生长激素促释放剂受体配体的研究进展

生长激素促释放剂是一种合成的小分子化合物,它通过生长激素促释放剂受体而起作用,该受体是一种新的G蛋白偶联受体。以前曾认为生长激素促释放剂受体是一种孤儿受体,直到近年来从人和鼠的胃中鉴定到Ghrelin的存在,而改变了这种看法。Ghrelin是包含28个氨基酸残基的肽,在3号位的丝氨酸位点有辛酰化基团。该肽是在X／A样细胞分泌颗粒中发现的,Ghrelin的发现表明促垂体分泌生长激素可能不止受到来自下丘脑的生长激素释放激素的调节,同时还可能受到来自胃和下丘脑的Ghrelin的调节。     

Purification and characterization of rat des-Gln14-Ghrelin, a second endogenous ligand for the growth hormone secretagogue receptor

Ghrelin, a peptide purified from the stomach, is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R) and potently stimulates growth hormone release from the pituitary. Ghrelin is modified with an n-octanoyl group at Ser(3). This modification is essential for the activity of ghrelin. Previously, it was not known whether other ligands for GHS-R existed. Here, we report the purification of the second endogenous ligand for GHS-R from rat stomach. This ligand, named des-Gln(14)-ghrelin, is a 27-amino acid peptide, whose sequence is identical to ghrelin except for one glutamine. Southern blotting analysis under low hybridization conditions indicates that no homologue for ghrelin exists in rat genomic DNA. Furthermore, genomic sequencing and cDNA analysis indicate that des-Gln(14)-ghrelin is not encoded by a gene distinct from ghrelin but is encoded by an mRNA created by alternative splicing of the ghrelin gene. This is the first example of a novel mechanism that produces peptide multiplicity. Des-Gln(14)-ghrelin has an n-octanoyl modification at Ser(3) like ghrelin, which is also essential for its activity. Des-Gln(14)-ghrelin-stimulated growth hormone releases when injected into rats. Thus, growth hormone release is regulated by two gastric peptides, ghrelin and des-Gln(14)-ghrelin.     

Structural similarity of ghrelin derivatives to peptidyl growth hormone secretagogues

Ghrelin is a 28-amino acid residue endogenous growth hormone secretagogue. Intensive investigations revealed that the N-terminus tetrapeptide, having octanoyl group at Ser(3), is the minimum active core. In this study, we further explored the structure-function relationships of the active N-terminus portion of ghrelin using a Ca(2+) mobilization assay. The smallest and most potent ghrelin derivative we have found so far is 5-aminopentanoyl-Ser(Octyl)-Phe-Leu-aminoethylamide, showing comparable activity to the natural molecule. In the process of modifying the active core, the ghrelin-derived short analogues emerged structurally close to peptidyl growth hormone secretagogues. The N-terminus modification suggested that Gly(1)-Ser(2) unit works as a spacer, forming adequate distance between N(alpha)-amino group and n-octanoyl group. Replacement of 3rd and 4th amino acid residues to D-isomer suggested that the N-terminal dipeptide contributes to shape the biologically active geometry by effecting conformation of residues in positions 3 and 4.     

The role of C-terminal part of ghrelin in pharmacokinetic profile and biological activity in rats

Ghrelin is an endogenous ligand for growth hormone secretagogue receptor 1a (GHS-R1a), and consists of 28 amino acid residues with octanoyl modification at Ser³. The previous studies have revealed that N-terminal part of ghrelin including modified Ser³ is the active core for the activation of GHS-R1a. On the other hand, the role of C-terminal (8-28) region in ghrelin has not been clarified yet. In the present study, we prepared human ghrelin, C-terminal truncated ghrelin derivatives and anamorelin, a small molecular GHS compound which supposedly mimics the N-terminal active core, and examined GHS-R1a agonist activity in vitro, pharmacokinetic (PK) profile and growth hormone (GH) releasing activity in rats. All compounds demonstrated potent GHS-R1a agonist activities in vitro. Although the lack of C-terminal two amino acids did not modify PK profile and GH releasing activity, the deletion of C-terminal 8 and 20 amino acids affected them, and ghrelin(1-7)-Lys-NH₂ exhibited very short plasma half-life and low GH releasing activity in vivo. In rat plasma, ghrelin(1-7)-Lys-NH₂ was degraded more rapidly than ghrelin, suggesting that C-terminal part of ghrelin protected octanoylation of Ser³ from plasma esterases. Subdiaphragmatic vagotomy significantly attenuated GH response to ghrelin but not to anamorelin. These results suggest that the C-terminal part of ghrelin has an important role in the biological activity in vivo. We also found that ghrelin stimulated GH release mainly via a vagal nerve pathway but anamorelin augmented GH release possibly by directly acting on brain in rats.     

