Growth Hormone Secretagogue Receptor Type 1a Research Articles

Ghrelin-O-acyltransferase (GOAT) acylates ghrelin in the hippocampus.

Ghrelin is an appetite-stimulating peptide hormone and produced in the stomach. Serine 3 on ghrelin must be acylated by the lipid transferase known as Ghrelin-O-acyltransferase (GOAT) in order for the peptide to become physiologically-active and bind to the cognate receptor, growth hormone secretagogue receptor type 1a (GHSR1a). GHSR1a has been known to be expressed in the feeding center of the hypothalamus. However, the interest in GHSR1a increased dramatically among researchers in various biomedical fields when GHSR1a mRNA was found wide-spread in the brain including the hippocampus. Current understanding is that GHSR1a has multifaceted functions beyond the regulation of metabolism. In the blood, a nonacylated form of ghrelin (des-acyl ghrelin) exists in far greater amounts. Des-acyl ghrelin can cross the blood-brain barrier (BBB), but it cannot bind to GHSR1a in the brain. Thus, the identification of the source for acyl ghrelin in the brain became the critical and urgent quest. Here, we discuss the presence of GOAT in the hippocampus and its ability to acylate ghrelin locally within the hippocampus. We will show that GOAT is localized specifically at the base of the dentate granule cell layer in the rat and wild-type mouse, but not in the GHSR1a knockout mouse. This evidence points the possibility that the expression of GHSR1a may be a prerequisite for the synthesis of GOAT in the hippocampus. We will also show that: (1) the activation of GHSR1a by acyl ghrelin upregulates the cAMP and CREB phosphorylation, (2) amplifies the NMDA receptor-mediated synaptic transmission by phosphorylating GluN1 subunit at Ser896/897, and (3) activates Fyn kinase and induces GluN2B phosphorylation at Tyr1336. In summary, GOAT is a critical molecule that acts as the master switch in the initiation of ghrelin-induced hippocampal synapse and neuron plasticity.

The NanoBiT-Based Homogenous Ligand-Receptor Binding Assay.

NanoLuc Binary Technology (NanoBiT) was recently developed by Promega, based on a large NanoLuc fragment (LgBiT) and two small complementation tags, the low-affinity SmBiT tag and the high-affinity HiBiT tag. In recent studies, we applied NanoBiT to ligand-binding assays of some G protein-coupled receptors via genetic fusion of a secretory LgBiT (sLgBiT) to the extracellular N-terminus of the receptors and covalent attachment of the low-affinity SmBiT tag to an appropriate position of their peptide ligands. The NanoBiT-based homogenous ligand-receptor binding assay is convenient for use and suitable for both the wild-type and mutant receptors, representing a novel tool for interaction mechanism studies of these receptors with their ligands. In the present chapter, we provide detailed protocols for setting up the NanoBiT-based homogenous binding assay using growth hormone secretagogue receptor type 1a (GHSR1a) and its endogenous agonist and antagonist as a representative model system.

GHS-R1a deficiency mitigates lipopolysaccharide-induced lung injury in mice via the downregulation of macrophage activity

Acute respiratory distress syndrome (ARDS) is a critical illness syndrome characterized by dysregulated pulmonary inflammation. Currently, effective pharmacological treatments for ARDS are unavailable. Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor type 1a (GHS-R1a), has a pivotal role in regulating energy metabolism and immunomodulation. The role of endogenous ghrelin in ARDS remains unresolved. Herein, we investigated the role of endogenous ghrelin signaling by using GHS-R1a-null (ghsr−/−) mice and lipopolysaccharide (LPS)-induced ARDS model. Ghsr−/− mice survived longer than controls after LPS-induced lung injury. Ghsr−/− mice showed lower levels of pro-inflammatory cytokines and higher oxygenation levels after lung injury. The peritoneal macrophages isolated from ghsr−/− mice exhibited lower levels of cytokines production and oxygen consumption rate after LPS stimulation. Our results indicated that endogenous ghrelin plays a pivotal role in initiation and continuation in acute inflammatory response in LPS-induced ARDS model by modulating macrophage activity, and highlighted endogenous GHS-R1a signaling in macrophage as a potential therapeutic target in this relentless disease.

