Regulation of ghrelin receptor by microbial and inflammatory signals in human osteoblasts.

Recently, it has been suggested that the anti-inflammatory hormone ghrelin (GHRL) and its receptor GHS-R may play a pivotal role in periodontal health and diseases. However, their exact regulation and effects in periodontitis are not known. The aim of this in-vitro study was to investigate the effect of microbial and inflammatory insults on the GHS-R1a expression in human osteoblast-like cells. MG-63 cells were exposed to interleukin (IL)-1ß and Fusobacterium nucleatum in the presence and absence of GHRL for up to 2 d. Subsequently, gene expressions of GHS-R1a, inflammatory mediators and matrix metalloproteinase were analyzed by real-time PCR. GHS-R protein synthesis and NF-κB p65 nuclear translocation were assessed by immunocytochemistry and immunofluorescence microscopy, respectively. IL-1ß and F. nucleatum caused a significant upregulation of GHS-R1a expression and an increase in GHS-R1a protein. Pre-incubation with a MEK1/2 inhibitor diminished the IL-1ß-induced GHS-R1a upregulation. IL-1ß and F. nucleatum also enhanced the expressions of cyclooxygenase 2, CC-chemokine ligand 2, IL-6, IL-8, and matrix metalloproteinase 1, but these stimulatory effects were counteracted by GHRL. By contrast, the stimulatory actions of IL-1ß and F. nucleatum on the GHS-R1a expression were further enhanced by GHRL. Our study provides original evidence that IL-1ß and F. nucleatum regulate the GHS-R/GHRL system in osteoblast-like cells. Furthermore, we demonstrate for the first time that the proinflammatory and proteolytic actions of IL-1ß and F. nucleatum on osteoblast-like cells are inhibited by GHRL. Our study suggests that microbial and inflammatory insults upregulate GHS-R1a, which may represent a protective negative feedback mechanism in human bone.

Regulation of ghrelin receptor by microbial and inflammatory signals in human osteoblasts

Abstract: Recently, it has been suggested that the anti-inflammatory hormone ghrelin (GHRL) and its receptor GHS-R may play a pivotal role in periodontal health and diseases. However, their exact regulation and effects in periodontitis are not known. The aim of this in-vitro study was to investigate the effect of microbial and inflammatory insults on the GHS-R1a expression in human osteoblast-like cells. MG-63 cells were exposed to interleukin (IL)-1β and Fusobacterium nucleatum in the presence and absence of GHRL for up to 2 d. Subsequently, gene expressions of GHS-R1a, inflammatory mediators and matrix metalloproteinase were analyzed by real-time PCR. GHS-R protein synthesis and NF-κB p65 nuclear translocation were assessed by immunocytochemistry and immunofluorescence microscopy, respectively. IL-1β and F. nucleatum caused a significant upregulation of GHS-R1a expression and an increase in GHS-R1a protein. Pre-incubation with a MEK1/2 inhibitor diminished the IL-1β-induced GHS-R1a upregulation. IL-1β and F. nucleatum also enhanced the expressions of cyclooxygenase 2, CC-chemokine ligand 2, IL-6, IL-8, and matrix metalloproteinase 1, but these stimulatory effects were counteracted by GHRL. By contrast, the stimulatory actions of IL-1β and F. nucleatum on the GHS-R1a expression were further enhanced by GHRL. Our study provides original evidence that IL-1β and F. nucleatum regulate the GHS-R/GHRL system in osteoblast-like cells. Furthermore, we demonstrate for the first time that the proinflammatory and proteolytic actions of IL-1β and F. nucleatum on osteoblast-like cells are inhibited by GHRL. Our study suggests that microbial and inflammatory insults upregulate GHS-R1a, which may represent a protective negative feedback mechanism in human bone.

Peptidomimetic growth hormone secretagogue derivatives for positron emission tomography imaging of the ghrelin receptor.