Contractile effects of ghrelin-related peptides on the chicken gastrointestinal tract in vitro

Ghrelin is an endogenous ligand for growth hormone secretagogue receptor (GHS-R), and it stimulates growth hormone (GH) release, food intake and gastrointestinal motility in mammals. Ghrelin has also been identified in the chicken, but this peptide inhibits food intake in the chicken. We examined the effects of ghrelin and related peptides on contractility of the isolated chicken gastrointestinal tract in vitro. Among ghrelin-related peptides examined (1 microM of rat ghrelin, human ghrelin, chicken ghrelin and growth hormone releasing peptide-6 (GHRP-6)), only chicken ghrelin was effective on contraction of the chicken gastrointestinal tract. Des-acyl chicken ghrelin was ineffective, suggesting that octanoylation at Ser3 residue of chicken ghrelin was essential for inducing the contraction. Amplitude of chicken ghrelin-induced contraction was region-specific: highest in the crop and colon, moderate in the esophagus and proventriculus, and weak in the small intestine. The contractile response to chicken ghrelin in the crop was not affected by tetrodotoxin (TTX), but that in the proventriculus was decreased by TTX and atropine to the same extents. D-Lys3-GHRP-6 (a GHS-R antagonist) caused a transient contraction and inhibited the effect of chicken ghrelin without affecting the high-K+-induced contraction. Chicken ghrelin potentiated electrical field stimulation-induced cholinergic contraction without affecting the responsiveness to bath-applied carbachol in the proventriculus. The location of GHS-R differs in the crop (smooth muscle) and proventriculus (smooth muscle and enteric neurons). These results indicate that ghrelin has contractile activity on gastrointestinal tract in the chicken in vitro, and the effect was region-specific. The action would be mediated through the GHS-R, which is highly sensitive to chicken ghrelin.     

Semisynthesis of human ghrelin: condensation of a Boc-protected recombinant peptide with a synthetic O-acylated fragment

The creation of peptide using a combination of recombinant expression and chemical synthesis can be a powerful tool for the production of a wide variety of polypeptides modified by phosphorylation, glycosylation, etc. We have developed a new method for the preparation of a recombinant peptide with a free N(alpha)-amino group and protected N(epsilon)-amino groups, and have used this method in the semisynthesis of human ghrelin. Ghrelin, a natural ligand for growth hormone secretagogue receptor, is a 28-residue peptide with an essential n-octanoyl modification on Ser3. A 7-residue N-terminal fragment of ghrelin containing the octanoyl modification was prepared by Fmoc chemistry. In the preparation of it, all reactions were performed on the 2-chlorotrityl resin. Additionally, TBDMS and tBu turned out to be the most effective protection groups for the Ser3 and the Ser2, Ser6, respectively. For preparation of a 21-residue C-terminal fragment, we established a two-step protease processing method for the partially protected segment. A recombinant precursor peptide was Boc protected and subsequently cleaved using two distinct proteases, OmpT and Kex2. The peptides were then coupled to each other and, after deprotection, resulted in fully active human ghrelin.     