Ghrelin signaling in dCA1 suppresses neuronal excitability and impairs memory acquisition via PI3K/Akt/GSK-3β cascades

Ghrelin is a circulating peptide hormone that promotes feeding and regulates metabolism in humans and rodents. The action of ghrelin is mediated by the growth hormone secretagogue receptor type 1a (GHSR-1a) that is widely distributed in the brain, including the hippocampus. Studies have demonstrated the critical role of hippocampal ghrelin/GHS-R1a signaling in synaptic physiology and memory. However, those findings are controversial, and the mechanism underlying ghrelin modulation of learning and memory is uncertain. Here, we report that micro-infusion of ghrelin in the CA1 region of the dorsal hippocampus during training specifically impairs memory acquisition. The activation of GHS-R1a and the subsequent PI3K/Akt/GSK3β signaling cascades are involved in this process. Moreover, we report that bath application of ghrelin suppresses the intrinsic excitability of dCA1 pyramidal neurons through activating GHS-R1a, and PI3K inhibitor LY294002 blocks ghrelin's effect. However, LY294002 fails to rescue ghrelin-induced LTP impairment. Our findings support an adverse effect of ghrelin-dependent activation of GHS-R1a on memory acquisition, and suggest that PI3K/Akt/GSK3β signaling-dependent repression of neuronal intrinsic excitability is an important novel mechanism underlying memory inhibition of ghrelin in the hippocampus.This article is part of the special Issue on ‘Stress, Addiction and Plasticity'.

Unusual orthologs shed new light on the binding mechanism of ghrelin to its receptor GHSR1a

The gastric peptide ghrelin has important functions in energy metabolism and cellular homeostasis by activating growth hormone secretagogue receptor type 1a (GHSR1a). The N-terminal residues of ghrelin orthologs from all vertebrates are quite conserved; however, in orthologs from Cavia porcellus and Phyllostomus discolor, Ser2 and Leu5 are replaced by a smaller Ala and a positively charged Arg, respectively. In the present study, we first demonstrated that the hydrophobic Leu5 is essential for the function of human ghrelin, because Ala replacement caused an approximately 100-fold decrease in activity. However, replacement of Leu5 by an Arg residue caused much less disruption; further replacement of Ser2 by Ala almost restored full activity, although the [S2A] mutation itself showed slight detriments, implying that the positively charged Arg5 in the [S2A,L5R] mutant might form alternative interactions with certain receptor residues to compensate for the loss of the essential Leu5. To identify the responsible receptor residues, we screened GHSR1a mutants in which all conserved negatively charged residues in the extracellular regions and all aromatic residues in the ligand-binding pocket were mutated separately. According to the decrease in selectivity of the mutant receptors towards [S2A,L5R]ghrelin, we deduced that the positively charged Arg5 of the ghrelin mutant primarily interacts with the essential aromatic Phe286 at the extracellular end of the sixth transmembrane domain of GHSR1a by forming cation−π and π−π interactions. The present study provided new insights into the binding mechanism of ghrelin with its receptor, and thus would facilitate the design of novel ligands for GHSR1a.

Development of a ghrelin receptor inverse agonist for positron emission tomography.

Imaging of Ghrelin receptors in vivo provides unique potential to gain deeper understanding on Ghrelin and its receptors in health and disease, in particular, in cancer. Ghrelin, an octanoylated 28-mer peptide hormone activates the constitutively active growth hormone secretagogue receptor type 1a (GHS-R1a) with nanomolar activity. We developed novel compounds, derived from the potent inverse agonist K-(D-1-Nal)-FwLL-NH2 but structurally varied by lysine conjugation with 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), palmitic acid and/or diethylene glycol (PEG2) to allow radiolabeling and improve pharmacokinetics, respectively. All compounds were tested for receptor binding, potency and efficacy in vitro, for biodistribution and -kinetics in rats and in preclinical prostate cancer models on mice. Radiolabeling with Cu-64 and Ga-68 was successfully achieved. The Cu-64- or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiotracer were specifically accumulated by the GHS-R1a in xenotransplanted human prostate tumor models (PC-3, DU-145) in mice. The tumors were clearly delineated by PET. The radiotracer uptake was also partially blocked by K-(D-1-Nal)-FwLL-NH2 in stomach and thyroid. The presence of the GHS-R1a was also confirmed by immunohistology. In the arterial rat blood plasma, only the original compounds were found. The Cu-64 or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiolabeled inverse agonists turned out to be potent and safe. Due to their easy synthesis, high affinity, medium potency, metabolic stability, and the suitable pharmacokinetic profiles, they are excellent tools for imaging and quantitation of GHS-R1a expression in normal and cancer tissues by PET. These compounds can be used as novel biomarkers of the Ghrelin system in precision medicine.

