors were studied. In pancreatic islets, ghrelin immunoreactivity was present in all cases and confined to beta-cells. Eleven of the 28 (39%) endocrine tumors were immunoreactive for ghrelin. In situ hybridization and RT-PCR confirmed the immunohistochemical data for both tumors and islets but also revealed ghrelin mRNA in 8 and 11 additional tumors, respectively. GHS-R 1a and 1b mRNAs were present in 7 of 28 and 14 of 28 tumors, respectively, studied by RT-PCR. These findings demonstrate that ghrelin production is not restricted to the stomach but is also present in pancreatic beta-cells and endocrine tumors (regardless of the type of pancreatic hormone produced, if any). Expression of GHS-R in some of the endocrine tumors studied indicates that autocrine/paracrine circuits may be active in neoplastic conditions.     

Distribution and regulation of chicken growth hormone secretagogue receptor isoforms

Chicken ghrelin has recently been isolated as a hormone which stimulates growth hormone and corticosterone secretion in chicken. Ghrelin mediates these actions in mammals by binding to the growth hormone secretagogue receptor (GHS-R). In this study, we describe the partial cloning of two chicken GHS-R (cGHS-R) isoforms: cGHS-R1a and cGHS-R1c. cGHS-R1a and cGHS-R1c cDNA show, respectively, 81 and 78% homology with the corresponding parts of the human GHS-R1a cDNA. In contrast to the human GHS-R1b isoform, which is truncated after transmembrane domain 5 (TM-5), the chicken GHS-R1c isoform lacks 16 amino acids in TM-6 suggesting that this isoform is not active in ghrelin signal transduction. The cystein residues, N-linked glycosylation sites and potential phosphorylation sites, found in the human GHS-R1a, were also conserved in both chicken isoforms. RT-PCR analysis demonstrated cGHS-R1a and cGHS-R1c mRNA expression in all tissues tested, except liver and pancreas, with highest levels in the pituitary and the hypothalamus. Intermediate levels of expression were detected, in descending order, in the ovary, telencephalon, heart, adrenal gland, cerebellum, and optic lobes whereas low expression was detected in the brainstem, lung, kidney, proventriculus, duodenum, and colon. Very low expression was found in skin, stomach, and muscle. cGHS-R1c was expressed in lower amounts than cGHS-R1a in all analysed tissues. Administration of 1 microM chicken ghrelin to pituitaries in vitro resulted in a down-regulation of both cGHS-R isoforms within 15 min, whereas after 1h levels returned to control values. Growth hormone and corticosterone down-regulated cGHS-R1a and cGHS-R1c mRNA expression within 60 min of exposure, whereas growth hormone-releasing factor 1-29 (1 microM) only reduced cGHS-R1a mRNA expression after 60min. Thyrotropin-releasing hormone (1 microM) did not alter cGHS-R expression.     

Ghrelin regulates proliferation and differentiation of osteoblastic cells

It has previously been reported that growth hormone secretagogues (GHS) may have a role in the regulation of bone metabolism in animals and humans. In this study we evaluated the effect of ghrelin, the endogenous ligand of GHS receptors, on the proliferation rate and on osteoblast activity in primary cultures of rat calvaria osteoblasts. In the same experiments, we compared the effects of ghrelin with those of hexarelin (HEXA) and EP-40737, two synthetic GHS with different characteristics. Both ghrelin and HEXA (10(-11)-10(-8) M) significantly stimulated osteoblast proliferation at low concentrations (10(-10) M). Surprisingly, EP-40737 demonstrated an antiproliferative effect at 10(-9)-10(-8) M, whereas lower concentrations had no effect on cell proliferation. Ghrelin and HEXA significantly increased alkaline phosphatase (ALP) and osteocalcin (OC) production. At variance with these peptides, EP-40737 did not significantly stimulate ALP and OC. The mRNA for GHS-R1a receptors and the corresponding protein were detected in calvarial osteoblasts by RT-PCR and Western blot respectively, indicating that ghrelin and GHS may bind and activate this specific receptor. We conclude that endogenous ghrelin and synthetic GHS modulate proliferation and differentiation of rat osteoblasts, probably by acting on their specific receptor.