Design and characterization of a fluorescent ghrelin analog for imaging the growth hormone secretagogue receptor 1a

Ghrelin is a 28-amino acid peptide hormone produced in the stomach. It binds to the growth hormone secretagogue receptor 1a (GHS-R1a), a class A G-protein-coupled receptor. In the present study, we describe the design, synthesis and characterization of a truncated, 18-amino acid analog of ghrelin conjugated to a fluorescent molecule, fluorocein isothiocyanate (FITC), through the addition of a lysine at its C terminus ([Dpr(octanoyl)(3), Lys(fluorescein)(19)]ghrelin(1-19)). Receptor binding affinity of this novel fluorescein-ghrelin(1-18) was similar to that of wild-type ghrelin and a synthetic GHS-R1a ligand, hexarelin. Live cell imaging in CHO/GHS-R1a cells demonstrated cell surface receptor labeling and internalization, and agonist activity of fluorescein-ghrelin(1-18) was confirmed by increased phosphorylation of ERK1/2. We also show that GHS-R1a protein is expressed primarily in the heart when compared to all other organs, suggesting high receptor density in the left ventricle. Finally, we demonstrate that fluorescein-ghrelin(1-18) binds specifically to heart tissue in situ, and its binding is displaced by both wt ghrelin and hexarelin. We have therefore developed a novel imaging probe, fluorescein-ghrelin(1-18), that can be used to image GHS-R1a in situ, for the purposes of investigating mechanisms of receptor trafficking or pharmacological agents that target GHS-R1a.     

Effect of chronic hyperghrelinemia on ingestive action of ghrelin

The stomach hormone ghrelin is the endogenous ligand for the growth hormone secretagogue receptor (GHS-R). Systemic administration of ghrelin will cause elevations in growth hormone (GH) secretion, food intake, adiposity, and body growth. Ghrelin also affects insulin secretion, gastric acid secretion, and gastric motility. Several reports indicate that repeated or continuous activation of GHS-R by exogenous GHSs or ghrelin results in a diminished GH secretory response. The purpose of this study was to examine the extent to which the acute stimulation of food intake by exogenous ghrelin is altered by chronic hyperghrelinemia in transgenic mice that overexpress the human ghrelin gene. The present findings show that the orexigenic action of exogenous ghrelin is not diminished by a chronic hyperghrelinemia and indicate that the food ingestive pathway of the GHS-R is not susceptible to desensitization. In contrast, the epididymal fat pad growth response, like the GH response, to exogenous ghrelin is blunted in ghrelin transgenic mice with chronic hyperghrelinemia.     

Ghrelin in the human myometrium

Background  

Ghrelin is a 28-amino acid octanolyated peptide, synthesised primarily in the stomach. It stimulates growth hormone release, food intake and exhibits many other diverse effects. Our group have previously determined that ghrelin inhibited human contractility in vitro. The aim of this study therefore, was to investigate the expression of ghrelin, its receptor, the growth hormone secretagogue receptor type 1 (GHS-R1), ghrelin O-acyltransferase (GOAT) which catalyses ghrelin octanoylation, prohormone convertase 1/3 (PC1/3) responsible for pro-ghrelin processing, in human myometrium, during pregnancy prior to labour, during labour and in the non-pregnant state. Modulation of ghrelin and ghrelin receptor expression in cultured myometrial cells was also investigated.     

Ghrelin--not just another stomach hormone

Growth hormone (GH) secretagogues (GHSs) are non-natural, synthetic substances that stimulate GH secretion via a G-protein-coupled receptor called the GHS-receptor (GHS-R). The natural ligand for the GHS-R has been identified recently; it is called ghrelin. Ghrelin and its receptor show a widespread distribution in the body; the greatest expression of ghrelin is in stomach endocrine cells. Administration of exogenous ghrelin has been shown to stimulate pituitary GH secretion, appetite, body growth and fat deposition. Ghrelin was probably designed to be a major anabolic hormone. Ghrelin also exerts several other activities in the stomach. The findings that ghrelin is produced in mucosal endocrine cells of the stomach and intestine, and that ghrelin is measurable in the general circulation indicate its hormonal nature. A maximal expression of ghrelin in the stomach suggests that there is a gastrointestinal hypothalamic-pituitary axis that influences GH secretion, body growth and appetite that is responsive to nutritional and caloric intakes.     