Pharmacological profile and clinical efficacy of anamorelin HCl (ADLUMIZ<sup>®</sup>Tablets), the first orally available drug for cancer cachexia with ghrelin-like action in Japan

Anamorelin hydrochloride (hereinafter referred to as anamorelin) is an orally active, small-molecule drug with a similar pharmacological action to ghrelin, an endogenous ligand of growth hormone secretagogue receptor type 1a (GHS-R1a). It was first approved in Japan for the treatment of cancer cachexia, characterized by weight loss and anorexia. Anamorelin stimulated the secretion of growth hormone (GH) from cultured rat pituitary cells and increased plasma GH levels by oral administration to rats, pigs and humans. When anamorelin was orally administered once daily for 6 days to rats, larger body weight gain associated with increased food consumption compared to the control group was observed from after the first dose. Anamorelin is a selective agonist for GHS-R1a and enhanced GHS-R1a-mediated pituitary GH secretion and increased food consumption, resulting in body weight gain. In the two Japanese phase II studies in patients with cancer cachexia associated with non-small cell lung cancer (NSCLC), improvement of lean body mass (LBM) and body weight losses and anorexia were demonstrated. The tumor types of target patients in the Japanese phase III study were colorectal, gastric, and pancreatic cancer. As a result, maintenance and increase of LBM and body weight as well as improvement of anorexia were observed, and the efficacy against cancer cachexia associated with colorectal, gastric, and pancreatic cancer was confirmed. There were no observed events considered to be significant safety risks. In conclusion, anamorelin is expected to provide a new therapeutic option for cancer cachexia for which no effective treatment has been available.

Single Amino Acid Replacement in G-7039 Leads to a 70-fold Increase in Binding toward GHS-R1a.

The growth hormone secretagogue receptor type 1a (GHS-R1a) is a class A rhodopsin-like G protein coupled receptor (GPCR) that is expressed in a variety of human tissues and is differentially expressed in benign and malignant prostate cancer. Previously, the peptidomimetic [1-Nal4 ,Lys5 (4-fluorobenzoyl)]G-7039 was designed as a molecular imaging tool for positron emission tomography (PET). However, this candidate was a poor binder (IC50 =69 nm), required a lengthy four-step radiosynthesis, and had a cLogP above 8. To address these challenges, we now report on changes targeted at the 4th position of G-7039. A 2-fluoropropionic acid (2-FPA) group was added on to Lys5 to determine the potential binding affinity of the [18 F]-2-FP radiolabeled analogue, which could be prepared by simplified radiochemistry. Lead candidate [Tyr4 ,Lys5 (2-fluoropropionyl)]G-7039 exhibited an IC50 of 0.28 nm and low picomolar activity toward GHS-R1a. Molecular docking revealed a molecular basis for this picomolar affinity.

Ghrelin fiber projections from the hypothalamic arcuate nucleus into the dorsal vagal complex and the regulation of glycolipid metabolism

