Mouse ghrelin-O-acyltransferase (GOAT) plays a critical role in bile acid reabsorption

Ghrelin is a unique peptide gut hormone that requires post-translational modification to stimulate both feeding and growth hormone release. Ghrelin O-acyltransferase (GOAT) was identified as a specific acyl-transferase for ghrelin, and recent genetic deletion studies of the Goat gene (Goat(-/-)) uncovered the role of ghrelin in the regulation of glucose homeostasis. To further understand the physiological functions of the GOAT/ghrelin system, we have conducted a metabolomic and microarray profile of Goat-null mice, as well as determined Goat expression in different tissues using the lacZ reporter gene. Serum metabolite profile analysis revealed that Goat(-/-) mice exhibited increased secondary bile acids >2.5-fold. This was attributed to increased mRNA and protein expression of the ileal sodium-dependent bile acid transporter (ISBT) in the intestinal and biliary tract. Increased expression of additional solute carrier proteins, including Slc5a12 (>10-fold) were also detected in the small intestine and bile duct. Goat staining was consistently observed in the pituitary glands, stomach, and intestines, and to a lesser extent in the gallbladder and pancreatic duct. This is the first report that the GOAT/ghrelin system regulates bile acid metabolism, and these findings suggest a novel function of GOAT in the regulation of intestinal bile acid reabsorption..     

The ghrelin cell: a novel developmentally regulated islet cell in the human pancreas

OBJECTIVES: Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), was recently identified in the stomach. Ghrelin is produced in a population of endocrine cells in the gastric mucosa, but expression in intestine, hypothalamus and testis has also been reported. Recent data indicate that ghrelin affects insulin secretion and plays a direct role in metabolic regulation and energy balance. On the basis of these findings, we decided to examine whether ghrelin is expressed in human pancreas. Specimens from fetal to adult human pancreas and stomach were studied by immunocytochemistry, for ghrelin and islet hormones, and in situ hybridisation, for ghrelin mRNA. RESULTS: We identified ghrelin expression in a separate population of islet cells in human fetal, neonatal, and adult pancreas. Pancreatic ghrelin cells were numerous from midgestation to early postnatally (10% of all endocrine cells). The cells were few, but regularly seen in adults as single cells at the islet periphery, in exocrine tissue, in ducts, and in pancreatic ganglia. Ghrelin cells did not express any of the known islet hormones. In fetuses, at midgestation, ghrelin cells in the pancreas clearly outnumbered those in the stomach. CONCLUSIONS: Ghrelin is expressed in a quite prominent endocrine cell population in human fetal pancreas, and ghrelin expression in the pancreas precedes by far that in the stomach. Pancreatic ghrelin cells remain in adult islets at lower numbers. Ghrelin is not co-expressed with any known islet hormone, and the ghrelin cells may therefore constitute a new islet cell type.     

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.     

Mice with chronically increased circulating ghrelin develop age-related glucose intolerance

Ghrelin is a gut peptide that stimulates food intake and increases body fat mass when administered centrally or peripherally. In this study, ghrelin was overexpressed in neurons using the neuron-specific enolase (NSE) promoter sequences and mouse ghrelin cDNA (NSE-Ghr). Ghrelin expression in NSE-Ghr brain tissues was increased compared with wild-type mice. Ghrelin expression was also increased to a much smaller extent in liver of these mice, but mRNA levels in stomach or duodenum did not differ from wild-type mice. Body weight and composition was analyzed in two lines of NSE-Ghr mice, one line with increased circulating bioactive ghrelin (L43) and one line without (L73). No increases in body weight, food intake, or fat mass were found. Energy expenditure was measured in L43 mice and did not differ from wild-type controls, whereas locomotor activity was increased in NSE-Ghr mice. Young NSE-Ghr mice had normal glucose tolerance; however, L43 NSE-Ghr mice, but not L73 mice, developed glucose intolerance at 32 wk of age. Despite the impaired glucose tolerance in L43 mice, insulin levels did not differ from those of wild-type mice. These findings suggest a role for ghrelin in age-associated impairments of glucose homeostasis.     

人类RELM-beta基因启动子的结构与功能分析

为克隆人类抵抗素样分子β（resistin-like molecule beta, RELMβ）基因的上游启动子序列,并观察其不同截短片段的启动子活性,以人类基因组DNA为模板,通过PCR扩增方法获得-871～+50 bp、-729～+50 bp、-471～+50 bp、-438～+50 bp、-371～+50 bp大小的RELMβ启动子片段,将其定向克隆入pGL3-Basic载体,构建荧光素酶报告基因载体,并制备转录因子CDX-2结合位点的突变或缺失体.在阳离子脂质体的介导下,报告基因载体分别瞬时转染人胚肾293细胞、结肠癌HCT116和SW480细胞、宫颈癌HeLa细胞.结果发现,各RELMβ启动子片段在293、HCT116、SW480细胞中均有活性,但在HeLa细胞中活性缺失;-471～-438 bp区存在RELMβ启动子的核心调控元件.针对该区域CDX-2转录因子结合位点进行突变,能导致RELMβ启动子活性显著降低;凝胶电泳迁移率实验表明,该区段能结合CDX-2.结果提示,成功克隆了具有活性的RELMβ启动子序列,CDX-2为其重要的转录因子,为研究RELMβ基因的转录调控机制奠定了实验基础.     