Structures and molecular forms of the ghrelin-family peptides

Ghrelin is an acylated peptide hormone produced mainly from the stomach. The major active products of the ghrelin gene in the stomach of rats, mice and humans are 28-amino acid peptides acylated at the serine-3 position with an n-octanoyl group (C8:0), called simply ghrelin. However, recent studies have revealed that the ghrelin gene can generate a variety of bioactive molecules besides ghrelin. These include acyl forms of ghrelin other than C8:0-ghrelin (i.e., n-decanoyl ghrelin or n-decenoyl ghrelin), des-acyl ghrelin, obestatin and ghrelin-associated peptides originated from the ghrelin gene. This review surveys the structures of the ghrelin peptides and molecular forms of ghrelin gene-derived products, and summarizes the knowledge about the functions of these peptides, with an emphasis on the acyl forms of the ghrelin peptide.     

In vitro and in vivo effects of ghrelin on luteinizing hormone and growth hormone release in goldfish

We studied the in vitro and in vivo effects of octanoylated goldfish ghrelin peptides (gGRL-19 and gGRL-12) on luteinizing hormone (LH) and growth hormone (GH) release in goldfish. gGRL-19 and gGRL-12 at picomolar doses stimulated LH and GH release from dispersed goldfish pituitary cells in perifusion and static incubation. Incubation of pituitary cells for 2 h with 10 nM gGRL-12 and 1 or 10 nM gGRL-19 increased LH-beta mRNA expression, whereas only 10 nM gGRL-19 increased GH mRNA expression. Somatostatin-14 abolished the stimulatory effects of ghrelin on GH release from dispersed pituitary cells in perifusion and static culture. The GH secretagogue receptor antagonist d-Lys(3)-GHRP-6 inhibited the ghrelin-induced LH release, whereas no effects were found on stimulation of GH release by ghrelin. Intracerebroventricular injection of 1 ng/g body wt of gGRL-19 or intraperitoneal injection of 100 ng/g body wt of gGRL-19 increased serum LH levels at 60 min after injection, whereas significant increases in GH levels were found at 15 and 30 min after these treatments. Our results indicate that, in addition to its potent stimulatory actions on GH release, goldfish ghrelin peptides have the novel function of stimulating LH release in goldfish.     

Morphological analysis of ghrelin and its receptor distribution in the rat pancreas

Ghrelin, a novel peptide isolated from stomach tissue of rats and humans, has been identified as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In addition to its secretion from the stomach, ghrelin is also expressed in the hypothalamic arcuate nucleus, intestine, kidney, placenta, and pancreas. GHS-R mRNA, on the other hand, is expressed in the hypothalamus, pituitary, heart, lung, liver, pancreas, stomach, intestine, and adipose tissue. Ghrelin is considered to have important roles in feeding regulation and energy metabolism as well as in the release of growth hormone (GH). Recent physiological experiments on the pancreas have shown that ghrelin regulates insulin secretion. However, sites of action of ghrelin in the pancreas are yet to be identified. In this study, to gain insight into the role of ghrelin in rat pancreatic islets, we used immunohistochemistry to determine the localization of ghrelin and GHS-R in islet cells. Double fluorescence immunohistochemistry revealed that weak GHS-R-like immunoreactivity was found in B cells containing insulin. GHS-R immunoreactivity overlapped that of glucagon-like immunoreactive cells. Moreover, both ghrelin and GHS-R-like immunoreactivities were detected mostly in the same cells in the periphery of the islets of Langerhans. These observations suggest that ghrelin is synthesized and secreted from A cells, and acts back on A cells in an autocrine and/or paracrine manner. In addition, ghrelin may act on B cells via GHS-R to regulate insulin secretion.     