ObjectivesThis study aimed to explore the involvement of the ghrelin pathway from the arcuate nucleus (ARC) to the dorsal vagal complex (DVC) and to determine its role in the regulation of glycolipid metabolism. MethodsThe protein and mRNA expression of ghrelin and growth hormone (GH) secretagogue receptor type 1a (GHSR-1a) were measured using immunohistochemistry and the polymerase chain reaction (PCR) method, respectively. Ghrelin fiber projections arising from the ARC and projecting into the DVC were investigated using retrograde tracing, combined with fluorescence immunohistochemical staining. The effects of electrical stimulation (ES) of the ARC on ghrelin-responsive, glucose-sensitive DVC neurons, glycolipid metabolism, and liver lipid enzymes were determined using electrical physiological method, biochemical analysis, quantitative real-time PCR (qRT-PCR) and Western blot analysis. ResultsGHSR-1a was expressed in the DVC neurons. Ghrelin fibers originating from the ARC projected into the DVC. ES of the ARC-activated the ghrelin-responsive glucose-excited (GE) and glucose-inhibited (GI) neurons in the DVC. ES of the ARC significantly elevated the serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and glucose levels; it reduced the serum high-density lipoprotein (HDLC) and insulin levels. Moreover, ES of the ARC increased liver acetyl-CoA carboxylase-1 (ACC-1) and decreased carnitine palmitoyltransferase-1 (CPT-1) expression, resulting in lipid accumulation in the liver. All the aforementioned effects were partially blocked by pretreatment with the ghrelin receptor antagonist [D-Lys-3]-GHRP-6 in the DVC and were reduced by vagotomy. ES of the ARC increased agouti-related protein (AgRP)/neuropeptide Y (NPY) expression in the ARC and ghrelin expression in the DVC. ConclusionGhrelin fiber projections arising from the ARC and projecting into the DVC play a role in the regulation of afferent glucose metabolism and glycolipid metabolism via the ghrelin receptor GHSR-1a in the DVC.

A Dairy-Derived Ghrelinergic Hydrolysate Modulates Food Intake In Vivo.

Recent times have seen an increasing move towards harnessing the health-promoting benefits of food and dietary constituents while providing scientific evidence to substantiate their claims. In particular, the potential for bioactive protein hydrolysates and peptides to enhance health in conjunction with conventional pharmaceutical therapy is being investigated. Dairy-derived proteins have been shown to contain bioactive peptide sequences with various purported health benefits, with effects ranging from the digestive system to cardiovascular circulation, the immune system and the central nervous system. Interestingly, the ability of dairy proteins to modulate metabolism and appetite has recently been reported. The ghrelin receptor (GHSR-1a) is a G-protein coupled receptor which plays a key role in the regulation of food intake. Pharmacological manipulation of the growth hormone secretagogue receptor-type 1a (GHSR-1a) receptor has therefore received a lot of attention as a strategy to combat disorders of appetite and body weight, including age-related malnutrition and the progressive muscle wasting syndrome known as cachexia. In this study, a milk protein-derivative is shown to increase GHSR-1a-mediated intracellular calcium signalling in a concentration-dependent manner in vitro. Significant increases in calcium mobilisation were also observed in a cultured neuronal cell line heterologously expressing the GHS-R1a. In addition, both additive and synergistic effects were observed following co-exposure of GHSR-1a to both the hydrolysate and ghrelin. Subsequent in vivo studies monitored standard chow intake in healthy male and female Sprague-Dawley rats after dosing with the casein hydrolysate (CasHyd). Furthermore, the provision of gastro-protected oral delivery to the bioactive in vivo may aid in the progression of in vitro efficacy to in vivo functionality. In summary, this study reports a ghrelin-stimulating bioactive peptide mixture (CasHyd) with potent effects in vitro. It also provides novel and valuable translational data supporting the potential role of CasHyd as an appetite-enhancing bioactive. Further mechanistic studies are required in order to confirm efficacy as a ghrelinergic bioactive in susceptible population groups.