The ghrelin receptor is a seven-transmembrane (7-TM) receptor known to have an increased level of expression in human carcinoma and heart failure. Recent work has focused on the synthesis of positron emission tomography (PET) probes designed to target and image this receptor for disease diagnosis and staging. However, these probes have been restricted to small-molecule quinalizonones and peptide derivatives of the endogenous ligand ghrelin. We describe the design, synthesis and biological evaluation of a series of 4-fluorobenzoylated growth hormone secretagogues (GHSs) derived from peptidic (GHRP-1, GHPR-2 and GHRP-6) and peptidomimetic (G-7039, [1-Nal4]G-7039 and ipamorelin) families in order to test locations for the insertion of fluorine-18 for PET imaging. The peptidomimetic G-7039 was found to be the most suitable for 18F-radiolabelling as its non-radioactive 4-fluorobenzoylated analogue ([1-Nal4,Lys5(4-FB)]G-7039), had both a high binding affinity (IC50 = 69 nM) and promising in vitro efficacy (EC50 = 1.1 nM). Prosthetic group radiolabelling of the precursor compound [1-Nal4]G-7039 using N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) delivered the PET probe [1-Nal4,Lys5(4-[18F]-FB)]G-7039 in an average decay-corrected radiochemical yield of 48%, a radio-purity ≥ 99% and an average molar activity of >34 GBq/µmol. This compound could be investigated as a PET probe for the detection of diseases that are characterised by overexpression of the ghrelin receptor.

Structure-Activity Study of Ghrelin(1-8) Resulting in High Affinity Fluorine-Bearing Ligands for the Ghrelin Receptor.

The ghrelin receptor, also known as the growth hormone secretagogue receptor 1a (GHS-R1a), is a G-protein-coupled receptor that is differentially expressed in healthy tissue and several cancers, including prostate, testicular, and ovarian. Selectively targeting the ghrelin receptor using fluorine-18 tagged entities would allow localization and visualization of ghrelin receptor expressing carcinomas using PET imaging. The endogenous ligand ghrelin, a 28 amino acid peptide with 3.1 nM affinity, has poor in vivo stability. Here we report on ghrelin(1-8) analogues bearing modifications at residues 1, 3, 4, and 8. The lead analogue, [Inp1,Dpr3(6-fluoro-2-naphthoate),1-Nal4,Thr8]ghrelin(1-8), possessed an IC50 value of 0.11 nM that is a 28-fold improvement compared to the natural ligand. A novel 6-fluoro-2-pentafluorophenyl naphthoate (PFPN) prosthetic group was synthesized to incorporate fluorine-18 for PET imaging. This is not only the highest affinity ghrelin analogue reported but also the shortest ghrelin analogue capable of binding GHS-R1a with better affinity than ghrelin(1-28).

Enhanced alcohol-drinking behavior associated with active ghrelinergic and serotoninergic neurons in the lateral hypothalamus and amygdala.

Central ghrelin is required for the rewarding properties of drug abuse. We investigated whether alcohol affects ghrelinergic, dopaminergic, and serotoninergic neurons and growth hormone secretagogue receptor 1A (GHS-R1A) levels in the reward system of the brain. Alcohol-naïve C57BL/6J mice received 2g/kg ethanol (EtOH) intraperitoneally (i.p.). Plasma ghrelin levels decreased between 1 and 4h. We investigated the effects of EtOH administration on plasma ghrelin levels in two different animal models at 1, 3, and 10months of age. Plasma ghrelin levels decreased following the EtOH treatment in 1- and 3-month-old short-term (1-day) alcohol vapor-exposed (STA) mice. In contrast, EtOH administration increased plasma ghrelin levels in 1- and 3-month-old long-term (20-day) alcohol vapor-exposed (LTA) mice. In vivo ghrelin release in the lateral hypothalamus (LH) increased in STA and LTA mice after the i.p. administration of EtOH. EtOH increased in vivo dopamine (DA), but not serotonin (5-HT) release in the LH of STA mice, and increased in vivo DA and 5-HT release in the LH of LTA mice. GHS-R1A mRNA expression and GHS-R1A protein levels in the LH were increased in LTA mice. The number of GHS-R1A-immunoreactive cells was greater in the LH and amygdala of LTA mice. These results support the neurobiological correlation between the development of drinking behavior and activation of ghrelinergic and serotonergic neurons in the LH. The activation of ghrelinergic systems in the amygdala may also induce an increase in 5-HT release in the LH during long-term alcohol intake.

GHSR DNA hypermethylation is a common epigenetic alteration of high diagnostic value in a broad spectrum of cancers.