Lysophospholipase I identified as a ghrelin deacylation enzyme in rat stomach

Ghrelin, discovered in rat stomach as an endogenous growth hormone secretagogue, is octanoylated at the Ser3 residue. Since this octanoylation is essential for the functions of ghrelin, the enzymes that catalyze acylation for ghrelin biosynthesis and deacylation (deactivation step) must be considered as important regulators. We found that rat stomach homogenate contained ghrelin deacylation activity, and we isolated the active fractions by column chromatography. After sequencing and expressing candidate proteins, the ghrelin deacylation enzyme in the stomach was identified as lysophospholipase I (LysoPLA I). The enzyme properties were examined using recombinant rat LysoPLA I expressed in Escherichia coli. K(m) and V(max) values were determined as 6.5 microM and 2.3 micromol/min/mg for ghrelin and 2.2 x 10(2) microM and 0.5 micromol/min/mg for lysophosphatidylcholine (LysoPC), respectively. The deacylation of both substrates was inhibited by methyl arachidonyl fluorophosphonate (MAFP), which is known as an irreversible inhibitor of LysoPLA I. These results reveal that LysoPLA I catalyzes the removal of n-octanoic acid from ghrelin to form des-acyl ghrelin. Identification of the ghrelin deacylation enzyme in the stomach and a deacylation inhibitor will be helpful in investigating ghrelin biosynthesis.     

Ghrelin receptor (GHS-R)-like receptor and its genomic organisation in rainbow trout, Oncorhynchus mykiss

Ghrelin, a GH-releasing and appetite-regulating peptide that is released from the stomach is an endogenous ligand for growth hormone secretagogue-receptor (GHS-R). Two types of GHS-R are accepted to be present, a functional GHS-R1a and GHS-R1b with unknown function. In this study, we identified cDNA that encodes protein with close sequence similarity to GHS-R and exon–intron organization of the GHS-R genes in rainbow trout, Oncorhynchus mykiss. Two variants of GHS-R1a proteins with 387-amino acids, namely DQTA/LN-type and ERAT/IS-type, were identified. In 3'-RACE PCR and genomic PCR, we also identified three GHS-R1b orthologs that are consisted of 297- or 300-amino acids with different amino acid sequence at the C-terminus, in addition to the DQTA/LN-type and ERAT/IS-type variations. Genomic PCR revealed that the genes are composed of two exons separated by an intron, and that two GHS-R1a and three GHS-R1b variants are generated by three distinct genes. GHS-R1a and GHSR-1b mRNA were predominantly expressed in the pituitary, followed by the brain. Identified DQTA/LN-type or ERAT/IS-type GHS-R1a cDNA was transfected into mammalian cells, and intracellular calcium ion mobilization assay was carried out. However, we did not find any response to rat ghrelin and a homologous ligand, des-VRQ trout ghrelin, of either receptor in vitro. We found that unexpected mRNA splicing had occurred in the transfected cells, suggesting that the full-length, functional receptor protein might not be generated in the cells. Gene structure and characterization of protein sequence identified in this study were closely similar to other GHS-R, but to conclude that it is a GHS-R for rainbow trout, further study is required to confirm activation of GHS-R1a by ghrelin or GHS. Thus we designated the identified receptor proteins in this study as GHS-R-like receptor (GHSR-LR).     

1H NMR structural analysis of human ghrelin and its six truncated analogs.

Human ghrelin, the first recognized natural ligand of growth hormone secretagogue growth hormone secretagogue receptors (GHS-Rs) (M. Kojima, H. Hosada, Y. Date, M. Nakazato, H. Matsuo, and K. Kangawa, Nature, 1999, Vol. 402, pp. 656-660), consists of 28 amino acids of which Ser3 is modified by n-octanoylation. This new peptide hormone has been implicated not only in regulation of the GH secretion but also in regulation of food intake. The discovery of ghrelin opens up more opportunities to study the relationship of ghrelin with metabolic diseases. Until now, only mass spectometry analysis has been reported on the structure of ghrelin. NMR analysis is a suitable way to study if any tertiary structure of unbound ghrelin is present in solution. NMR studies were carried out on human ghrelin and its five truncated analogs. The full-length ghrelin and its fragments exhibited random coil behavior in aqueous solution. Additional studies were carried out on the shortest active segment of human ghrelin, which consists of the first five amino acids of the ghrelin sequence (M. A. Bednarek, S. D. Feighner, S.-S. Pong, K. K. McKee, D. L. Hreniuk, M. V. Silva, V. A. Warrem, A. D. Howard, L. H. Y. Van der Ploeg, and J. V. Heck, Journal of Medical Chemistry, 2000, Vol. 43, pp. 4370-4376), to compare the spectral features with their counterparts in the full-length ghrelin. The NMR data showed behavior similar to ghrelin except for two additional nuclear Overhauser effects (NOEs) between the Phe4 NH and the protons of the beta-methylene of Ser3. CD on human ghrelin and its short active analog in water were indicative of random coil peptides. Molecular modeling based on NMR data was carried out to probe which structural features were similar to growth hormone-releasing peptide-6 (GHRP-6), a hexapeptide that binds to GHS-R releasing GH and stimulating food intake. Modeling suggested some similarities, but they were not of a nature to account for binding properties of these compounds.     