MK-0677, a Ghrelin Agonist, Alleviates Amyloid Beta-Related Pathology in 5XFAD Mice, an Animal Model of Alzheimer's Disease.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by cognitive deficits, neuroinflammation, and neuronal death. The primary pathogenic cause is believed to be the accumulation of pathogenic amyloid beta (Aβ) assemblies in the brain. Ghrelin, which is a peptide hormone predominantly secreted from the stomach, is an endogenous ligand for the growth hormone secretagogue-receptor type 1a (GHS-R1a). MK-0677 is a ghrelin agonist that potently stimulates the GHS-R1a ghrelin receptor. Interestingly, previous studies have shown that ghrelin improves cognitive impairments and attenuates neuronal death and neuroinflammation in several neurological disorders. However, it is unknown whether MK-0677 can affect Aβ accumulation or Aβ-mediated pathology in the brains of patients with AD. Therefore, we examined the effects of MK-0677 administration on AD-related pathology in 5XFAD mice, an Aβ-overexpressing transgenic mouse model of AD. MK-0677 was intraperitoneally administered to three-month-old 5XFAD mice. To visualize Aβ accumulation, neuroinflammation, and neurodegeneration, thioflavin-S staining and immunostaining with antibodies against Aβ (4G8), ionized calcium-binding adaptor molecule 1 (Iba-1), glial fibrillary acidic protein (GFAP), neuronal nuclear antigen (NeuN), and synaptophysin were conducted in the neocortex of 5XFAD and wild-type mice, and to evaluate changes of phosphorylated cyclic adenosine monophosphate (cAMP) response element binding protein (pCREB) levels, immunostaining with antibody against pCREB was performed in dentate gyrus of the hippocampus of 5XFAD and wild-type mice. The histological analyses indicated that MK-0677-treated 5XFAD mice showed reduced Aβ deposition, gliosis, and neuronal and synaptic loss in the deep cortical layers, and inhibited the decrement of pCREB levels in dentate gyrus of the hippocampus compared to vehicle-treated 5XFAD mice. Our results showed that activation of the ghrelin receptor with MK-0677 inhibited the Aβ burden, neuroinflammation, and neurodegeneration, which suggested that MK-0677 might have potential as a treatment of the early phase of AD.

DTPA-(PABn)-Leu5]-des-acyl ghrelin(1-5) as a new carrier of radionuclides and potential precursor of radiopharmaceuticals.

Ghrelin is a peptide consisting of 28 aminoacids and an octadecyl side chain (acyl group) binding to the growth hormone secretagogue receptor type 1a (GHS-R1a). Its des-acylated form, des-acyl ghrelin (DAG) binds to the corticotropin releasing factor receptor type 2a (CRF2a) located on endocrine cancer cells such as the prostate carcinoma cell line DU 145. The aim of this study is to develop a new DAG-based carrier of radionuclides with potential application in therapy. Trunctated C-terminal five aminoacids chain of the DAG peptide (H2N-Gly-Ser-Ser-Phe-Leu-COOH) was linked to DTPA to obtain [DTPA-(PABn)-Leu5]-DAG(1-5). For therapeutic application the lutetium-177 (177Lu) radionuclide was coordinated to the peptide. To determine biological and chemical properties of newly synthesized radiopharmaceutical, two iodine-131 (131I)-labelled compounds were used: [131I]-Tyr4-DAG(1-5) and full length [131I]-DAG(1-28) together with their nonradioactive forms: DAG(1-28) and DAG(1-5). Identical HPLC elution profiles of [177Lu-DTPA-(PABn)-Leu5]-DAG(1-5) before and after incubation with human serum proved its stability. The lipophilicity profile of [177Lu-DTPA-(PABn)-Leu5]-DAG(1-5) was log DO/W=-2.68±0.05, pH 7.4. Receptor affinity of the nonradioactive conjugate [Lu-DTPA-(PABn)-Leu5]-DAG(1-5) was IC50 (21.06 nmol/l), as shown against the [131I]-DAG(1-28) used as a competitor. The 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide assay indicated the significant cytotoxicity of the newly synthesized compounds, similar to that of [131I]-Tyr4-DAG(1-5). The results obtained suggest the potency of the [DTPA-(PABn)-Leu5]-DAG(1-5) as a new carrier of radionuclides in radiopharmacy.

Ghrelin Attenuates Retinal Neuronal Autophagy and Apoptosis in an Experimental Rat Glaucoma Model.