Identification of a single molecular trait that is determinant of common malignancies may serve as a powerful diagnostic supplement to cancer type-specific markers. Here, we report a DNA methylation mark that is characteristic of seven studied malignancies, namely cancers of lung, breast, prostate, pancreas, colorectum, glioblastoma and B cell chronic lymphocytic leukaemia (CLL) (n = 137). This mark was defined by substantial hypermethylation at the promoter and first exon of growth hormone secretagouge receptor (GHSR) through bisulfite pyrosequencing. The degree of aberrant methylation was capable of accurate discrimination between cancer and control samples. The highest sensitivity and specificity of cancer detection was achieved for cancers of pancreas, lung, breast and CLL yielding the area under the curve (AUC) values of 1.0000, 0.9952, 0.9800 and 0.9400, respectively. Narrowing to a single CpG site within the gene's promoter or four consecutive CpG units of the highest methylation levels within the first exon improved the detection power. GHSR hypermethylation was detected already at the early stage tumors. The accurate performance of this marker was further replicated in an independent set of pancreatic cancer and control samples (n = 78). These findings support the candidature of GHSR methylation as a highly accurate pan-cancer marker.

Novel evidence of ghrelin and growth hormone segretagogue receptor expression by human ocular tissues.

AIM OF THE STUDY: The gastrointestinal peptide hormone ghrelin (Ghr) was discovered in 1999 as the endogenous ligand for the growth hormone secretagogue receptor (GHSR-1a). It is a pleiotropic peptide that modulates a wide spectrum of biological activities, such as growth hormone (GH) release, feeding stimulation, adiposity and cardiovascular actions. The presence of Ghr mRNA in the iris and ciliary body (CB) epithelium was recently demonstrated in animal models, where a possible myorelaxing effect on the iris muscles has been suggested. Based on these observations, the aim of our study was to investigate the Ghr and GHSR-1a expression and localization in the normal human eye. MATERIAL: Five different ciliary body/iris samples from normal eyes were subjected to Western blot analysis. Immunohistochemical detection was performed on three enucleated eyes. Twenty aqueous humor (AqH) samples obtained from patients submitted to cataract surgery were analyzed with an ELISA for the presence of Ghr. RESULTS: Ghr and GHSR-1a were co-expressed by the pigmented epithelium (PE) of the CB, by the retinal pigmented epithelium (RPE) and by the anterior limiting layer (ALL) of the iris. No reaction was detected at the subepithelial level in the ciliary or pupillae smooth muscle cells. The AqH samples were positive for the presence of Ghr. CONCLUSION: This study provides the first evidence that Ghr and GHSR-1a are expressed in the human eye by specific cells. The understanding of the functional role of Ghr at the human eye level needs more efforts and investigation, but a hypothetical action on the GH retinal synthesis and/or on the circadian clock system could be suggested.

Characterization of a far-red analog of ghrelin for imaging GHS-R in P19-derived cardiomyocytes.

Ghrelin and its receptor, the growth hormone secretagogue receptor (GHS-R), are expressed in the heart, and may function to promote cardiomyocyte survival, differentiation and contractility. Previously, we had generated a truncated analog of ghrelin conjugated to fluorescein isothiocyanate for the purposes of determining GHS-R expression in situ. We now report the generation and characterization of a far-red ghrelin analog, [Dpr(3)(octanoyl), Lys(19)(Cy5)]ghrelin (1-19), and show that it can be used to image changes in GHS-R in developing cardiomyocytes. We also generated the des-acyl analog, des-acyl [Lys(19)(Cy5)]ghrelin (1-19) and characterized its binding to mouse heart sections. Receptor binding affinity of Cy5-ghrelin as measured in HEK293 cells overexpressing GHS-R1a was within an order of magnitude of that of fluorescein-ghrelin and native human ghrelin, while the des-acyl Cy5-ghrelin did not bind GHS-R1a. Live cell imaging in HEK293/GHS-R1a cells showed cell surface labeling that was displaced by excess ghrelin. Interestingly, Cy5-ghrelin, but not the des-acyl analog, showed concentration-dependent binding in mouse heart tissue sections. We then used Cy5-ghrelin to track GHS-R expression in P19-derived cardiomyocytes. Live cell imaging at different time points after DMSO-induced differentiation showed that GHS-R expression preceded that of the differentiation marker aMHC and tracked with the contractility marker SERCA 2a. Our far-red analog of ghrelin adds to the tools we are developing to map GHS-R in developing and diseased cardiac tissues.

Localization of ghrelin and its receptor in the reproductive tract of Holstein heifers.