Ghrelin: a striking example of neuroendocrine peptide pleiotropy

Ghrelin, a peptide predominantly produced by the stomach, has been discovered as a natural ligand of the growth hormone secretagogue receptor (GHS-R) type 1a. Shortly there after, it attracted enormous interest since it appeared as the first peripheral orexigenic factor. Besides, ghrelin exerts other neuroendocrine metabolic and non-endocrine actions (e.g. cardiovascular activities) that may rely on the widespread distribution of ghrelin and its receptor (GHS-R). The existence of several GHS-R subtypes and evidences that neuroendocrine and metabolic but not all other ghrelin actions are dependent on acylation on serine 3 add further complexity to the system whose major physiological role remains to be definitely elucidated. Ghrelin knockout(-/-) mice are neither anorectic nor dwarf though GHS-R-/- are slightly underweight and do not respond to ghrelin with increased GH secretion or appetite. Thus, the continuation of the fascinating ghrelin story as well as its potential pathophysiological implications in endocrinology and internal medicine remain open avenues for future investigations.     

Structural divergence of human ghrelin. Identification of multiple ghrelin-derived molecules produced by post-translational processing

Ghrelin, a novel 28-amino acid peptide with an n-octanoyl modification at Ser(3), was isolated from rat stomach and found to be an endogenous ligand for the growth-hormone secretagogue receptor (GHS-R). This octanoyl modification is essential for ghrelin-induced GH release. We report here the purification and identification of human ghrelin from the stomach, as well as structural analysis of the human ghrelin gene and quantitation of changes in plasma ghrelin concentration before and after gastrectomy. Human ghrelin was purified from the stomach by gel filtration and high performance liquid chromatography, using a ghrelin-specific radioimmunoassay and an intracellular calcium influx assay on a stable cell line expressing GHS-R to test the fractions. In the course of purification, we isolated human ghrelin of the expected size, as well as several other ghrelin-derived molecules. Classified into four groups by the type of acylation observed at Ser(3); these peptides were found to be non-acylated, octanoylated (C8:0), decanoylated (C10:0), and possibly decenoylated (C10:1). All peptides found were either 27 or 28 amino acids in length, the former lacking the C-terminal Arg(28), and are derived from the same ghrelin precursor through two alternative pathways. The major active form of human ghrelin is a 28-amino acid peptide octanoylated at Ser(3), as was found for rat ghrelin. Synthetic octanoylated and decanoylated ghrelins produce intracellular calcium increases in GHS-R-expressing cells and stimulate GH release in rats to a similar degree. Both ghrelin and the ghrelin-derived molecules were found to be present in plasma as well as stomach tissue. Plasma levels of immunoreactive ghrelin after total gastrectomy in three patients were reduced to approximately half of their pre-gastrectomy values, after which they gradually increased. This suggests that the stomach is the major source of circulating ghrelin and that other tissues compensate for the loss of ghrelin production after gastrectomy.     

Ghrelin stimulates interleukin-8 gene expression through protein kinase C-mediated NF-kappaB pathway in human colonic epithelial cells

Ghrelin, a newly identified gastric peptide, is known for its potent activity in growth hormone (GH) release and appetite. Although ghrelin is involved in several other responses such as stress and intestinal motility, its potential role in intestinal inflammation is not clear. Here, we show that expression of ghrelin and its receptor mRNA is significantly increased during acute experimental colitis in mice injected intracolonically with trinitrobenzene sulfate (TNBS). We found by PCR that ghrelin receptor mRNA is expressed in non-transformed human colonic epithelial NCM460 cells. Exposure of NCM460 cells stably transfected with ghrelin receptor mRNA to ghrelin, increased IkappaBalpha phosphorylation and its subsequent degradation. In addition, ghrelin stimulated NF-kappaB-binding activity and NF-kappaB p65 subunit phosphorylation, and induced IL-8 promoter activity and IL-8 protein secretion. Furthermore, our data show that ghrelin-induced IkappaBalpha and p65 phosphorylation was markedly reduced by pharmacological inhibitors of intracellular calcium mobilization (BAPTA/AM) and protein kinase C (GF 109203X). Pretreatment with BAPTA/AM or GF109203X also significantly attenuated ghrelin-induced IL-8 production. Together, our results strongly suggest that ghrelin may be a proinflammatory peptide in the colon. Ghrelin may participate in the pathophysiology of colonic inflammation by inducing PKC-dependent NF-kappaB activation and IL-8 production at the colonocyte level.     

Somatostatin suppresses ghrelin secretion from the rat stomach

Ghrelin is an acylated peptide that stimulates food intake and the secretion of growth hormone. While ghrelin is predominantly synthesized in a subset of endocrine cells in the oxyntic gland of the human and rat stomach, the mechanism regulating ghrelin secretion remains unknown. Somatostatin, a peptide produced in the gastric oxyntic mucosa, is known to suppress secretion of several gastrointestinal peptides in a paracrine fashion. By double immunohistochemistry, we demonstrated that somatostatin-immunoreactive cells contact ghrelin-immunoreactive cells. A single intravenous injection of somatostatin reduced the systemic plasma concentration of ghrelin in rats. Continuous infusion of somatostatin into the gastric artery of the vascularly perfused rat stomach suppressed ghrelin secretion in both dose- and time-dependent manner. These findings indicate that ghrelin secretion from the stomach is regulated by gastric somatostatin.