Ghrelin, a natural ligand for the growth hormone secretagogue receptor type 1a (GHSR-1a), may protect retinal neurons against glaucomatous injury. We therefore characterized the underlying mechanism of the ghrelin/GHSR-1a-mediated neuroprotection with a rat chronic intraocular hypertension (COH) model. The rat COH model was produced by blocking episcleral veins. A combination of immunohistochemistry, Western blot, TUNEL assay, and retrograde labeling of retinal ganglion cells (RGCs) was used. Elevation of intraocular pressure induced a significant increase in ghrelin and GHSR-1a expression in retinal cells, including RGCs and Müller cells. Western blot confirmed that the protein levels of ghrelin exhibited a transient upregulation at week 2 after surgery (G2w), while the GHSR-1a protein levels were maintained at high levels from G2w to G4w. In COH retinas, the ratio of LC3-II/LC-I and beclin1, two autophagy-related proteins, were increased from G1w to G4w, and the cleavage product of caspase3, an apoptotic executioner, was detected from G2w to G4w. Intraperitoneal injection of ghrelin significantly increased the number of surviving RGCs; inhibited the changes of LC3-II/LC-I, beclin1, and the cleavage products of caspase3; and reduced the number of TUNEL-positive cells in COH retinas. Ghrelin treatment also reversed the decreased levels of p-Akt and p-mTOR, upregulated GHSR-1a protein levels, and attenuated glial fibrillary acidic protein levels in COH retinas. All these results suggest that ghrelin may provide neuroprotective effect in COH retinas through activating ghrelin/GHSR-1a system, which was mediated by inhibiting retinal autophagy, ganglion cell apoptosis, and Müller cell gliosis.

Endogenous ghrelin-O-acyltransferase (GOAT) acylates local ghrelin in the hippocampus.

Ghrelin is an appetite-stimulating peptide. Serine 3 on ghrelin must be acylated by octanoate via the enzyme ghrelin-O-acyltransferase (GOAT) for the peptide to bind and activate the cognate receptor, growth hormone secretagogue receptor type 1a (GHSR1a). Interest in GHSR1a increased dramatically when GHSR1a mRNA was demonstrated to be widespread in the brain, including the cortex and hippocampus, indicating that it has multifaceted functions beyond the regulation of metabolism. However, the source of octanoylated ghrelin for GHSR1a in the brain, outside of the hypothalamus, is not well understood. Here, we report the presence of GOAT and its ability to acylate non-octanoylated ghrelin in the hippocampus. GOAT immunoreactivity is aggregated at the base of the dentate granule cell layer in the rat and wild-type mouse. This immunoreactivity was not affected by the pharmacological inhibition of GHSR1a or the metabolic state-dependent fluctuation of systemic ghrelin levels. However, it was absent in the GHSR1a knockout mouse hippocampus, pointing the possibility that the expression of GHSR1a may be a prerequisite for the production of GOAT. Application of fluorescein isothiocyanate (FITC)-conjugated non-octanoylated ghrelin in live hippocampal slice culture (but not in fixed culture or in the presence of GOAT inhibitors) mimicked the binding profile of FITC-conjugated octanoylated ghrelin, suggesting that extracellularly applied non-octanoylated ghrelin was acylated by endogenous GOAT in the live hippocampus while GOAT being mobilized out of neurons. Our results will advance the understanding for the role of endogenous GOAT in the hippocampus and facilitate the search for the source of ghrelin that is intrinsic to the brain.

Role of LPS-elicited signaling in triggering gastric mucosal inflammatory responses to H. pylori: modulatory effect of ghrelin.

Infection with Helicobacter pylori is a primary culprit in the etiology of gastric disease, and its cell-wall lipopolysaccharide (LPS) is recognized as a potent endotoxin responsible for triggering a pattern of the mucosal inflammatory responses. The engagement by the LPS of gastric mucosal Toll-like receptor 4 (TLR4) leads to initiation of signal transduction events characterized by the activation of mitogen-activated protein kinase (MAPK) cascade, induction of phosphoinositide-specific phospholipase C (PLC)/protein kinase C (PKC)/phosphatidylinositol 3-kinase (PI3K) pathway, and up-regulation in Src/Akt. These signaling events in turn exert their influence over H. pylori-elicited excessive generation of NO and PGE2 caused by the disturbances in nitric oxide synthase and cyclooxygenase isozyme systems, increase in epidermal growth factor receptor transactivation, and the induction in matrix metalloproteinase-9 (MMP-9) release. Interestingly, the extent of gastric mucosal inflammatory response to H. pylori is influenced by a peptide hormone, ghrelin, the action of which relays on the growth hormone secretagogue receptor type 1a (GHS-R1a)-mediated mobilization of G-protein dependent transduction pathways. Yet, the signals triggered by TLR-4 activation as well as those arising through GHS-R1a stimulation converge at MAPK and PLC/PKC/PI3K pathways that form a key integration node for proinflammatory signals generated by H. pylori LPS as well as for those involved in modulation of inflammation by ghrelin. Hence, therapeutic targeting these signals' convergence and integration node could provide a novel and attractive opportunities for developing more effective treatments of H. pylori-related gastric disease.