The aim of this experiment was to localize the mRNA and protein of ghrelin and its active receptor, growth hormone secretagogue 1A (GHS-R1A), within the reproductive tract of dairy cattle. Ghrelin is an orexigenic hormone that has been identified as a potent regulator of energy homeostasis. Recent evidence suggests that ghrelin may also serve as a metabolic signal to the reproductive tract. Ghrelin and GHS-R1A have been identified in the reproductive tract of several species, including humans, mice, and rats. However, ghrelin and GHS-R1A expression have not been described within bovine reproductive tissues. Therefore, the ampulla, isthmus, uterine body, corpus luteum, and follicles were harvested from 3 Holstein heifers (15.91±0.07 mo of age) immediately following exsanguination. Duodenum and hypothalamus were collected as positive controls for ghrelin and GHS-R1A, respectively. Tissues were fixed in 10% formalin and embedded in paraffin for microscopy. Additional samples were stored at -80°C for detection of mRNA. Ghrelin and GHS-R1A mRNA and protein were observed in all tissue types within the reproductive tract of dairy heifers; however, expression appeared to be cell specific. Furthermore, ghrelin protein appeared to be localized to the cytoplasm, whereas GHS-R1A protein was found on the plasma membrane. Within the reproductive tissues, ghrelin mRNA and protein were most abundantly expressed in the ampulla of the oviduct. Concentrations of GHS-R1A were lower than those of ghrelin but differed between tissues. This is one of the first studies to provide molecular evidence for the presence of ghrelin and GHS-R1A within the entire reproductive tract. However, implications for fertility remain to be determined.

Synthesis and in vivo evaluation of (S)-6-(4-fluorophenoxy)-3-((1-[11C]methylpiperidin-3-yl)methyl)-2-o-tolylquinazolin-4(3H)-one, a potential PET tracer for growth hormone secretagogue receptor (GHSR).

The peptide hormone ghrelin mediates through action on its receptor, the growth hormone secretagogue receptor (GHSR), and is known to play an important role in a variety of metabolic functions including appetite stimulation, weight gain, and suppression of insulin secretion. In light of the fact that obesity is one of the major health problems plaguing the modern society, the ghrelin signaling system continues to remain an important and attractive pharmacological target for the treatment of obesity. In vivo imaging of the GHSR could shed light on the mechanism by which ghrelin affects feeding behavior and thus offers a new therapeutic perspective for the development of effective treatments. Recently, a series of piperidine-substituted quinazolinone derivatives was reported to be selective and potent GHSR antagonists with high binding affinities. Described herein is the synthesis, in vitro, and in vivo evaluation of (S)-6-(4-fluorophenoxy)-3-((1-[(11)C]methylpiperidin-3-yl)methyl)-2-o-tolylquinazolin-4(3H)-one ([(11)C]1), a potential PET radioligand for imaging GHSR.

[Relationship between Ghrelin and growth hormone secretagogue receptor expression and catch-up growth in rats with intrauterine growth restriction].

OBJECTIVE: To study the relationship between Ghrelin and growth hormone secretagogue receptor (GHSR) expression and the catch-up growth in rats with intrauterine growth restriction (IUGR). METHODS: The rat model of IUGR was established by food restriction during pregnancy. The small for gestational age (SGA) and appropriate for gestational age (AGA) rat pups from the pregnant rats were used as the experimental group. The AGA rat pups from the pregnant rats without food restriction served as the control group. The samples from the stomach fundus and hypothalamus were taken postnatal days 0, 20 and 40. Ghrelin mRNA and GHSR mRNA expression were determined by real-time fluorescence quantitative PCR (real-time FQ-PCR). Ghrelin protein and GHSR protein expression were examined by immunohistochemistry (IHC). RESULTS: At postnatal day 0, both Gherlin mRNA and protein levels in the stomach fundus were significantly higher, while GHSR mRNA expression in the hypothalamus were significantly lower in SGA rats from food restriction group than those in AGA rats from restriction and control groups. At postnatal day 20, the ghrelin protein expression in the stomach of fundus, and GHSR mRNA and protein expression in the hypothalamus in SGA catch-up rats were significantly higher than those in SGA non-catch-up growth rats and AGA rats from the control group. At postnatal day 40, there were no significant differences among SGA catch-up growth rats, SGA non-catch-up growth rats and normal AGA rats. CONCLUSIONS: Ghrelin-GHSR might be involved in the physiological regulation and pathological process in IUGR rats. It is also possibly involved in the regulation of catch-up growth in the early life of SGA rats.

Expression of the orexigenic peptide ghrelin and the type 1a growth hormone secretagogue receptor in sheep oocytes and pre-implantation embryos produced in vitro.