Maintenance of a normal meal-induced decrease in plasma ghrelin levels in children with Prader-Willi syndrome.

Ghrelin is a 28-amino acid peptide recently identified in the stomach as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R1a). Ghrelin is a potent stimulator of GH secretion. It was recently shown that circulating ghrelin levels in humans rise shortly before and fall shortly after every meal, and that ghrelin administration increases voluntary food intake. The hypothesis that ghrelin hypersecretion might contribute to genetic obesity has never been investigated. In this context, Prader-Willi syndrome is the most common form of human syndromic obesity. As ghrelin affects appetite as well as GH secretion and both are abnormal in PWS, it has been surmised that these alterations might be due to ghrelin dysregulation. The aim of the study was to investigate whether ghrelin is suppressed by the meals differently in PWS children than in PWS adults. Overnight circulating fasting ghrelin levels and ghrelin levels 120 min after breakfast were assayed in 7 PWS children (10.2 +/- 1.7 yr), 7 subjects with morbid obesity (10.3 +/- 1.3 yr), and 5 normal controls (8.4 +/- 1.4 yr). Because of the data spread, no statistical difference was observed in fasting ghrelin levels between PWS and control children (p = NS); anyway, fasting ghrelin levels were significantly lower in obese children than in the other groups (p < 0.05 vs. control and PWS children). Ghrelin levels were slightly suppressed by the meal in control subjects (mean fasting ghrelin: 160.2 +/- 82 pg/ml; after the meal, 141.2 +/- 57 pg/ml, p = NS); the meal failed to suppress ghrelin levels in obese children (mean fasting ghrelin: 126.4 +/- 8.5 pg/ml; after the meal, 119.1 +/- 8.3 pg/ml, p = NS). Interestingly, the meal markedly suppressed ghrelin levels in PWS children (mean fasting ghrelin: 229.5 +/- 70.4 pg/ml; after the meal, 155.8 +/- 34.2 pg/ml, p < 0.01). In conclusion, since a lack of decrease in circulating ghrelin induced by the meal was previously reported in PWS adults, the finding of a meal-induced decrease in ghrelin levels in our population of young PWS would imply that the regulation of the ghrelin system involved in the orexigenic effects of the peptide is operative during childhood, although it progressively deteriorates and is absent in adulthood when hyperphagia and obesity progressively worsen.     

Growth hormone secretagogues and ghrelin: an update on physiology and clinical relevance

The pulsatile release of growth hormone (GH) by the anterior pituitary is stimulated by small synthetic molecules termed GH secretagogues (GHS). The receptor for GHS (GHS-R) belongs to the family of G-protein-coupled receptors. An endogenous specific ligand of 28 amino acids has recently been purified from rat stomach, it has been termed 'ghrelin'. Ghrelin demonstrates potent and reproducible GH-releasing activity, as well as significant prolactin-, ACTH- and cortisol-releasing activity. However, its major physiological relevance may relate to energy homeostasis. Peripheral daily administration of ghrelin caused weight gain by reducing fat utilization in mice and rats. In man, intravenous ghrelin was shown to stimulate food intake. The pathophysiological role and the potential clinical use of ghrelin are reviewed.