Is Ghrelin Synthesized in the Central Nervous System?

Ghrelin is an octanoylated peptide that acts via its specific receptor, the growth hormone secretagogue receptor type 1a (GHSR-1a), and regulates a vast variety of physiological functions. It is well established that ghrelin is predominantly synthesized by a distinct population of endocrine cells located within the gastric oxyntic mucosa. In addition, some studies have reported that ghrelin could also be synthesized in some brain regions, such as the hypothalamus. However, evidences of neuronal production of ghrelin have been inconsistent and, as a consequence, it is still as a matter of debate if ghrelin can be centrally produced. Here, we provide a comprehensive review and discussion of the data supporting, or not, the notion that the mammalian central nervous system can synthetize ghrelin. We conclude that no irrefutable and reproducible evidence exists supporting the notion that ghrelin is synthetized, at physiologically relevant levels, in the central nervous system of adult mammals.

Influence of ghrelin on rat pituitary GH3 cell line proliferation

Introduction. Human ghrelin is the endogenous ligand of the growth hormone secretagogue receptor type 1a (GHSR1a). It is suggested that ghrelin is involved in pituitary adenomas pathogenesis. There are inconsistent data regarding the effect of ghrelin on cell proliferation. In this study the outcome of ghrelin in the rat pituitary adenoma GH3 cell line on morphology and proliferation ratio was evaluated. The ghrelin receptor (Ghsr) mRNA expression in GH3 cell line was established as well, because it was found that heterogeneous expression pattern characterized physiological and pathological conditions of tissues of different origin.Material and Methods. Suitable experimental model pituitary tumor (rat GH3 cell line) was stimulated with ghrelin in the final concentrations 10–12 M, 10–9 M and 10–6 M. Reverse transcription followed by real time polymerase chain reaction was used for ghrelin receptor gene transcript detection. The morphology as well as cell cycle of those cells were analyzed using Axio Vert.A1 Microscope (Zeiss) and BD FACSCalibur™ flow cytometer (Beckton Dickinson), respectively. The percentages of cells in the G0/G1, S, G2/M cycle phases were evaluated using the ModFit™ software (Verity Software, Inc., USA). Results. Ghsr mRNA presence was confirmed in GH3 cells. Ghrelin did not affect conspicuously GH3 cells morphology, however the ghrelin‑induced proliferation index increase was caused by both decline of G0/G1 phases cells count and increase those being in S+G2/M (p < 0.05). Conclusions. In conclusion, this study indicates that ghrelin stimulates GH3 cells proliferation and may play role in pituitary tumorigenesis via an autocrine/paracrine pathway.

Influence of ghrelin on rat pituitary GH3 cell line proliferation

Introduction. Human ghrelin is the endogenous ligand of the growth hormone secretagogue receptor type 1a (GHSR1a). It is suggested that ghrelin is involved in pituitary adenomas pathogenesis. There are inconsistent data regarding the effect of ghrelin on cell proliferation. In this study the outcome of ghrelin in the rat pituitary adenoma GH3 cell line on morphology and proliferation ratio was evaluated. The ghrelin receptor (Ghsr) mRNA expression in GH3 cell line was established as well, because it was found that heterogeneous expression pattern characterized physiological and pathological conditions of tissues of different origin.Material and Methods. Suitable experimental model pituitary tumor (rat GH3 cell line) was stimulated with ghrelin in the final concentrations 10–12 M, 10–9 M and 10–6 M. Reverse transcription followed by real time polymerase chain reaction was used for ghrelin receptor gene transcript detection. The morphology as well as cell cycle of those cells were analyzed using Axio Vert.A1 Microscope (Zeiss) and BD FACSCalibur™ flow cytometer (Beckton Dickinson), respectively. The percentages of cells in the G0/G1, S, G2/M cycle phases were evaluated using the ModFit™ software (Verity Software, Inc., USA). Results. Ghsr mRNA presence was confirmed in GH3 cells. Ghrelin did not affect conspicuously GH3 cells morphology, however the ghrelin-induced proliferation index increase was caused by both decline of G0/G1 phases cells count and increase those being in S+G2/M (p < 0.05). Conclusions. In conclusion, this study indicates that ghrelin stimulates GH3 cells proliferation and may play role in pituitary tumorigenesis via an autocrine/paracrine pathway.