Recent studies have implicated the peripheral actions of ghrelin in reproductive tissues. Here, we provide evidence that both ghrelin and its receptor GHSR-1a (the type 1a growth hormone secretagogue receptor) are expressed at both mRNA and protein levels in sheep oocytes and pre-implantation embryos produced in vitro. Real-time RT-PCR experiments confirmed that ghrelin mRNA levels varied depending on developmental stage, with the highest expression in metaphase II (MII) oocytes, higher expression at the 2-cell stage, and minimal expression in germinal vesicle (GV) oocytes, 4- and 8-cell stages, and in the blastocyst. The levels of GHSR-1a mRNA decreased from GV to MII, increased immediately at the 2-cell stage and then remained stable until the blastocyst stage. Ghrelin protein was detected mainly in the cytoplasm close to the plasma membrane in both inner cell mass and trophectoderm cells, while GHSR-1a protein was most abundant in the plasma membrane. In conclusion, the presence of the ghrelin signalling system within the sheep oocytes and pre-implantation embryos opens up the possibility of a potential regulatory role of this novel molecule in reproductive function.

Therapeutic effects of ghrelin and growth hormone releasing peptide 6 on gastroparesis in streptozotocin-induced diabetic guinea pigs in vivo and in vitro.

BACKGROUND: Diabetic gastroparesis is a disabling condition with no consistently effective treatment. In normal animals, both ghrelin and its synthetic peptide, growth hormone releasing peptide 6 (GHRP-6), increase gastric emptying. Thus, we investigated the potential therapeutic significance of ghrelin and GHRP-6 in diabetic guinea pigs with gastric motility disorders. METHODS: A diabetic guinea pig model was produced by intraperitoneal (i.p.) injection of streptozotocin (STZ, 280 mg/kg). Diabetic guinea pigs were injected i.p. with ghrelin or GHRP-6 (10 - 100 microg/kg), and the effects on gastric emptying were measured after intragastric application of phenol red. The effect of atropine or a growth hormone secretagogue receptor (GHS-R) antagonist, D-Lys(3)-GHRP-6, on the gastroprokinetic effects of ghrelin or GHRP-6 (100 microg/kg) was also investigated. Further, the in vitro effects of ghrelin or GHRP-6 (0.01 - 10 micromol/L) on spontaneous or carbachol-induced contractile amplitude in gastric fundic circular strips taken from diabetic guinea pigs were examined. Growth hormone secretagogue receptor transcripts in the fundic strips of diabetic guinea pigs were detected by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: We established a guinea pig model of delayed gastric emptying. Ghrelin (20, 50, or 100 microg/kg) and GHRP-6 (20, 50, or 100 microg/kg) accelerated gastric emptying in diabetic guinea pigs with gastroparesis (n = 6, P < 0.05). In the presence of atropine, which delayed gastric emptying, ghrelin and GHRP-6 (100 microg/kg) failed to accelerate gastric emptying (n = 6, P < 0.05). D-Lys(3)-GHRP-6 also delayed gastric emptying induced by the GHS-R agonist (n = 6, P < 0.05). Ghrelin and GHRP-6 increased the carbachol-induced contractile amplitude in gastric fundic strips taken from diabetic guinea pigs (n = 6, P < 0.05). RT-PCR confirmed the presence of GHS-R mRNA in the strip preparations. CONCLUSIONS: Ghrelin and GHRP-6 increased gastric emptying in diabetic guinea pigs with gastroparesis, potentially, by activating the peripheral cholinergic pathways in the enteric nervous system.

Ghrelin promotes thymopoiesis during aging.

The decline in adaptive immunity, T lymphocyte output, and the contraction of the TCR repertoire with age is largely attributable to thymic involution. The loss of thymic function with age may be due to diminished numbers of progenitors and the loss of critical cytokines and hormones from the thymic microenvironment. We have previously demonstrated that the orexigenic hormone ghrelin is expressed by immune cells and regulates T cell activation and inflammation. Here we report that ghrelin and ghrelin receptor expression within the thymus diminished with progressive aging. Infusion of ghrelin into 14-month-old mice significantly improved the age-associated changes in thymic architecture and thymocyte numbers, increasing recent thymic emigrants and improving TCR diversity of peripheral T cell subsets. Ghrelin-induced thymopoiesis during aging was associated with enhanced early thymocyte progenitors and bone marrow-derived Lin(-)Sca1(+)cKit(+) cells, while ghrelin- and growth hormone secretagogue receptor-deficient (GHS-R-deficient) mice displayed enhanced age-associated thymic involution. Leptin also enhanced thymopoiesis in aged but not young mice. Our findings demonstrate what we believe to be a novel role for ghrelin and its receptor in thymic biology and suggest a possible therapeutic benefit of harnessing this pathway in the reconstitution of thymic function in immunocompromised subjects.