Insulinotropic action of bombesin-like peptides mediated by gastrin-releasing peptide receptors in steers.

The present study characterizes the receptor that mediates the insulinotropic action of bombesin-like peptides (BLP) in ruminants. Eight Holstein steers were randomly and intravenously injected with synthetic bovine gastrin-releasing peptide (GRP; 0.9 nmol/kg BW), neuromedin B (NMB; 0.9 nmol/kg BW), or neuromedin C (NMC; 0.9 nmol/kg BW), each alone or combined with the antagonist of GRP receptors N-acetyl-GRP-OCHCH (N-GRP-EE; 22.5 nmol/kg BW) or the antagonist of GH secretagogue receptor type 1a (GHS-R1a) [D-Lys]-GHRP-6 (21.5 nmol/kg BW). Blood samples were collected at -10, 0 (just before injection), 5, 10, 15, 20, 30, 45, 60, 75, and 90 min relative to injection time. Levels of injected peptides, insulin, and glucose in plasma were analyzed. Results showed that the peak of insulin levels was seen at 5 min after injection of NMC or GRP. Plasma glucose was observed in 2 phases; a significant rise followed a remarkable fall after NMC or GRP administration compared with injection of the vehicle ( < 0.05). On a same molar basis, effects of GRP on insulin and glucose were more potent than those of NMC ( < 0.05). The NMC-induced changes of insulin and glucose were completely blocked by N-GRP-EE, but [D-Lys]-GHRP-6 did not block any of these changes. Administration of NMB or N-GRP-EE alone did not change the circulating levels of insulin or glucose during any of the sampling time points ( > 0.05). These results indicated that the insulinotropic action of BLP is mediated by GRP receptors but not through a ghrelin/GHS-R1a pathway and that BLP may be involved in the regulation of glucose homeostasis in ruminants.

Obesity in MENX Rats Is Accompanied by High Circulating Levels of Ghrelin and Improved Insulin Sensitivity.

Ghrelin, the natural ligand of the growth hormone secretagogue receptor type 1a (GHS-R1a), is mainly secreted from the stomach and regulates food intake and energy homeostasis. p27 regulates cell cycle progression in many cell types. Here, we report that rats affected by the multiple endocrine neoplasia syndrome MENX, caused by a p27 mutation, develop pancreatic islet hyperplasia containing elevated numbers of ghrelin-producing ε-cells. The metabolic phenotype of MENX-affected rats featured high endogenous acylated and unacylated plasma ghrelin levels. Supporting increased ghrelin action, MENX rats show increased food intake, enhanced body fat mass, and elevated plasma levels of triglycerides and cholesterol. Ghrelin effect on food intake was confirmed by treating MENX rats with a GHS-R1a antagonist. At 7.5 months, MENX-affected rats show decreased mRNA levels of hypothalamic GHS-R1a, neuropeptide Y (NPY), and agouti-related protein (AgRP), suggesting that prolonged hyperghrelinemia may lead to decreased ghrelin efficacy. In line with ghrelin's proposed role in glucose metabolism, we find decreased glucose-stimulated insulin secretion in MENX rats, while insulin sensitivity is improved. In summary, we provide a novel nontransgenic rat model with high endogenous ghrelin plasma levels and, interestingly, improved glucose tolerance. This model might aid in identifying new therapeutic approaches for obesity and obesity-related diseases, including type 2 diabetes.